ORI GIN AL ARTICLE

Recognition of GPCRs by Peptide Ligands and MembraneCompartments theory: Structural Studies of EndogenousPeptide Hormones in Membrane Environment

Ramasubbu Sankararamakrishnan

Published online: 8 June 2006� Springer Science+Business Media, Inc. 2006

Abstract One of the largest family of cell surface proteins, G-protein coupled receptors

(GPCRs) regulate virtually all known physiological processes in mammals. With seven

transmembrane segments, they respond to diverse range of extracellular stimuli and represent a

major class of drug targets. Peptidergic GPCRs use endogenous peptides as ligands. To

understand the mechanism of GPCR activation and rational drug design, knowledge of three-

dimensional structure of receptor–ligand complex is important. The endogenous peptide

hormones are often short, flexible and completely disordered in aqueous solution. According to

‘‘Membrane Compartments Theory’’, the flexible peptide binds to the membrane in the first

step before it recognizes its receptor and the membrane-induced conformation is postulated to

bind to the receptor in the second step. Structures of several peptide hormones have been

determined in membrane-mimetic medium. In these studies, micelles, reverse micelles and

bicelles have been used to mimic the cell membrane environment. Recently, conformations of

two peptide hormones have also been studied in receptor-bound form. Membrane environment

induces stable secondary structures in flexible peptide ligands and membrane-induced peptide

structures have been correlated with their bioactivity. Results of site-directed mutagenesis,

spectroscopy and other experimental studies along with the conformations determined in

membrane medium have been used to interpret the role of individual residues in the peptide

ligand. Structural differences of membrane-bound peptides that belong to the same family but

differ in selectivity are likely to explain the mechanism of receptor selectivity and specificity

of the ligands. Knowledge of peptide 3D structures in membrane environment has potential

applications in rational drug design.

Keywords Lipid-peptide interactions Æ Ligand docking Æ Induced conformation ÆMembrane-mimetic Æ Micelles Æ Bicelles Æ Membrane protein structure ÆReceptor-ligand interactions Æ NMR spectroscopy Æ GPCR activation Æ GPCR modeling

R. Sankararamakrishnan (&)Department of Biological Sciences and Bioengineering, Indian Institute of Technology,Kanpur 208 016, Indiae-mail: rsankar@iitk.ac.in

Biosci Rep (2006) 26:131–158DOI 10.1007/s10540-006-9014-z

123

Abbreviations

Aib Alpha-aminoisobutyric acid

aPP Avian pancreatic polypeptide

bPP Bovine pancreatic polypeptide

CCK Cholecystokinin

CD Circular Dichroism

CNS Central nervous system

DCPC Dicaproylphosphatidylcholine

DHPC Dihexanoylphosphatidylcholine

DMPC Dimyristoylphosphatidylcholine

DMPG Dimyristoyl phosphoglycerol

DMSO Dimethyl sulfoxide

DOP-R d opioid receptor

DPC Dodecylphosphocholine

DynA Dynorphin A

FTIR Fourier transform infrared

GPCR G Protein-coupled receptor

GRP Gastrin-releasing peptide

GRP-R GRP receptor

HFA Hexafluoroacetone

Hyp Hydroxyproline

IR-ATR spectroscopy Infrared-attenuated total reflection spectroscopy

KOP-R j opioid receptor

Lenk Leucine-enkephalin

LMPC Lyso-myristoylphosphatidylcholine

MD Molecular dynamics

Menk Methionine-enkephalin

MOP-R l opioid receptor

NKA Neurokinin A

NKB Neurokinin B

NMB Neuromedin B

NMB-R NMB receptor

NOE Nuclear overhauser effect

NOP-R Nociceptin/orphanin FQ receptor

NPc Neuropeptide cNPAF Neuropeptide AF

NPFF Neuropeptide FF

NPK Neuropeptide K

NPY Neuropeptide Y

NT Neurotensin

NTR Neurotensin receptor

PACAP Pituitary adenylate cyclase-activating polypeptide

PDB Protein Data Bank

PP Pancreatic polypeptide

PYY Peptide YY

SDS Sodium dodecylsulfate

SP Substance P

TFE Trifluoroethanol

132 Biosci Rep (2006) 26:131–158

123

TM Transmembrane

TRNOE Transferred NOE

VIP Vasoactive intestinal peptide

VR Vasopressin receptor

Introduction

G protein-coupled receptors (GPCRs) constitute the most diverse and the largest family of

transmembrane proteins and represent a major class of drug targets [1, 2]. More than 30% of

the clinically marketed drugs are modulators of GPCR function and these drugs have thera-

peutic benefits across a broad spectrum of human diseases. Human genome project has

identified more than 700 genes that belong to the GPCR superfamily [1]. Loss- and gain-of-

function mutations in GPCR-encoding genes have been identified as the cause of an increasing

number of retinal, endocrine, metabolic, and developmental disorders [3]. Bioactive peptides,

chemoattractants, neurotransmitters, hormones, phospholipids, photons, divalent cations,

odorants and taste ligands are some of the diverse array of GPCR external ligands [1, 2]. Upon

activation by ligands, GPCRs can mediate a variety of intracellular responses via G proteins,

such as regulation of ion channels, hormone secretion, enzyme activity and gene expression.

The common structural features shared by all members of the GPCR superfamily include

seven membrane-spanning domains, a putative extracellular ligand binding domain and an

intracellular domain responsible for interaction with G-proteins or other intracellular signaling

proteins [4]. Knowledge of the three-dimensional structure of GPCRs and their ligands are

essential for understanding of their function. Unfortunately, elucidation of GPCR structures,

like other integral membrane proteins, is extremely difficult to determine experimentally.

High-resolution X-ray structure of bovine rhodopsin [5] is the only GPCR structure available

for homology modeling. Rhodopsin is unique among GPCRs in that its ligand, retinal, is

covalently bound and the structure is determined in inactivated state. Rhodopsin structure has

been successfully used as a template and the mechanism by which the agonist–receptor

complex activates the G protein has been proposed based on the modeled GPCR [6]. Other

studies indicate that rhodopsin-based homology models do not satisfy the available experi-

mental data [7, 8]. De novo modeling techniques are also being developed that do not rely on

the rhodopsin structure [9]. The current status of GPCR modeling and the progress made in this

area in the context of drug discovery is discussed in a recent review article [10].

Among several classes of GPCRs, receptors that use endogenous peptides as ligands are

known as peptidergic GPCRs [11]. This group is diverse and at least 35 different families and

their ligands have been identified. Majority of peptidergic GPCRs fall into the category of class

A (or rhodopsin-like) GPCRs and the rest can be classified as class B (secretin family). In

peptide hormones, residues that govern specificity and elicit activity form ‘message’ segment.

The residues of the message segment are evolutionarily conserved for a given peptide family

and are responsible for triggering all the receptors of that family. The variable region directs the

message to the individual receptor subtypes within a family and is called ‘address’ segment.

Message segment may lie either in the C-terminal or N-terminal end of peptide hormones.

Conformational features of endogenous peptide ligands could control receptor binding/

selectivity and influence their biological activity. Structural studies of such compounds will be

useful for rational drug design. Short and linear endogenous peptide hormones are usually very

flexible and have been shown to assume random conformations in aqueous medium (see

Biosci Rep (2006) 26:131–158 133

123

below). X-ray structures of some peptide ligands have been determined [12–14] and the

peptide conformations in GPCR-bound states are likely to be different from the structures

determined in solid-state. Investigating the bioactive conformation of flexible peptide ligands

is an active area of research. This review will focus on the structural studies of several peptide

hormones in membrane environment in which their receptors reside.

Membrane Compartments Theory

Peptide hormones can interact with its receptor site in at least two ways. There could be a direct

interaction from the aqueous phase with the extracellular loops of GPCRs. There is also a

possibility of preadsorption of peptide hormones to the target cell membranes followed by

subsequent interaction with the receptor. Kaiser and Kezdy studied biologically active peptides

that act on cell surfaces and demonstrated the role of amphiphilic cell surface environment in

influencing the structure of the peptides [15]. By increasing the amphiphilicity of the structurally

important regions of the molecules that is complementary to the cell surfaces, biological activity

of the peptides could be enhanced. Models of peptide hormone neuropeptide Y were designed by

Minakata et al. [16] to investigate the role of the hydrophobic and hydrophilic domains of

potential amphiphilic a-helix in this peptide. Their experiments demonstrated that amino acids in

the hydrophobic side of the amphiphilic a-helix could be replaced without much loss in activity

provided that the hydrophobicity of amino acids was not changed. McLean et al. [17] synthesized

cyclic, conformationally restricted disulfide analogs of NPY to investigate the role of the

amphipathic helix. The synthesized peptides contained various lengths of amphiphilic helical

region of the peptide. It was shown that the peptide with larger amphiphilic helical region had

significant interactions with lipids and higher potency in pig spleen receptors.

Robert Schwyzer and his colleagues studied several regulatory peptides on the surface of

artificial lipid bilayer membranes and developed the concept proposed by Kaiser and Kezdy

[15]. The peptide compounds were found to interact with the bilayers [18] and the resulting

conformational preferences in the membrane-bound state were correlated with their bioac-

tivities. Thus, while conformational space of the short peptide is generally poorly defined in

aqueous solution, it is strongly affected by the local environment. Based on these studies,

Schwyzer postulated ‘‘membrane compartments theory’’, a two-step model for the peptide–

receptor interaction [18, 19]. In this concept, it is the membrane-bound conformation of the

ligand that is recognized by the receptor. In the first step, it is proposed that cell membrane

induces preferred conformations and orientations of the peptide by guiding important residues

into different components of the cell membrane (hydrophobic, interface or bulk water envi-

ronments). In the second step, the peptide undergoes two-dimensional diffusion on the

membrane surface to the receptor where receptor recognition and binding occur. Experimental

support for Schwyzer’s theory came from the work of Moroder et al. on cholecystokinin

peptide [20]. A fully active CCK analogue was covalently linked to 1,2-dimyristoyl-3-mer-

captoglycerol. It was demonstrated that the lipophilic CCK adduct inserted rapidly into

phospholipid bilayers. Binding experiments showed that lipo-CCK derivative competes with

the unmodified CCK compound for the receptor binding and this confirmed a two-dimensional

membrane-bound migration of the ligand to the receptor.

Membrane Environment: Micelles, Reverse Micelles and Bicelles

Organic solvents such as methanol and TFE have been used in the conformational studies of

peptide hormones since they were considered to mimic the influences of membranes [21–23].

134 Biosci Rep (2006) 26:131–158

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Aqueous mixtures of methanol, TFE, HFA and DMSO were used in the structural studies of

opioid peptides [24, 25]. These authors argued that the viscosity of these solvent mixtures used

in their study is higher than that of pure water, but comparable to that of cytoplasm and hence

could play the role of effective environmental constraints on peptide conformation. Several

model systems have been used to mimic the cell membrane environment to determine the

structure of membrane-bound peptides [26, 27]. Novel classes of model membrane media

using lipopeptides and amphipathic polymers (‘‘Amphipols’’) are also being developed [28].

DPC is one of the well-characterized model membrane systems used frequently in high-

resolution NMR studies of peptide–membrane interactions. It is zwitterionic and present as a

predominant constituent of animal cell membrane [29]. More than two decades ago, Kurt

Wuthrich and his colleagues used DPC in solution NMR studies and demonstrated that the

conformation of micelle-bound melittin is quite closely related to that of phosphatidylcholine

bilayers [30]. DPC mimics the anisotropic environment of a lipid membrane and forms a stable

micelle which provides motional and kinetic properties desirable for solution NMR. The

electrostatic and hydrophobic components of the DPC micelles approximate a cell membrane.

Similarly, SDS has long been used as a membrane mimetic to study lipid-binding proteins.

Like DPC, this detergent also has favorable properties for NMR structural studies. The micelle

formed by SDS is relatively small and spherical, resulting in reasonable correlation time and

manageable line widths for studies utilizing solution NMR. As a result, structure determination

of smaller membrane proteins in micelles with solution NMR methods seems to be feasible

now [31, 32]. Recently, it has been demonstrated that the structure of a micelle-bound peptide

hormone, PACAP, is similar to its GPCR-bound form [33]. The conformational differences

between these two states were limited to a few residues in the N-terminal region. Thus

structures of micelle-bound GPCR-peptide ligands are believed to closely resemble that of

peptides that encounter cell membranes in vivo and hence structural studies in micelles can

provide wealth of information about the possible bioactive conformations of endogenous

peptide hormones.

Apart from DPC and SDS micelles, properties of bicelles and reverse micelles have also

been exploited in NMR studies as membrane mimetic. Reverse micelles of bis(2-ethyl-

hexyl)sulfosuccinate sodium salt (AOT) micelles have been used to mimic cell membranes. In

this system, the external shell points to the bulk hydrophobic solvent and the water is confined

in the inner cavity of the aggregate. This property mimicking the biological environments

helped to study the structures of opioid peptide hormones [34, 35]. Fiori et al. [34] have argued

that due to the large shell of ordered water molecules, reverse surfactant micelles could better

mimic the electrostatic and hydrophobic gradients of the biological membrane interface as

well as of receptor surfaces. They have claimed that the conformations of endomorphin-1

induced in AOT reverse micelles are likely to represent the best available approximation to the

real structure in the membrane-bound or receptor-bound state.

Conformations of an endogenous peptide have also been studied in binary bilayered

mixed micelles or ‘‘bicelles’’ [36]. They are aqueous lipid-detergent assemblies in which

discrete bilayer fragments are edge stabilized by certain detergents. Bicelles represent an

intermediate morphology between lipid vesicles and classical mixed micelles. They combine

attractive properties of both of these model membrane systems, while eliminating some of the

drawbacks of the both [37]. Homogenous mixing can be easily achieved in bicelles than lipid

vesicles due to the fact that they are non-compartmentalized, optically transparent and effec-

tively monodisperse. Since bicelles have a much lower detergent content than classical mixed

micelles, they maintain some key bilayer properties. Several complementary physical tech-

niques have been used to demonstrate that mixtures of DMPC and DHPC exhibit the phos-

pholipid domain structure and dicoidal shape predicted in bicellar models [38]. The bicelle

Biosci Rep (2006) 26:131–158 135

123

system used to study the structure of a opioid peptide hormone is composed of long-chain

DMPC localized in the planar section and short-chain DCPC stabilizing the torus of the disc

[36]. Due to the diamagnetic susceptibility of their phospholipids components, these bicelles

spontaneously align in the magnetic field with the bilayer normal perpendicular to the direction

of the magnetic field. A small molar raito of DMPC and DCPC was used in the studies of the

opioid ligand enkephalin. It was observed that the resulting model membranes have a small

diameter and undergo fast tumbling motion in the magnetic field. This allowed Marcotte et al.

[36] to determine the structure of bicelle-associated peptides by solution NMR techniques.

In this review, we have discussed conformational studies of some of the flexible endoge-

nous peptide hormones in membrane or membrane mimetic environments. Structures of

chemically modified peptides, non-peptide agonists or antagonists of peptidegeric GPCRs or

structural studies in hydrophobic solvents are discussed elsewhere [39]. The following sections

are organized as follows. A brief overview of each of the peptide/peptide family is given. Their

biological significance, expression profile and preferred GPCRs are discussed. Peptide struc-

tures determined in membrane environment are compared with the structures from aqueous

solution. Membrane-induced structure and its biological activity are correlated wherever it is

possible.

Structures of Peptide Hormones in Membrane Environment

Cholecystokinin Peptides

Cholecystokinin (CCK) is believed to be the most widespread and abundant neuropeptide in

CNS. Experimental and clinical studies have clearly shown that CCK participates in the

neurobiology of anxiety, depression, psychosis, cognition and nociception [40]. The family of

carboxyamidated CCK peptides (CCK-58, CCK-39, CCK-33, CCK-22 and CCK-8) is derived

from a 115-amino acid precursor molecule prepro-CCK [41]. CCK peptide hormones act on

two pharmacologically distinct GPCRs namely, CCK type 1 and type 2 receptors (CCK1-R

and CCK2-R) [42] to stimulate the secretion of pancreatic amylase, gallbladder contraction,

regulation of feeding behavior and gastric emptying. CCK1-R and CCK2-R are readily dis-

tinguished on the basis of their relative affinity for the natural ligands, their differential

distribution and their molecular structure. The CCK1-R has a higher affinity for the sulfated

CCK than non-sulfated CCK [43]. CCK2-R discriminates poorly between the sulfated and

non-sulfated CCK analogs [44]. The carboxyamidated C-terminal heptapeptide of CCK, sul-

fated at Tyr (7th residue from the C-terminus; see Table 1), is the minimal sequence necessary

for a full activation of CCK1-R, whereas for the CCK2-R, only carboxyamidated C-terminal

tetrapeptide is required [42]. Chimeric and mutagenesis studies suggest that CCK peptide

agonists interact with the extracellular domain of CCK receptors [40]. These studies report that

CCK1-R and CCK2-R have distinct binding sites despite the high sequence homology and

shared affinity for CCK-8.

Structures of the N-terminus (residues 1–47) and the third extracellular loop (residues 329–

358) of CCK1-R have been determined in the zwitterionic environment of DPC micelles [45,

64]. Interactions of these regions with the ligand CCK-8 have also been reported in the same

studies. Analysis of intermolecular NOEs and MD simulations indicated a preferred mode of

ligand binding; the N-terminus of the ligand is found in close proximity with the extracellular

portion of the first TM helix, while the C-terminus projected toward the sixth TM segment.

Similarly, the conformational features of third extracellular loop of CCK2-R (residues 352–

379) and its interactions with CCK8 were determined by high-resolution NMR in a membrane

136 Biosci Rep (2006) 26:131–158

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Tab

le1

Endogen

ous

pep

tide

horm

ones

whose

stru

cture

sw

ere

det

erm

ined

inm

embra

ne-

mim

etic

med

ium

Pep

tide:

mem

bra

ne-

mim

etic

med

ium

Am

ino

acid

sequen

cea

Ref

eren

ceP

DB

b

Chole

cyst

oki

nin

pep

tides

CC

K8

:DP

CD

YM

GW

MD

F-N

H2

[45

]1

D6

GC

CK

15

:DP

CS

HR

ISD

RD

[SO

4]Y

MG

WM

DF

-NH

2[4

6]

N.A

.N

euro

pep

tide

Yfa

mil

yN

PY

:DP

CY

PS

KP

DN

PG

ED

AP

AE

DL

AR

YY

SA

LR

HY

INL

ITR

QR

Y-N

H2

[47

]1

F8

P[A

la31,

Pro

32]N

PY

:DP

CY

PS

KP

DN

PG

ED

AP

AE

DL

AR

YY

SA

LR

HY

INL

AP

RQ

RY

-NH

2[4

8]

1IC

Yb

PP

:DP

CA

PL

EP

EY

PG

DN

AT

PE

QM

AQ

YA

AE

LR

RY

INM

LT

RP

RY

-NH

2[4

9]

1L

JVP

YY

:DP

CY

PA

KP

EA

PG

ED

AS

PE

EL

SR

YY

AS

LR

HY

LN

LV

TR

QR

Y-N

H2

[50

]1

RU

UO

pio

idp

epti

des

Men

k:N

eutr

alb

icel

les

YG

GF

M[3

6]

1P

LW

Men

k:

-vel

ych

arg

edb

icel

lsY

GG

FM

[36

]1

PL

XD

yn

A:D

PC

YG

GF

LR

RIR

PK

LK

WD

NQ

-OH

[51

]N

.A.

En

dom

orp

hin

-1:S

DS

YP

WF

-NH

2[3

4]

N.A

.O

rexi

np

epti

des

Ore

xin

-A:S

DS

EP

LP

DC

CR

QK

TC

SC

RL

YE

LL

HG

AG

NH

AA

GIL

TL

-NH

2[6

2]

N.A

.O

rexin

-B:S

DS

RS

GP

PG

LQ

GR

LQ

RL

LQ

AS

GN

HA

AG

ILT

M-N

H2

[52

]N

.A.

Ta

chyk

inin

fam

ily

Ele

do

isin

:D

PC

EP

SK

DA

FIG

LM

-NH

2[5

3]

1M

XQ

Kas

sin

in:

DP

CD

VP

KS

DQ

FV

GL

M-N

H2

[54

]1

MY

US

P:D

PC

/SD

SR

PK

PQ

QF

FG

LM

-NH

2[5

5,

56

]N

.A.

NK

A:

DP

CH

KT

DS

FV

GL

M-N

H2

[57

]1

N6

TN

KA

:SD

SH

KT

DS

FV

GL

M-N

H2

[58

]N

.A.

NK

B:D

PC

DM

HD

FF

VG

LM

-NH

2[5

9]

1P

9F

NK

B:S

DS

DM

HD

FF

VG

LM

-NH

2[5

8]

N.A

.N

Pc:

DP

CD

AG

HG

QIS

HK

RH

KT

DS

FV

GL

M-N

H2

[60

]N

.A.

Oth

ers

NT

:GP

CR

pE

LY

EN

KP

RR

PY

IL-O

H[6

1]

N.A

.P

AC

AP

:GP

CR

HS

DG

IFT

DS

YS

RY

RK

QM

AV

KK

-NH

2[3

3]

1G

EA

NP

FF

:SD

SA

GE

GL

NS

QF

WS

LA

AP

QR

F-N

H2

[23

]N

.A.

NM

B:S

DS

GN

LW

AT

GH

FM

-NH

2[2

2]

1C

98

aC

on

serv

edre

sid

ues

acro

ssth

em

emb

ers

of

the

sam

efa

mil

yar

esh

ow

nin

bo

ld;

hel

ical

reg

ion

issh

aded

ing

ray

bT

hre

e-le

tter

PD

B[6

3]

cod

esar

eg

iven

;N

.A–

–no

tav

aila

ble

Biosci Rep (2006) 26:131–158 137

123

mimetic solvent system composed of DPC micelles. NOE analysis indicates interactions of

CCK8 with the extracellular end of TM helix 7. Comparison of the results suggests an alternate

mode of binding for CCK8 to CCK1-R and CCK2-R. Structural studies are supported by many

site-directed mutagenesis experiments [65–67]. Conformational features of the C-terminal

carboxyamidated CCK15 were determined by NMR in DPC micelles [46]. The C-terminal

octapeptide of CCK15 consisted of a well-defined pseduohelix that was nearly identical to the

non-sulfated CCK8 structure (Fig. 1) in the same solvent system [45]. No clear conformational

preference was observed for the N-terminal residues of CCK15.

Neuromedin B

Neuromedin B (NMB) belongs to the family of bombesin-like peptides. Gastrin-releasing

peptide (GRP) and bombesin-related peptides are other members of this family [68]. The key

physiological functions of bombesin-like peptides include autonomic regulation, GI function,

Fig. 1 Structures of some of the endogenous peptide ligands determined in membrane mimetic environments.For each peptide, N-terminus is shown on the top and four-letter PDB code is given at the bottom (see alsoTable 1). Shown in the figure are enkephalin (1PLW), cholecystokinin (1D6G), neuromedin B (1C98),neurokinin A (1N6T), PACAP (1GEA) and neuropeptide Y (1F8P)

138 Biosci Rep (2006) 26:131–158

123

appetite control and growth regulation [68–70]. NMB has a distribution in brain, pituitary and

is also found in nerves innervating the esophagus and intestines. Bombesin hormones are also

expressed in cancers [71]. The distinct distribution of NMB and GRP suggests that the two

peptides may exhibit separate function in the brain. NMB and GRP bind to their own cognate

receptors (NMB-R and GPR-R) and they belong to the same seven-membrane-spanning do-

main GPCR superfamily. Two other bombesin receptors have also been cloned and the four

subtypes are highly homologous sharing an overall homology of 50–60% at the amino acid

level. NMB binds to NMB-R with highest affinity and to GRP-R with lowest affinity. Similarly

GRP binds to GRP-R with highest affinity and to NMB-R with lowest affinity. NMB, GRP and

other bombesin-like peptides share similar amino acids in their amidated C-terminal regions

[70] and this region plays an important role in receptor-binding and related pharmacological

effects [72]. NMB is encoded in a prepro-NMB gene [73]. The NMB Preprohormone is a 76-

amino acid precursor which consists of a signal peptide, NMB and carboxyl terminal extension

peptides with respective lengths of 24, 32 and 17 amino acids. Structure–function relationships

of NMB and its affinities to NMB-R and GRP-R have been studied extensively by Jensen et al.

[74–76].

The structure of tetradecapeptide bombesin has been studied in aqueous solution using 1H

NMR spectroscopy. The chemical shifts indicate that the molecule adopts a random coil

conformation [77]. IR and IR-ATR spectroscopy studies on bombesin and NMB in phos-

pholipids bilayer suggested that they adopt a-helical conformation in the C-terminal message

region and a random coil was predicted for the four N-terminal residues. Based on these

studies, Erne and Schwyzer [78] have proposed peptide’s interactions with the membrane. CD,

fluorescence and MD simulations studies also indicate a helix-like conformation for NMB with

its Trp residue in the apolar surface [79] (Table 1).

Three-dimensional structure of NMB in SDS micelles has been determined using NMR

spectroscopy [22]. Analysis of NOE data, 3JHNa coupling constants and distance geometry

calculations support a 310-helical structure from residues Trp4 to Phe9 and a random structure

is suggested for the N-terminal region (Table 1). Further analysis showed that the interactions

between NMB and SDS micelles are mainly weak hydrophobic interactions and the aromatic

rings are not deeply inserted into the SDS micelles. The side chains of these aromatic residues

(Trp4, His8 and Phe9) orient toward the same direction (Fig. 1) and they are of primary

importance in binding to NMB receptor [72].

Neuropeptide AF

Neuropeptide AF (NPAF) and a related octadecapeptide neuropeptide FF (NPFF) are members

of the large family of neuropeptides known as morphine-modulating peptides [80]. These

FMRFamide (Phe-Arg-Met-Phe-NH2) related peptides have been implicated in pain modu-

lation and depending on the route of administration, they have been shown to possess pro- and

anti-opioid activity [80, 81]. Cloning of NPFF gene revealed that the gene encodes for both

NPFF and NPAF [82–84]. Elshourbagy et al. [85] have cloned a novel human orphan GPCR

for which both NPAF and NPFF have high affinity. The expression levels of this receptor was

detected in many tissues [86]. Another receptor subtype was isolated, characterized and was

shown to bind NPFF and related peptides in the nanomolar and subnanomolar range [87].

The structure of NPAF was studied in trifluoroethanol/water (TFE/H2O) solutions and in the

presence of SDS micelles by Miskolzie and Kotovych [23]. In both solvent systems, the central

part of the peptide assumes an a-helical conformation (Table 1). The helical region contains a

hydrophobic cluster, which serves to bind NPAF to the micelle. NPAF’s association with the

micelles was further confirmed by spin labeling studies. Going from TFE/H2O solution to SDS

Biosci Rep (2006) 26:131–158 139

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micellar solution a change in conformation was observed in the C-terminal tetrapeptide. This

region is much more structured in micelles consisting of an inverse c-turn and a b-turn. Based

on the conformational changes in membrane mimetic micellar medium, it was suggested that

the last four C-terminal amino acids are important in receptor recognition.

Neuropeptide Y Family

The neuropeptide Y family of hormones includes neuropeptide Y (NPY), peptide YY (PYY)

and pancreatic polypeptide (PP) [88]. All NPY family members are 36-amino acids long, C-

terminally amidated and are found in central and peripheral nervous systems [89]. They display

high sequence homology and have been suggested to arise from a common ancestor gene [90].

NPY influences several physiological parameters including blood pressure, food intake, sexual

behavior and circadian rhythm [91–94]. The other two peptides play significant roles in the

regulation of digestion and behavior [95, 96]. All members of NPY family of hormones bind to

Y receptors that belong to GPCR superfamily [97]. Among the subtypes of Y receptors, NPY

and PYY display high affinity for the Y1, Y2 and Y5 receptor subtypes. NPY and PYY bind to

Y4 receptors, but PP shows relative selectivity for Y4 receptors [98]. All the Y receptors seem

to use similar signal transduction pathways primarily activating Gi which causes inhibition of

adenylate cyclase. Structural studies of NPY peptide hormones have been initiated more than

two decades ago. The crystal structure of aPP displays an extended N terminus type II po-

lyproline helix (residues 1–8) back-folded onto the C-terminal a-helix (residues 14–31) [12].

This fold, commonly referred to PP fold, was subsequently observed in solution also [99]. The

importance of amphiphilicity in C-terminal helix has been demonstrated by Minakata et al. [16].

Membrane compartments theory proposes that it is the membrane-bound conformation of the

peptide hormone that is recognized by the receptor and hence membrane binding will be an

important step for receptor recognition. Oliver Zerbe and his colleagues have studied the

structures of all the three peptide hormones (NPY, PYY and PP) in membrane-mimetic medium

and compared them with that of solution. Structures of each peptide hormone in DPC micelles

and in solution have been compared with the other two and the differences in the binding

affinities for different subtypes of Y receptors have been correlated.

Neuropeptide Y (NPY)

Three-dimensional structure of NPY in DPC micelles was calculated using NMR technique at

pH 6 [47] and it is different from the PP fold, but similar to the solution structure determined

for human NPY [100] and porcine NPY [101]. In micellar medium, N terminus of NPY (Tyr1

to Pro13) is unstructured and the C-terminal region from Ala14 to Tyr36 forms an a-helix

(Table 1 and Fig. 1). Comparison of micelle-bound and solution structures reveals a confor-

mational reorientation of C-terminal pentapeptide. Arg33 and Arg35 in this region are believed

to participate in electrostatic interactions with the receptor [102]. Spin labeling studies showed

that the amphipathic helical segment is parallel to the interface with the hydrophobic residues

of the helix facing towards the micelle surface. In solution, these hydrophobic residues were

believed to be involved in dimer formation. NMR relaxation data indicate that the unstructured

N-terminus is completely flexible in aqueous solution. Results of spin labeling studies suggest

that C-terminus is oriented towards the micelle surface. Based on these studies, Bader et al.

[47] speculated that partitioning of Tyr36-amide at the interface together with the anchoring of

the helix to the membrane via hydrophobic residues Ile31/Thr32 will provide proper posi-

tioning and pre-orientation of key interaction residues Arg33 and Arg35 relative to the

membrane. Such positioning/orientation are likely to facilitate the receptor recognition.

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[Ala31, Pro32] NPY

The mutants [Ala31, Aib32]NPY, [Ala31, Pro32]NPY and [Ala31, Hyp32] bind selectively to

the Y5 receptor with affinity in sub-lM range [103]. Structure and dynamics of [Ala31,

Pro32]NPY bound to DPC micelles were examined by 1H NMR and analysis of 15N-relaxation

data [48]. NOE data, chemical shift analysis and NMR relaxation measurements showed a

highly flexible N-terminus, a stable helical segment consisting of residues Leu17 to Leu30 and

a decrease in the stability of C-terminus (Table 1). The nearly complete lack of NOEs in the C-

terminus results in a highly disordered segment in that region. However, Tyr36-amide comes

into proximity of the membrane as shown by the spin-label experiments [48]. Membrane

anchoring is no longer mediated by Ile28/Asn29 as observed in NPY but rather shifts to Asn29/

Leu30. Thus compared to the wild type NPY, an increased rigidity in the central helix and a

much more flexible C-terminal pentapeptide are observed in the mutant. The pentapeptide

Ala31 to Arg35 can be considered as a flexible loop being anchored to the membrane through

residues Asn29/Leu30 as well as through Tyr36-NH2. In NPY, the functionally important

Arg33 and Arg35 are in regular helical turn and membrane anchoring takes place through

residues 32–36. These basic residues are more flexible in the mutant. Here again based on the

structural studies, Bader et al. [48] speculated that the selectivity of [Ala31, Pro32]NPY

mutant might be due to spatial charge complementarity between the positively charged Arg

residues from the ligand and the negatively charged residues from the receptor.

Pancreatic Polypeptide (PP)

Solution structure bPP shows a clear PP-fold [99] in which the N-terminal polyproline helix is

bent back onto the C-terminal a-helix and the interface in between consists of hydrophobic

contacts. Although NPY and PP share a high sequence homology, structural studies of human

and porcine NPY [100, 101] revealed that N-termini remains unordered, free and flexible in

solution. Any structural differences in the conformations of membrane-bound NPY and bPP

are likely to explain their preferences for different Y receptors. Lerch et al. [49] have studied

the structure of bPP in membrane mimetic DPC micelles and its dynamics in both DPC as well

as in solution. They have found that bPP exists in the form of dimer in solution and the stability

of its PP-fold was attributed to a combination of inter- and intra-molecular aromatic ring-

stacking interactions. Based on NOE and NOSEY data, such dimer formation is excluded in

the case of micelle-bound bPP and the typical PP-fold is lost upon binding to membrane. In

contrast to micelle-bound NPY structure [47], spin-labeling studies show that several N-

terminal residues in bPP (Ala3, Glu4, Glu6, Gly9 and Ala12) are in the vicinity of membrane–

water interface (Table 1). This difference might be due to the presence of an aromatic residue

(Tyr7) that are usually highly enriched at the membrane interface in transmembrane proteins

[104]. Micelle-bound bPP shows a helical structure between Met17 and Leu31 and the helix is

oriented parallel to the micelle surface. On the basis of deuterium exchange and spin-label

experiments, it was concluded that binding of the helical region between residues 17–25 is

similar for bPP and NPY. However, although structurally stable, residues 26–30 was found to

be tilted away from the membrane–water interface. NOE data reveal that the C-terminal

residues 33–35, important for receptor activation, are close to the micelle surface or partly

buried. It should be noted that Pro34 in bPP and Pro32 in [Ala31, Pro32] NPY [48] mutants

result in the disruption and shortening of the a-helical segment in the respective peptides and

introduces flexibility in this region. Importance of C-terminal region for achieving high affinity

has been demonstrated in experimental studies. Reduced binding affinities for PP are observed

if C-terminal amide is modified or Tyr36 is deleted. Similar to NPY, electrostatic interactions

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have been suggested to play an important role for initial recognition of the receptor and Arg25,

Arg26 and/or Arg33, Arg35 have been speculated to interact with the acidic residues of the

receptor (Table 1). N-terminal residues of bPP are also likely to be significant in the binding

the Y4 receptor and the peptide’s efficacy.

Peptide YY (PYY)

PYY and NPY have very similar binding profiles with nanomolar inhibition constants at

various Y receptor subtypes [98] and hence they are expected to display similar conformations

in the particular environment in which they are recognized. High-resolution NMR spectros-

copy was used by Lerch et al. [50] to study the structures and backbone dynamics of porcine

PYY in the solution state and when bound to DPC micelles. The NMR structures calculated in

solution clearly display the typical PP-fold similar to the crystal structure of aPP [12] or the

solution structure of bPP [99]. In DPC-bound state, the N-terminus of PYY is unstructured and

an a-helix between Leu17 and Val31 is present in all NMR-derived structures (Table 1). An

interesting feature is that the helix displays a slight curvature with the hydrophobic residues

pointing towards the interface. As observed in other structures of NPY peptides, the a-helix

displays a pronounced amphipathicity and anchors to the DPC micelles via intercalation of

hydrophobic side chains into the micelle interior. No such association with the micelle surface

is observed for the flexible N-terminus. A 310-helix is observed for residues 33–35 in more

than 50% of the NMR-derived structures. Thus the overall solution structure of PYY is similar

to that of bPP [99] while the micelle-bound state shows the general features as described for

the other members of the NPY family [47–49]. However, the following features are observed

only between NPY and PYY. (a) Identical residues of NPY and PYY are forming the mem-

brane–binding interface. (b) The C-terminal pentapeptide is much more rigid with its con-

formation highly similar to the one observed for NPY. (c) Tyr36-amide is partitioned at the

water–membrane interface in both NPY and PPY and (d) N-terminus freely diffuses into

solution. Apart from the conformational differences in the C-terminus between bPP and PYY,

the Tyr36-amide is held at larger distance from the interface and N-terminus is loosely

associated with the micelle surface in bPP [49]. When Arg33 and/or Arg35 are replaced by

alanine in human NPY, binding affinities at the human Y1 receptor drop by more than four

orders of magnitude [105]. Considering the high degree of sequence homology between PYY

and NPY, especially in the C-terminal half, the almost identical conformation in the

C-terminal pentapeptide of micelle-bound NPY and PYY is likely to be of biological rele-

vance. Based on these studies, Zerbe and his colleagues have proposed that PP-fold found in

the solution for PYY is of little relevance for binding to the Y receptor subtypes.

Neurotensin

Neurotensin (NT) is an endogenous tridecapeptide found in the CNS as well as in the gas-

trointestinal tract [106]. It exerts potent CNS effects including hypothermia, anti-nociception,

modulation of dopamine neurotransmission, and stimulation of anterior pituitary hormone

secretion [107]. A hypothesis speculating a role for NT in schizophrenia states that a hypo-

functioning neurotensin system in the limbic region of the brain is involved with the patho-

physiology of schizophrenia [108]. NT mediates its effects through two cell surface receptors

NTR1 and NTR2, both are members of the family of GPCRs [109–111] In addition to the full

length peptide, the C-terminal fragment 8–13 also has been found to interact with NTR1 with

high affinity [109, 112].

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Structure of NT was investigated in aqueous solution, methanol and SDS micelles [113,

114]. No discernable secondary structure was observed in water and methanol. The confor-

mational ensemble of the peptide is observed to narrow in SDS micelle with local extended

structure [114]. Conformation of NT(8–13) in its GPCR-bound form has been studied using 2D

solid-state NMR experiments [61]. Results on solid-phase NT(8–13) immobilized in a deter-

gent environment indicate that the peptide remains largely unstructured in the absence of the

receptor. In the presence of NTR1, this agonist changes from a disordered state into a defined

b-strand conformation (Table 1).

Opioid Peptides

Endogenously produced opioid peptides and exogenously administered opioid drugs, such as

morphine, are not only among the most effective analgesics known but also highly addictive

drugs of abuse [115]. Other effects produced by the opioid peptides include respiratory

depression, euphoria, feeding, the release of hormones, and inhibition of gastrointestinal transit

[116, 117]. Opioid ligands activate opioid receptors that belong to the large superfamily of

seven TM GPCRs [118]. From the pharmacological responses to the repertoire of opioid

ligands, a variety of opioid receptor subtypes have been uncovered by extensive pharmaco-

logical studies. At least four different opioid receptors have been cloned (MOP-R, KOP-R,

DOP-R and NOP-R; www.iuphar.org). Opioid receptors are about 60% identical to each other

with TM helices having the greatest homology [119]. Endogenous opioid peptides are derived

from four precursor molecules; pro-opioimelanocortin, pro-dynorphin, pro-enkephalin and

pro-nociceptin/orphanin FQ and are expressed in CNS and in various glands throughout the

body [119, 120]. Except for nociceptin/orphanin FQ, all ‘typical’ opioid peptides derived from

the other precursors have the tetrapeptide sequence Tyr-Gly-Gly-Phe at their N-terminus and

they have different affinities for MOP-R, DOP-R and KOP-R [118]. Nociceptin/orphanin FQ

contains a Phe instead of the N-terminal Tyr, a residue necessary for high-affinity binding to

the classic opioid receptors [121]. There are also amidated tetrapeptides like endomorphins,

which are structurally unrelated to the typical opioid peptides, but show highest affinity and

selectivity for the MOP-R [122]. Structural studies of several opioid peptides in different

solvents have been reported [24, 25, 123]. A recent article discusses conformational analysis of

opioid peptides in the solid states and the membrane environments [124]. In this review,

structures of three endogenous opioid peptides in membrane-mimetic environment are dis-

cussed.

Enkephalin

Enkephalins bind preferentially to DOP-R with a significant affinity for MOP-R [125]. These

neuropeptides have the amino acid sequences Tyr-Gly-Gly-Phe-Met (Methionine-enkephalin;

Menk) and Tyr-Gly-Gly-Phe-Leu (Leucine-enkephalin; Lenk). In order to understand the

‘‘bioactive’’ conformation, enkephalins have been investigated using diverse experimental

techniques in model membranes [126–131]. NMR, IR and Raman spectroscopy experiments

carried out in water exhibited enkephalin conformations characteristic of an extended random-

coil polypeptide with no distinguishable secondary structure [132–134]. In membrane-like

medium [PC/PS vesicles, lyso PC or SDS micells or reverse bis(2-ethylhexyl) sulfosuccinate

micelles] a bend structure, more often a b-turn structure, is observed for Lenk and Menk [35, 129,

132, 135]. X-ray structures of enkephalins and analogues showed extended or bend structures

depending on the crystallization solvent and degree of hydration (for a review, see [14]).

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Recently, Marcotte et al. [36] investigated the conformation of Menk in fast-tumbling

bicelles using multidimensional 1H NMR technique. Bicelles used in this study were com-

posed of long-chain DMPC localized in the planar section and short-chain DCPC stabilizing

the torus of the discs. To investigate the effect of phospoholipid headgroups on the confor-

mation, Menk was also studied in negatively charged bicelles in which 10 mol% of DMPC

was replaced by DMPG. Structures of Menk determined in zwitterionic and negatively charged

bicelles were divided into five groups according to the peptide backbone conformation. Rel-

ative orientation of two aromatic residues was also considered when analyzing the structures

(Fig. 1). All the Menk structures are observed to be in bent form, and the side chains of

residues Tyr1, Phe4 and Met5 form a hydrophobic patch (Table 1). Once in contact with the

membrane, the formation of this hydrophobic patch is expected to enhance the peptide’s

interaction with the apolar region of the bilayer with their amide region close to the surface.

Many NOEs used to calculate the structures of Menk were similar in both membrane systems

and hence the calculated structures show similarities. An important conclusion from this study

is that different conformers are possible for enkephalins in a membrane environment. Based on

the structural grouping from two different bicellar systems, it is suggested that Menk would

adopt conformations suitable for binding to both MOP-R and DOP-R. Analysis of NOE

distance restraints derived from the two different systems suggests that variations in the

biological membrane composition would have an effect on the conformation adopted by

enkephalins.

Dynorphin A (DynA)

DynA is an endogenous ligand selective for KOP-R and has 17 amino acids [136–138]. Its

potential as an analgesic has made it an interesting target for research since its discovery more

than two decades ago. The 13-residue N-terminal fragment [DynA(1–13)] has been shown to

have the same pharmacological properties as its parent peptide [136] and hence this shorter

fragment is being used in many experimental studies in place of the native peptide. Details of

receptor binding characteristics and structure–activity relationships of DynA and related

peptides are discussed in the review by Naqvi et al. [139] Conformation of dynorphin has been

investigated in various solvents by spectroscopic methods [21, 140, 141]. FTIR, NMR and CD

studies of DynA(1–13) have shown a largely unstructured peptide in solution [140, 141]. IR-

ATR spectroscopy and capacitance measurements studies suggested a helical structure for

DynA(1–13) on contact with the neutral membranes [142]. The N-terminal message segment is

functionally important and more hydrophobic and the address segment is more hydrophilic and

charged. This amphiphilic ‘‘primary’’ structure of DynA(1–13) and hydrophobic forces have

been suggested to be the principal factors for strong binding and orientation at the membrane–

water interface [142].

Tessmer and Kallick have reported a detailed study of DynA bound to DPC micelles using

NMR spectroscopy and distance geometry calculations [51]. The structures determined in this

study indicate a well-defined a-helix from Phe4 to Pro10. Two types of b-turns are observed in

the C-terminal region from Trp14 to Gln17 (Table 1). The structures are less well determined

at the N-terminus and from residues Lys11 to Leu13. This structure supports the model that N-

terminal helical segment of DynA binds perpendicularly to the membrane surface [142]. Since

considerable amount of ‘‘secondary amphiphilicity’’ is also displayed by the DynA helix,

binding parallel to the membrane surface is also not ruled out. Fluorescence emission spec-

troscopy studies have been used to find the importance of Trp14 in the C-terminal b-turn

region [143]. MD simulation studies of DynA and its fragments in explicit bilayers have

demonstrated the importance of aromatic residues in orienting the peptide hormone within the

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bilayers [144–146]. Several basic residues present in this hormone interact favorably with

different components of the lipid to stabilize the peptide–lipid complex.

Endomorphin-1

Endomorphin-1 is a tetrapeptide and an atypical opioid ligand with three aromatic residues

[122]. Conformational preferences of this small peptide has been studied in reverse micelles of

bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) [34]. In reverse micelles, the external shell

points toward a bulk hydrophobic solvent and the water is confined in the inner cavity of the

aggregate. This medium has been considered to mimic most closely the biological environ-

ments and used in the structural studies of Lenk [35] and endomorphin-1 [34]. Structure of

endomorphin-1 determined in reverse AOT micelles has been compared with the preferred

fold in aqueous solution as well as in SDS micelles [34]. In aqueous solution two families of

structures were obtained and they differed mainly in the orientation of the backbone. All

structures adopted a slightly bent conformation and the turn is not rigid and less well defined.

Stabilization of backbone conformation is attributed to the hydrophobic microenvironment

built up in aqueous solution by the aromatic groups of Tyr1 and Phe4 residues (Table 1). Trp3

side chain is oriented toward the bulk solvent. In AOT reverse micelles, two major conformers

were observed with one of them having cis-configuration in Tyr1-Pro2 imide bond. NOE data

between the peptide and AOT molecule confirms the deep insertion of Tyr-1 aromatic ring into

the lipid layer of the reverse micelle. The peptide backbone of both the conformers shows a

bent structure involving the two central residues Pro2 and Trp3. The most populated conformer

in SDS micelles was found to be significantly less compact and the backbone assumed a

more stretched conformation than in the AOT system. In SDS as well as in AOT systems, the

aromatic groups have the same arrangements. Side chains of Tyr1 and Trp3 point to the same

direction opposite to that of Phe4 aromatic group and this arrangement is different in water.

Thus a reordering of endomorphin structure takes place from water to the membrane-mimetic

medium. The relative orientation of aromatic groups has been predicted to dictate the

l-receptor selectivity.

Orexin Peptides

The recently discovered orexin peptides have been shown to be involved in sleep-wakefulness

regulation as well as in feeding [147, 148]. Both peptides are derived from a 130 amino acid

precursor protein, prepro-orexin which is encoded by a gene localised to human chromosome

17q21. These peptide hormones bind to two GPCRs (orexin-1 and -2 receptors) to exert their

biological functions. Orexin A acts on both receptors equally, whereas orexin-B has a higher

affinity for orexin-2 [147]. Orexin-A has an N-terminal pyroglutamyl residue. Both the neu-

ropeptides are C-terminally amidated and share 46% sequence identity. It has been shown that

orexin-containing neurons in lateral hypothalamic area and dorsomedial hypothalamic nucleus

play an important role in integrating the complex physiology underlying feeding behavior

[149]. The absence of orexin peptides in the patient results in narcolepsy, a chronic sleep

disorder [150]. Structures of both the orexin peptides have been determined in SDS micelles

[52, 62] as well as in solution [151].

NMR studies show that orexin-A has two helical segments (Asp5 to Gln9 and Leu16 to

Gly22; see Table 1). The peptide segment Leu16 to Gly22 is observed to be helical in solution

also (PDB ID: 1WSO and 1R02). It is speculated that the shorter N-terminal helical segment

might have resulted due to the two disulfide bridges involving cysteines within and outside this

helix. The second helix (Leu16 to Gly22) has been shown to be amphipathic and is believed to

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be important in membrane binding. This structure has been used to explain the results of

studies using truncated orexin-A analogues and alanine substitution mutagenesis experiments

[152, 153]. Substitutions along the hydrophobic face of the helix (Leu16, Leu19 and Leu20)

have been shown to significantly reduce the potency of Orexin-A (15–33) [152]. Due to

spectral overlap and lack of many NOE restraints, it was difficult to determine the structure of

functionally important C-terminal region in SDS micelles. Miskolzie and Kotovych [62] have

predicted that this segment might adopt a turn-like or short helical structure. In contrast to the

micelle structure, C-terminal region of orexin-A is clearly helical in solution (PDB codes:

1WSO and 1R02).

The shorter Orexin-B peptide has also been shown to have two helical segments in SDS

micelles. The longer helix overlaps with the second helix of orexin-A. A short a-helix is also

present towards the peptide’s C-terminal region which was not observed in orexin-A. A similar

structure for orexin-B, two a-helices connected with a short linker, was also deduced in H2O

and 30% trifluoroethanol solutions [151]. The last two amino acids (Thr and Met) in the

C-terminus are unstructured in micelles and it is argued that the conformational freedom of

these receptor binding residues is essential for its biological activity [52].

Pituitary Adenylate Cyclase-Activating Polypeptide

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38 amino acid peptide

(PACAP38) that belongs to the PACAP/Glucagon superfamily [154]. There are nine bio-

active peptides in this superfamily including secretin and vasoactive intestinal peptide

(VIP). Members of this superfamily are related in structure by the N-terminal amino acids.

The structure of biologically active region of PACAP, corresponding to the N-terminal 27-

amino acid sequence (PACAP27), has been totally preserved during the evolution, from

amphibians to mammals [154, 155]. The human VIP peptide is 70% identical to PACAP27.

Both PACAP and VIP have a widespread distribution and are known to affect the neural,

circulatory, gastrointestinal, endocrine and immune systems [154]. PACAP and VIP have

equal affinity for two GPCRs, VPAC1 and VPAC2. In addition, PACAP binds to PACAP-

specific receptor, PAC1 [155, 156]. All three receptors belong to Class B GPCRs, also

known as secretin family of GPCRs. Class B GPCRs comprise a moderately sized N-

terminal domain of ~100 to 160 amino acid residues connected to the 7-TM a-helical region

[157].

PACAP, VIP and secretin have been shown to interact with biomimetic phospholipid

monolayers and bilayers at physiological concentrations. All the three peptides undergo

conformational transition from predominantly random coil in aqueous solution to a-helix in

phospholipids [158]. It is proposed that molecular interactions between the peptides and the

membrane medium protect the peptides from hydrolytic attack and enzyme degradation in vivo

[159, 160].

Recently, Inooka et al. [33] determined the conformation of PACAP21, bound to PACAP-

specific receptor by NMR spectroscopy. PACAP21 acts as an agonist with moderate affinity to

the receptor and displayed a large dissociation rate. PACAP21 bound to the receptor is

composed of three secondary structure elements (Fig. 1): an N-terminal extended segment

(residues 1–3), two consecutive b-turns (residues 3–7) and a C-terminal a-helix (residues 8–

21). The conformation of PACAP27 bound to DPC micelles was also determined and com-

pared with the receptor-bound PACAP21 structure. The micelle-bound PACAP27 consisted of

a longer C-terminal helix (residues 5–27) and a disordered N-terminal tail. A loosely packed

hydrophobic core (formed by residues Ile5, Phe6, Tyr10, Tyr13 and Met17) and a compact

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hydrophobic cluster (formed by residues Tyr22, Leu23 and Val26) interact with the micelle

surface and give an amphipathic character to the helix (Table 1).

The a-helical conformation of the C-terminal region of the receptor-bound PACAP21 is

strikingly similar to that of micelle-bound PACAP27. The N-terminal structure of receptor-

bound PACAP21 is unique and not observed in the micelle-bound form. The b-coil formed by

the two b-turns is responsible for producing a hydrophobic patch that results in a totally

different sidechain arrangement in the receptor-bound peptide. The functional significance of

the hydrophobic patch is supported by several experimental studies [155]. The results of this

study demonstrate that the structural differences observed between the receptor-bound and

micelle-bound PACAP is limited to the few N-terminal residues. This strongly supports the use

of micelles to study the structures of peptide hormones. Based on this study, Inooka et al. [33]

proposed a two-step ligand transportation model, very similar to the membrane compartments

theory proposed by Schwyzer [18, 19].

Tachykinin Peptides

One of the largest peptide families described in the animal kingdom, tachykinins have been

isolated from invertebrate and vertebrate tissues. Tachykinins have the characteristic C-ter-

minal pentapeptide Phe-Xaa-Gly-Leu-Met-NH2, where Xaa is either Phe/Tyr (aromatic

tachykinins) or Val/Ile (aliphatic tachykinins) [161]. All natural tachykinins are amidated at

the C-terminus and deamidated peptides are biologically inactive [162]. The biological activity

of tachykinins is due to their interactions with three GPCRs––NK1, NK2 and NK3––which

share considerable sequence homology and are heterogeneously distributed within each spe-

cies [163–165]. The influence of some key amino acids on receptor selectivity and activity in

the tachykinin sequences have been clarified in the recent experimental studies [166]. Some of

the biological responses induced by tachykinins include stimulation of extravascular smooth

muscle, powerful vasodilation, hypertensive action, nociception and neuroimmunomodulation.

Several reports suggest that these peptides are involved in the development of different dis-

eases such as bronchial asthma, inflammatory bowel syndrome and psychiatric disorders [167].

They are also implicated in neurodegenerative disorders like Alzheimer’s disease, schizo-

phrenia, Parkinson’s disease and epilepsy [168, 169]. Tachykinins possess a widespread dis-

tribution in the central and peripheral nervous system. They also have a limited and species-

dependent distribution in non-neuronal structures [163]. The three classical members of the

mammalian tachykinin family are Substance P, Neurokinin A (NKA) and Neurokinin B

(NKB). In addition, the N-terminally extended forms of NKA, named neuropeptide K (NPK)

and neuropeptide c(NPc), are also biologically active peptides. SP, NKA, NPK and NPc are

encoded by a single gene, preprotachykinin A, by alternative RNA splicing [170, 171]. NKB is

derived from a different gene, preprotachykinin B [172]. SP, NKA and NKB are the primary

endogenous ligands for NK1, NK2 and NK3 receptors respectively. However, a certain degree

of cross reactivity is observed for the tachykinin peptides among the three receptor subtypes

[163]. Other non-mammalian tachykinin peptides include eledoisin of mollusk origin, kassinin

and physalaemin of amphibian origin [161]. Affinity of eledoisin and kassinin for the mam-

malian tachykinin receptors is weak and their selectivity is less pronounced compared to other

tachykinins. For a detailed account of different occurrences, species distributions and local-

izations of the numerous members of the tachykinin peptide family and extensive pharma-

cological studies on the non-mammalian tachykinins, readers are referred to the recently

published review article by Severini et al. [161]. Structures of both mammalian and

non-mammalian tachykinin peptides have been studied in membrane mimetic solvents/lipo-

somes and are briefly described below.

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Non-Mammalian Tachykinins

Grace et al. [53] have investigated the structure of eledoisin in different solvents using CD and

NMR spectroscopic techniques. CD spectrum in aqueous solution shows that eledoisin is

primarily unstructured. When bound with DPC micelles, structure of eledoisin is helical be-

tween residues 4–11 (Table 1). Analysis of chemical shift values, NOE data and structures

calculated with NMR-derived distance constraints, all suggest a structural equilibrium between

310- and regular a-helix. Helical structure in the functionally important C-terminal segment is

associated with the peptide’s poor binding property of eledoisin to the NK1 receptor. The

upper half of the helix is the hydrophobic C-terminus (Phe-7, Ile-8, Leu-10, Met-11) and the

hydrophilic lower half is comprised of Ser-3, Lys-4 and Asp-5. Grace et al. [53] have pos-

tulated that the hydrophobic C-terminus interacts with the TM region of the receptor with the

binding pocket formed by Phe-7, Leu-10 and Met-11. The hydrophilic lower half has been

suggested to play important role in the affinity and selectivity of the peptide with Lys-4 and

Asp-5 as anchoring points. Similarly, upon binding to DPC micelles, a helical conformation is

induced in the central core and the C-terminal region of kassinin [54]. Although less defined,

N-terminal residues of this peptide seem to display some degree of order.

Neurokinin A (NKA)

Structure of NKA has been investigated in two different membrane mimetic systems by NMR.

Chandrashekar and Cowsik have studied NKA in DPC micelles [57]. Whitehead et al. have

used SDS micelles to understand the influence of membrane medium to induce a secondary

structure for NKA [58]. CD results suggest that NKA undergoes a conformational transition

from a prevalently random coil state in water to a-helical state when SDS and DPC micelles

are added [57]. Analysis of chemical shifts and NOE data indicate a helical structure for

residues 4–10 when bound to DPC (Table 1). Three-dimensional structures generated using

NMR results show that NKA has a preference for 310-helix over regular a-helix. A turn

structure is likely to be the conformation of the first three N-terminal residues (Fig. 1).

Identification of such a folded structure in N-terminus has been suggested to represent an

essential feature of NK2 binding. Structural determination of NKA in SDS micelles indicates a

helical structure from residues 6 to 10 [58]. The helical core of NKA seems to be better defined

in DPC than in SDS.

Neurokinin B (NKB)

Mantha et al. [59] studied NKB using CD and NMR techniques in different solvents. Primarily

unstructured NKB in aqueous solution acquired helical conformation upon addition of solvents

SDS and DPC. The likely structure emerged from NOE data and structure calculations is a 310-

helix for residues 1–3, followed by a predominantly a-helical structure for residues 4–10

(Table 1). This structure is suggested to exist in equilibrium with another where regular a-helix

extends from Met2 to Met10. It should thus be noted that the N-terminal address domain of

NKB retains a substantial conformational order in DPC and it may represent an essential

feature of NK3 binding. It is proposed that the helix stabilization through an increase in helix

length results in reduction in the flexibility of the C-terminal message domain and this situ-

ation is determined to be unfavorable for NK1 receptor binding. Structural studies of NKB in

SDS micelles show a helical structure from residues 4 to 10, thus a reduced helical content as

observed in NKA [58].

148 Biosci Rep (2006) 26:131–158

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Neuropeptide c (NPc)

NPc is an N-terminally extended form of NKA and NKA corresponds to the residues 12–21 of

C-terminal region of NPc. CD experiments on this peptide were performed in phosphate

buffer, in the presence of SDS micelles and in DMSO and they revealed that NPc is flexible in

polar solvents [173]. A tendency to adopt a helical structure was observed in hydrophobic

environment. In DMSO, NPc was shown to adopt a b-turn structure from residues 6 to 12 and a

random structure on the N- and C-terminal fragments [173]. Lee et al. [174] investigated NPcin 200 mm SDS micelles from two different sources. NMR studies suggested that the structure

of NPc from goldfish contains a stable a-helix from His12 to Met 21, while mammalian NPchas a short helix from Ser16 to Met21. Cowsik and coworkers [60] have reported the structural

studies of NPc in different solvents. CD studies revealed an a-helical conformation in the

presence of anionic DMPG vesicles. Analysis of chemical shifts and NOE data indicate a

helical secondary structure from residues 13 to 21 in the presence of DPC and a type II¢ b-turn

precedes the helical structure from His9 to Arg11 (Table 1). A b-turn was also observed for

goldfish NPc from residues 9 to 11 in the presence of SDS micelles [174]. In DPC micelles, a

b-turn-like structure is observed in the N-terminal region between Gly3 and Ile7 [60]. The

identification of folded conformation in N-terminus has been described to be an essential

feature for NK2-binding. Since the region His12 to Met21 corresponds to NKA, the role of N-

terminal extension for NPc on NK2 receptor potency and selectivity has been discussed in

several papers. From structural point of view, Chandrasekhar et al. suggest that the N-terminal

domain may make additional contacts with the NK2 receptor and/or influence the C-terminal

conformation [60]. Presence of a-helix and conservation of primary and secondary structure in

the C-terminus of NK2-agonists have given rise to a hypothesis that the biological activity and

receptor activation are mediated by the C-terminal ‘‘message domain’’ of tachykinins.

Structures of NKA, NKB and NKc all have a-helical segments with the hydrophobic-half in

the C-terminus. The conserved hydrophobic residues Phe, Leu and Met in this region (see

Table 1) are proposed to form anchoring points within the TM region of the receptor and are

expected to contribute a major portion of the binding energy [57, 59, 60]. The hydrophilic-half

of the helix might possibly interact with the extracellular regions of the receptor and thus play

an important role in the NK2 receptor binding.

Substance P (SP)

Structure of substance P has been investigated by several groups in different solvents [55, 56,

175–178]. SP favors an extended chain conformation in water [56, 176]. This peptide has been

studied in both SDS and DPC micelles. CD spectroscopy showed a preferential a-helical

conformation for this peptide upon addition of SDS in aqueous solution [175]. Young et al.

[177] found that in the presence of 15 mM SDS micelles, SP undergoes a conformational

equilibrium between an a- and 310-helix involving the mid-region (Pro4 to Phe8) of the

peptide. Conformations of SP were determined in the presence of zwitterionic DPC as well as

anionic SDS micelles by Keire and Fletcher [55]. Both structures were found to be similar with

a turn structure involving residues 6–9. Cowsik et al. [56] observed that DPC micelles induce

an amphiphilic helical conformation in the mid-region from residues 5 to 8 of SP and C-

terminal residues remain extended (Table 1). Structure of SP bound to DMPC vesicles indi-

cates that the N-terminus remains flexible in the membrane-bound state [178]. The calculated

structure using transferred NOE-derived restraints had a sequence of non-standard turns fol-

lowing each other in a helix-like manner. The conserved C-terminal hydrophobic residues

Biosci Rep (2006) 26:131–158 149

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(Table 1) are localized on the same side of the peptide as has been observed in the DPC-bound

peptide [56]. Neutron diffraction and amide-exchange experiments have been carried out to

investigate the interactions of SP and NKA with the micelles or phospholipids bilayers [179–

181]. Amide-exchange experiments in SDS micelles suggest interactions of Phe8, Phe9 and

Gly10 residues in the interfacial region of the lipid micelle headgroups [179]. Neutron dif-

fraction experiments clearly demonstrate that SP interacts with both zwitterionic and anionic

membranes and the C-terminus of the peptide is positioned at a depth below the membrane

surface [180]. A model of binding of NK1 agonists have been evolved based on the several

structural studies described above. According to this model, the structurally less-defined

C-terminal hydrophobic residues interact with the TM region of the receptor.

Structure of NK1-preferring SP differs from the other tachykinin peptides NKA, NKB,

NKc, eledoisin and kassinin in the C-terminal region. Extension of helix length at the

C-terminus alters the positions of hydrophobic and hydrophilic side chains for these peptides.

Additionally, an increase in helix length results in increase in the stability of the helix and thus

the flexibility of the message domain will be reduced in the NK2/NK3 selective agonists.

These factors contribute to the poor binding property to the NK1 receptor. Such a hypothesis is

also supported by the structures of NK1 antagonists which have b-turn structure at the

C-terminus [58].

Conclusions

The number of endogenous peptide structures determined in membrane environment is stea-

dily growing over the years. It is clear that the micelles and other model systems that mimic

the cell membranes induce a stable conformation in otherwise flexible peptide ligands. The

question is whether this stable conformation is really the bioactive conformation that binds to

the peptidergic GPCRs. In each study discussed above, the structure obtained in the mem-

brane-mimetic medium is correlated with its biological activity. Site-directed mutagenesis and

spectroscopic results have been analyzed in the context of membrane-induced structure.

Peptide conformations determined in membrane-mimetic medium have been used to test

whether ligands with very similar binding profiles at the receptor subtypes are expected to

display similar conformations. Such approaches are useful to find the selectivity and specificity

of peptide ligands for a particular receptor subtype.

It is also promising to note that structures of two peptide hormones, PACAP and

neurotensin, have been determined in GPCR-bound state [33, 61]. This is a formidable task

towards understanding the GPCR-peptide ligand interaction. The methods used to obtain

the structures of these peptide hormones in receptor-bound form are complimentary in

some respect. Solid-state NMR method was used to determine the conformation of high-

affinity agonist neurotensin bound to its receptor [61]. The detection of the receptor-bound

ligand’s signal depends upon the relative size of the ligand and natural abundance back-

ground (receptor, lipids etc.). Double-quantum filtering extended into two dimensions was

used to achieve this in the study of uniformly labeled NT bound to NTR. Functional

receptors were reconstituted in lipids to obtain a high peptide/receptor molar ratio. This

assured that NMR signals predominantly resulted from peptide-bound receptor molecules.

The chemical shift assignments from these experiments were then used to construct the

model of NT.

TRNOE spectroscopy method was used to determine the structure of GPCR-bound PACAP

[33]. Since rapid exchange between bound and free ligands is a prerequisite for the successful

application of this method, PACAP21 (C-terminal truncated form of PACAP) was used.

150 Biosci Rep (2006) 26:131–158

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PACAP21 has a large dissociation rate and moderate affinity towards PACAP receptor. PA-

CAP27 is a high affinity ligand with slow dissociation rate. Receptor binding and receptor

activation show that both are full agonists. In the 2D TRNOE spectrum of PACAP21 in the

presence of purified receptor, both TRNOE and NOE crosspeaks were observed. Conforma-

tions of PACAP21 in the receptor-bound and free forms are reflected respectively in TRNOE

and NOE crosspeaks. The high affinity ligand PACAP27 was used to selectively eliminate the

TRNOE crosspeaks and the difference spectrum yielded only TRNOE-related crosspeaks. The

distance restraints derived from these experiments were used to calculate the conformation of

GPCR-bound peptide structure. This study also demonstrated that the conformational differ-

ences between micelle-bound and receptor-bound structures are limited to a few residues in the

N-terminus. We can hope that more GPCR-bound ligand structures will be determined using

one of the two methods described above.

In a recent study, Charles Sanders and his colleagues obtained a 2D TROSY spectrum of

vasopressin receptor (VR), a 371 residue GPCR, under optimized conditions [182]. They

searched for conditions for expressing VR in a manner that avoids formation of inclusion

bodies. Using optimum pH and temperature, the spectrum of LMPC-solubilized VR displayed

over 250 amide peaks out of an expected 349 peaks. The data suggested a high degree of

global and/or local mobility associated with the amides. The authors speculated that this might

be due to the mobile extra loops and termini. Although, this study looked at the receptor level,

its ability to characterize the extramembrane domains of intact GPCRs and their interactions

with cognate ligands will be an important development in understanding the mechanism of

GPCR activation.

With the exception of small peptide hormones (Menk, Lenk, endomorphin-1 and neuro-

tensin fragement), structures of all peptide ligands studied in membrane-mimetic medium

contained helical regions and displayed primary or secondary amphiphilicity. Three groups

of residues clearly influenced the interactions of peptides with the membrane medium;

aromatic (NMB, NPY family, opioid peptides), basic (NPY family, DynA) and hydrophobic

(all the hormones discussed above). They are also believed to play important role in the

interactions with their respective receptors. When they encounter the membrane, the sites of

first contact for the peptide hormones will be the chemically heterogeneous membrane

interface. With rich possibilities for non-covalent interactions with peptides, interfaces play

significant role in the folding and stability of the peptide ligands. White and his colleagues

determined a hydrophobicity scale, composed of experimentally determined transfer free

energies for each amino acid for studies of water-to-interface free energies of unfolded

peptide chains [183]. A whole-residue hydrophobicity scale for partitioning into n-octanol

was also established [183]. These scales included contribution of the peptide bonds and side

chains. It is obvious from these studies that the aromatic residues are exceptionally favorable

in the interface, which is consistent with the observations from membrane protein crystal

structures [184]. Similarly, the positively charged amino acids, lysine and arginines, can have

‘‘snorkeling’’ type of special interactions with the membrane interface [104]. The depth of

penetration of folded peptides will be strongly affected by hydrophobicity-hydrophilicity of

the peptide sequence [183]. Thus the aromatic, positively charged and hydrophobic residues

all seem to have important implications in the peptide’s structure, stability and interactions

with the membrane.

With more and more membrane-induced structures of peptide ligands available, the next

challenging task would be to dock these ligands in GPCR models. It should be noted that none

of the peptidergic GPCR structure has been determined in either free state or complex with the

ligand. Experimental studies have identified the important residues from the extracellular loops

and residues within the TM regions that bind to the ligand. Hence a reliable GPCR model has

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to be built to dock the ligand in its binding site that satisfies the results obtained by site-

directed mutagenesis, spectroscopy and other experiments. Loop modeling remains one of the

main problems in GPCR protein modeling [185–187]. A carefully built receptor–ligand

complex model will help to understand the mechanism by which the ligand–receptor complex

activates the G-protein and thus can aid in the rational drug design and development.

A combination of computer modeling, molecular dynamics studies combined with experi-

mental studies could be used to achieve this goal. This will be one of the main focus areas of

the GPCR researchers for many more years to come.

Acknowledgements I would like to thank all members of my lab for discussions. Priyanka Prakash Srivastavais acknowledged for her help while writing this review. This research is supported by Council of Scientific andIndustrial Research (No. 37(1199)/04/EMR-II).

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