Structural Basis for Ligand Recognition and Activation of RAGE

Structure

Article

Structural Basis for Ligand Recognitionand Activation of RAGEMichael Koch,1 Seth Chitayat,2 Brian M. Dattilo,2 Andre Schiefner,3 Joachim Diez,4 Walter J. Chazin,2,*and Gunter Fritz1,5,*1Department of Biology, University of Konstanz, 78457 Konstanz, Germany2Departments of Biochemistry and Chemistry, Center for Structural Biology, Vanderbilt University, Nashville, TN 37232-8725, USA3Lehrstuhl fur Biologische Chemie, Technische Universitat Munchen, 85350 Freising-Weihenstephan, Germany4Swiss Light Source at Paul Scherrer Institute, 5232 Villigen PSI, Switzerland5Department of Neuropathology, University of Freiburg, 79106 Freiburg, Germany

*Correspondence: [email protected] (W.J.C.), [email protected] (G.F.)DOI 10.1016/j.str.2010.05.017

SUMMARY

The receptor for advanced glycation end products(RAGE) is a pattern recognition receptor involvedin inflammatory processes and is associated withdiabetic complications, tumor outgrowth, and neuro-degenerative disorders. RAGE induces cellular sig-naling events upon binding of a variety of ligands,such as glycated proteins, amyloid-b, HMGB1, andS100 proteins. The X-ray crystal structure of theVC1 ligand-binding region of the human RAGE ecto-domainwas determined at 1.85 A resolution. The VC1ligand-binding surface was mapped onto the struc-ture from titrations with S100B monitored by hetero-nuclear NMR spectroscopy. These NMR chemicalshift perturbations were used as input for restraineddocking calculations to generate a model for theVC1-S100B complex. Together, the arrangement ofVC1 molecules in the crystal and complementarybiochemical studies suggest a role for self-associa-tion in RAGE function. Our results enhance under-standing of the functional outcomes of S100 proteinbinding to RAGE and provide insight into mecha-nistic models for how the receptor is activated.

INTRODUCTION

The receptor for advanced glycation end products (RAGE) is

a member of the immunoglobulin (Ig) superfamily of cell surface

receptors (Neeper et al., 1992; Schmidt et al., 1992). RAGE

signaling plays a central role in the inflammatory response (Hof-

mann et al., 1999), mediating aspects of innate immunity (Tian

et al., 2007), acute and chronic inflammatory disorders (Orlova

et al., 2007), and certain cancers (Taguchi et al., 2000). Ligand

binding to the ectodomain of RAGE is tightly coupled to the

recruitment of intracellular factors such as Diaphanous-1 to the

cytoplasmic domain, ultimately leading to the proliferation of

intracellular signals (Hudson et al., 2008b). Activated RAGE is

known to recruit extracellular signal–regulated kinase–1 and –2

1342 Structure 18, 1342–1352, October 13, 2010 ª2010 Elsevier Ltd

(ERK-1/2) (Ishihara et al., 2003), resulting in downstream activa-

tion of NF-kB via the MAP kinase pathway.

RAGE mediates physiological and pathological effects

through interaction with a diverse set of ligands, which remark-

ably, are each associated with a specific disease. The first

identified RAGE ligand was advanced glycation end products

(AGEs), which form by nonenzymatic glycation of proteins and

lipids and accumulate as a result of normal aging and inflamma-

tory processes, particularly in diabetes (Yamagishi et al., 2003).

RAGE is up-regulated in Alzheimer disease, and soluble

amyloid-b has been shown to bind to the receptor inducing

oxidative stress in neurons and the production of proinflamma-

tory cytokines in microglia (Yan et al., 2003). RAGE in the

endothelium mediates amyloid-b transport across the blood

brain barrier into the central nervous system, promoting

amyloid-b plaque formation (Deane et al., 2003). Activation of

RAGE on neurons by high mobility group protein 1 (HMGB1)

induces reorganization of the cytoskeleton and neurite extension

(Hori et al., 1995). However, in cancer cells, RAGE-HMGB1 inter-

action stimulates tumor invasiveness and growth (Taguchi et al.,

2000). Moreover, HMGB1-DNA complexes trigger the associa-

tion of RAGE with Toll-like receptor 9, which is essential for the

immune response to pathogens (Tian et al., 2007). Several

S100 proteins bind and activate RAGE. In fact, the first native

RAGE ligand discovered was EN-RAGE (S100A12), which initi-

ates and sustains the inflammatory response (Hofmann et al.,

1999). S100B binding to RAGE is among the best characterized,

having been shown to inducemultimerization of the receptor and

promote neurite extension and neuronal survival (Huttunen et al.,

2000; Leclerc et al., 2007; Ostendorp et al., 2007).

The specific association of RAGE with disease pathogenesis

has resulted in a growing interest in RAGE as a therapeutic

target. The initial steps toward validating RAGE as a target

involved the use of the extracellular ligand-binding region of

RAGE (sRAGE), which effectively competes with the receptor

for ligands. For example, sRAGE blockage of receptor activation

in cell culture has been useful for studies in mouse models for

various diseases (Deane et al., 2003; Park et al., 1998; Yan

et al., 2003). Amodel for advanced atherosclerosis shows severe

plaque development in the aorta, the formation of which is sup-

pressed in mice treated with exogenous sRAGE (Park et al.,

1998). In a mouse model for enterocolitis, administration of

sRAGE prevented immune cell infiltration into the colon and

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Structure

Structure of Human RAGE

sepsis, presumably by blocking the S100A12-RAGE interaction

and consequent signaling (Hofmann et al., 1999). Alternative

splicing of RAGE mRNA results in an endogenous secretory

form of RAGE (esRAGE). Levels of esRAGE are largely

decreased in inflammatory and neurodegenerative disorders

(Emanuele et al., 2005; Ghidoni et al., 2008; Sternberg et al.,

2008; Wittkowski et al., 2007), implying a regulatory function

for esRAGE.

In order to better understand the molecular basis for RAGE

signaling and its role in cellular physiology, and to lay a founda-

tion for exploiting RAGE as a therapeutic target, we have

undertaken a comprehensive analysis of the structure and

ligand-binding properties of human sRAGE. sRAGE is

composed of a V-type immunoglobulin (Ig) domain and two

C-type Ig domains (C1 and C2) (Schmidt et al., 1992). Previous

characterization of sRAGE revealed the C-terminal Ig domain

(C2) to be structurally independent of the first N-terminal two Ig

domains (V and C1), which together form an integrated structural

unit, VC1 (Dattilo et al., 2007). VC1 has been shown to bind

AGE-modified BSA and S100B (Allmen et al., 2008; Dattilo

et al., 2007). Here, we report a high-resolution crystal structure

of the tandem VC1 domain pair and link its unique structural

features to its binding properties. Heteronuclear NMR spectros-

copy is used to map the VC1-binding site for S100B and the

S100B-binding site for VC1. The NMR data are then used in

combination with molecular docking calculations to generate

a model of the complex. These results shed new light on models

for RAGE activation.

RESULTS

VC1 Forms an Integrated Structural UnitThe tandem RAGE VC1 domains were crystallized and the X-ray

structure was refined at 1.85 A resolution to a Rcryst of 20.9%

and Rfree of 24.0% (Table 1). VC1 is found to form a bent elon-

gated structure with an angle of 145� between the two Ig

domains (Figure 1A). A specific interface between the domains

is observed, which includes several interdomain hydrogen

bonds and hydrophobic interactions (Figure 1A). Among these

H-bonds, Gln119 in V engages in a pseudo b sheet hydrogen-

bonding pattern with Tyr150 in C1, and the carboxylate oxygen

atom of Glu94 in V forms an H-bond with the hydroxyl hydrogen

of Tyr150 in C1. In addition, a water molecule bridges the amide

nitrogen of Ile120 in C1 domain with the side chains of Gln119

and Arg29 in V. Hydrophobic contacts at the interface are medi-

ated between side chain atoms of Pro215 from the C1 F-G loop

and the side chain of Tyr118, as well as between the side chain of

Tyr150 from the C1 B-C loop and Ile91. Notably, all residues at

the VC1 interface are strictly conserved (see Figure S1 available

online). These characteristics confirm that the two domains form

an integrated structural unit (Dattilo et al., 2007).

On the basis of sequence alignments, the N-terminal domain

of RAGE (V) had been assigned to the V-set type of Ig molecules.

However, once the structure was determined, it revealed

features typical of I-set topology (Harpaz and Chothia, 1994),

which is characteristic for cell adhesion molecules (Casasnovas

et al., 1997; Feinberg et al., 2001; Freigang et al., 2000; Kasper

et al., 2000; Su et al., 1998) or muscle proteins (Holden et al.,

1992; Zou et al., 2006). Strands A, C, C0, F, andG form one sheet,

Structure 18, 1342–1

whereas strands B, D, and E form the second sheet (Figure 1).

The region in the V domain corresponding to C00 strand in V-set

Ig domains instead forms an extended loop encompassing

three glycine residues (Gly68, Gly69, and Gly70) and two proline

residues (Pro66 and Pro71). The frequency of glycine and proline

residues is inconsistent with formation of a b strand. High

B-factors in this region suggest that the loop might be adopting

variable conformations, which is not unusual for a short Gly and

Pro-rich sequence. Although more correctly classified as an I

domain, the V nomenclature will be retained here in order to

maintain consistency with previous literature.

The C1-domain fits to the C1-set of Ig molecules, with strands

A, B, D, and E forming the back b sheet and strands C, C0, F, andG forming the front sheet of the b sandwich. However, the RAGE

C1-domain has a unique topology distinct from other Ig fold sets:

it contains two additional b strands, A0 and G0, which belong

neither to the front nor to the back sheet. Both strands form an

additional parallel b sheet stabilizing the C terminus of the

domain (Figures 1A and 1B). Further variation is observed for

strands D and E. In comparison to most C1-type Ig domains,

strand D is elongated by two residues encompassing Arg178

and Arg179, as well as the D-E loop, which is slightly longer

through the inclusion of Pro181-Gly184. The E-F loop, which

includes Thr195-Thr205, is also elongated by residues Arg198-

Asp201. This loop has a unique interaction that stabilizes the C

terminus of C1 involving a hydrogen bond between the Arg198

side chain and the Pro232 main chain carbonyl oxygen.

A well-ordered 10-residue C-terminal extension from C1

(Glu231-Leu240) is observed in the high-resolution structure

(Figures 1A and 1B). This extension constitutes at least in part

the linker to the C2 domains, which NMR studies have shown

is flexible (Dattilo et al., 2007). A solution NMR structure of the

isolated C2 domain has been determined (PDB 2ENS), but the

N terminus is not well defined. In our structure, the extension is

stabilized by a contact between Leu240 and the C1-domain of

a neighboring molecule in the crystal. Hence, some uncertainty

remains over the exact length of the linker between the C1 and

C2 domains.

VC1 Contains a Highly Basic SurfaceOne of the most unusual properties of RAGE is its ability to

engage very diverse ligands of different size, fold, or symmetry.

The structure of the VC1 ligand-binding domain provides a

means to assess what specific surface properties enable this

unique characteristic. Our analysis revealed large positively

charged patches on the V domain (Figure 2), which are formed

by highly conserved Arg and Lys residues. A patch of positively

charged electrostatic surface was also identified on the C1

domain. Thus, the entire V domain and a major part of C1 form

a large area of electropositive surface (Figure 2) that fits well to

the acidic (negative) character of the diverse RAGE ligands,

including AGE-modified proteins, amyloid-b, and S100 proteins.

Comparison of molecular surfaces displaying the isoelectro-

static field potential of structural homologs neural adhesion

molecules TAG-1/axonin-1 (Freigang et al., 2000) and N-CAM

(Kasper et al., 2000), as well as a structural model of the closest

homolog ALCAM, reveals that the electrostatic potential of VC1

is significantly stronger (Figure 2), which suggests RAGE

contains an electrostatic trap for negatively charged ligands.

352, October 13, 2010 ª2010 Elsevier Ltd All rights reserved 1343

Table 1. Crystallographic Data and Refinement Statistics

Native Peak Zn-MAD Inflection Remote

Data collection

Wavelength 1.0082 1.2820 1.2825 1.0082

Resolution (A) 47–1.85 (2.0–1.85)a 47–2.6 (2.7–2.6)a 47–2.6 (2.7–2.6)a 47–2.4 (2.1–2.0)a

Space group P21212 P21212 P21212 P21212

Cell dimensions (A) 74.85, 119.96, 28.89 74.76, 119.90, 28.87 74.76, 119.90, 28.87 74.76, 119.90, 28.87

Total observations 153,044 (23,037) 57,942 (15,395) 57,858 (15,401) 124,565 (33,842)

Unique reflections 28,559 (4,704) 6,158 (1,619) 6,138 (1,622) 15,819 (4,599)

Completeness (%) 99.7(99.8)a 99.9 (100.0)a 99.9 (100.0)a 99.7 (99.7)a

Redundancy 6.6 (6.0)a 3.7 (3.8)a 3.7 (3.8)a 3.7 (3.4)a

Rsymb (%) 8.2 (57.1)a 6.0 (19.1)a 5.5 (16.5)a 7.6 (32.9)a

I / sI 12.2 (2.6)a 16.8 (6.7)a 17.6 (7.4)a 11.3 (3.6)a

Phasing power acentric 1.391 1.382 0.419

FOM acentric/centric 0.63279/0.44639

Refinement

Resolution (A) 1.85 2.0 2.0 2.0

Rcrystc / Rfree

d (%) 20.9/24.0 (24.7/26.0)a

No. atoms

Protein 1938

Ligand/ion 4

Water 229

B-factors

Protein 25.6

Ligand/ion 44.8

Water 47.0

R.m.s deviations

Bond angles (�) 1.99

Bond lengths (A) 0.018

Ramachandran plot

Most favored (%) 87.8

Additionally allowed (%) 11.2

Generously allowed (%) 1.2aNumbers in parentheses apply to the highest-resolution shell.b Rsym = Shkl Sj j Ij � < I > j Shkl Sj j Ij j.c Rcryst = Shklj Fobs – Fc j / ShklFobs, where Fobs and Fc are observed and calculated structure factors, respectively.d Five percent randomly selected reflections were excluded from refinement and used for the calculation of Rfree.

Structure


Self-Association of RAGERecently, it was shown that RAGE forms oligomers on the

plasma membrane (Xie et al., 2008), and it was suggested that

oligomerization is mediated by the C1 domain (Xie et al., 2007).

Indeed, in the crystal structure, there are side-by-side contacts

between VC1 molecules mainly involving residues in the C1

domain. The solvent accessible surface area buried by this

interface is 980 A2 (Figure 3A). The contact surface contains

several charged residues and is connected by a tight network

of hydrogen bonds and polar interactions (Figures 3B and 3C).

The guanidine of Arg178 side chain forms a salt bridge with the

carboxylate group of Glu1680 and a hydrogen bond to the

carbonyl oxygen of Pro1630 of the neighboring molecule

(Figure 3C). The Gln176 side chain oxygen forms two hydrogen

bonds to the N3 of Arg178, which positions the guanidine group

for optimal interaction with the neighboring molecule (Figure 3C;


Figure S2). These interactions are reminiscent of the critical salt

bridge formed between Arg and Glu/Asp residues at the inter-

face between Ig domains in the active dimer of the receptor

tyrosine kinase KIT (Yuzawa et al., 2007) or between the

membrane-proximal Ig-domains of the VEGF receptor (Yang

et al., 2010). The extensive set of stabilizing interactions between

C1 domains in adjacent molecules in the crystal structure is

consistent with the proposal of RAGE aggregation in the plasma

membrane. In addition to H-bonds and salt bridges, a Zn2+ ion is

found at the intermolecular interface. The Zn2+ is coordinated by

residues His180, Glu182, and His1580 from the adjacent VC1

molecule (Figure 3B). These residues, like most involved in at

the C1-C1 interface, are conserved across species (Figure S1),

which implies a role for Zn2+ in receptor multimerization. We

note that Zn2+ can induce dimerization of the class II major

histocompatibility complex, leading to cooperative binding of a

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Figure 1. Structure of the Tandem VC1

Domains of RAGE

(A) Stereo ribbon diagram VC1 with the V domain

in green and the C1 domain in magenta. The two

Ig domains adopt a fixed orientation that is

stabilized by hydrogen bonds and hydrophobic

contacts.

(B) Topology diagram of VC1 with the strands

colored as in (A). The structure of the V domain

shows it belongs to the I-set of Ig domains,

whereas C1 remains in the C1 set. Note that a

unique parallel b sheet is formed by strands A0

and G0, which stabilizes the C terminus of the C1

domain.

See also Figure S1.

Structure


superantigen ligand (Li et al., 2007). Moreover, homophilic inter-

actions of receptor Ig-type ectodomains play a central role in

ligand-binding and cellular activation (Stuttfeld and Ballmer-

Hofer, 2009; Xu and Jin, 2010; Yang et al., 2010).

In order to further investigate self-association of VC1 and a

potential role for Zn2+, we performed dynamic light scattering

experiments. The data show that VC1 is monomeric in the

absence of Zn2+ and at low pH. The tendency for VC1 to self-

Structure 18, 1342–1352, October 13, 2010 ª

associate is heavily influenced by Zn2+

and increased pH into the physiological

range (Figures 4A–4C). The degree of

self-association of VC1 is concentration

dependent (Figure 4D), which is consis-

tent with self-association being a specific

binding phenomenon. The concentration

dependence of self-association is pro-

moted by the presence of Zn2+ and

shows a saturation behavior that could

be fit by a hyperbolic function yielding

a Kd app of 3.8 ± 1.3 mM.

An NMR-Based Model for theComplex of VC1 with the RAGELigand S100BStructural details about ligand binding to

RAGE have been limited to a single study

thathighlightedseveral residues in theVdomain that are important

forbindingofAGEs (Matsumotoetal., 2008).Wehave investigated

the structural basis for binding of the ligand S100B, combining

two-dimensional heteronuclear NMR experiments to identify the

binding interface on both VC1 and S100B and HADDOCK-based

docking calculations to generate a model of the complex.

Tomap the S100B-binding site onRAGE, a series of 2D 15N-1H

HSQC NMR experiments were acquired as Ca2+-S100B was

Figure 2. Surface Analysis of VC1

Surface representation of the tandem VC1

domains and its closest structural homologs TAG

1/axonin-1 (Ig domains 3,4), neuronal cell adhe-

sion molecule (N-CAM Ig domains 2,3), and

activated leukocyte adhesion molecule (ALCAM

Ig domains 1,2). Positive electrostatic isopotential

surfaces at 0.8 kT/e are shown in blue. RAGE VC1

exhibits an unusually large positive electrostatic

potential. The isosurface shows a coherent poten-

tial across both Ig domains extending far into

space. The figure was prepared using PyMOL

(http://www.pymol.org) and APBS (Baker et al.,

2001).

See also Figure S2.

2010 Elsevier Ltd All rights reserved 1345

http://www.pymol.org

Figure 3. Packing of VC1 in Crystal Lattice

(A) Ribbon diagram of the VC1 assembly observed in the crystal. The molecules interact mainly via the C1 domain.

(B and C) Contact between molecules involve a bridging Zn2+ ion (B) and a salt bridge and hydrogen bonds (C). Stereo view around the bridging Zn2+ ion (B)

depicts a Fo-Fcmap calculated after refinement of a coordinate file missing the Zn2+ and the side chains of the coordinating residues; the map is shown in purple

at 3.0 s in and in cyan at 12.0 s, respectively.

See also Figure S1.

Structure


titrated into a solution of 15N-enriched V domain (Figure 5B). The

isolated V domain was used in NMR experiments rather than

sRAGE or VC1 because, first, the majority of the S100B binding

affinity has been shown to be localized to this domain (Dattilo

et al., 2007), second, S100B is a homodimer and working with

the smallest complex was important to facilitate the NMR anal-

ysis, and third, this enables direct comparison to a published

study with AGE ligands (Matsumoto et al., 2008). Broadening of

all NMR signals over the course of the titration was observed as

expected for formation of the V-S100B complex. However, care-

ful analysis of the data revealed differential line broadening

effects. As demonstrated elsewhere (Takeuchi and Wagner,

2006), resonances exhibiting enhanced line broadening corre-

spond to residues experiencing the largest perturbation upon

binding of the ligand, and these can be correlated to the ligand-

binding sites. The binding site was mapped by tracking 52 reso-

nancesdistributed throughout theVdomain that could bedirectly

assigned from the published chemical shift assignments (Matsu-

moto et al., 2008). Quantification of normalized intensities for

these resonances identified a subset of residues that are selec-

tively broadened upon addition of Ca2+-S100B (Figure 5B).

When these changes aremapped onto the VC1 structure, they

reveal that binding of Ca2+-S100B occurs to one face of the

molecule (Figure 5C). Interestingly, these residues map to the

highly basic surface of VC1 (Figure 5A), which is complementary

to the acidic nature of S100B (Figure 5E). Notably, electrostatic

forces have been shown to modulate ligand interactions with

other Ig-like domains, such as in the CD2-CD58 protein complex

(Wang et al., 1999). Moreover, the S100B-binding site appears

to correspond to the site used by RAGE for binding AGE-BSA

(Matsumoto et al., 2008). Key residues in AGE binding include

Arg48, Arg98, and a patch around Arg104, all of which are per-

turbed upon binding of Ca2+-S100B. Additional residues include

Val35, Lys39, Val89, Glu94, Phe97, Ala101, Asn105, Glu108, and


Thr109 (Figure 5C). Mutagenesis experiments on V residues

serve to validate this as the ligand-binding surface (Matsumoto

et al., 2008). The similarity of binding sites for different RAGE

ligands has been inferred previously (Matsumoto et al., 2008),

but this work combined with the previous NMR studies on

binding of AGE ligands, provides the first direct evidence in

support of this hypothesis.

In order to map the VC1 binding surface on S100B, a series of

2D 15N-1H HSQC NMR experiments were acquired over the

course of a reciprocal titration of RAGE VC1 into a solution of15N-enriched Ca2+-S100B. The effects observed in these

spectra were similar to those seen for the S100B titration into

the V domain (i.e., differential line broadening for a specific set

of S100B resonances). The residues affected include His42,

Val52, Asn62, Asp69, Phe70, and Ala78, which cluster to a

specific surface on S100B. We note that this surface, and in

particular His42, Val52, and Ala78, were shown previously to

be important for interactions with other S100B-specific ligands

such as NDR kinase (Bhattacharya et al., 2003).

Because residues that are selectively perturbed upon forma-

tion of the complex could be identified for both RAGE-V and

S100B, we proceeded to generate a model of the complex.

The program HADDOCK (de Vries et al., 2007; Dominguez

et al., 2003) was used for these calculations. A representative

structure was selected on the basis of the lowest HADDOCK

score and is shown in Figures 5D–5F. The models show S100B

uses a concave, negatively charged surface to clamp around

a complementary positively charged face of the RAGE V domain

(Figure 5E). A surface area greater than 1400 A2 is buried by

formation of the complex. Analysis of the model revealed a

number of possible polar hydrogen bonds and salt bridges in

the interface. Thus, the model suggests that clustering of basic

residues on the binding surface of VC1 and highly acidic surface

of S100B are the major driving forces in formation of the

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Figure 4. Self Association of RAGE VC1Monitored by Dynamic Light

Scattering

(A) VC1 exists as a monomer at pH 5.2.

(B and C) Increase of pH to 6.5 and 7.5 as well as the addition of Zn2+ leads to

a shift toward oligomeric states.

(D) VC1 oligomer formation at pH 7.6 was dependent on protein concentration

(-) and is strongly increased by the presence of Zn2+ (C). The Zn2+ and

concentration-dependent oligomerization was fit to a hyperbolic function

yielding an apparent dissociation constant (Kdapp) of 3.8 ± 1.3 mM.

See also Figure S2.

Structure



complex. In order to validate the result obtained by HADDOCK,

we performed a second set of calculations without NMR

restraints using the computational docking program HEX

(Ritchie and Kemp, 2000; Ritchie et al., 2008). The models

obtained in this manner were remarkably similar to the models

obtained using the NMR restraints in HADDOCK (Figure S3).

Analysis of these models of the VC1-S100B complex reveals

a strong electrostatic component to binding, consistent with

the proposed electrostatic trap contributing significantly to

RAGE ligand binding.

DISCUSSION

Initiation of signal cascades by ligand-induced receptor oligo-

merization has been proposed as a general mechanism of

receptor activation. In some models, multimeric ligands activate

their receptors by recruitment of receptor molecules. Hence,

association of the extracellular ligand-binding domains drives

the colocalization of the cytoplasmic domains, which is needed

for signal transduction. In these models, the geometry of the

ligand will control activity because the cytoplasmic domains

need to be brought into close proximity in a specific orientation.

This model has been invoked for receptor tyrosine kinases (Yu-

zawa et al., 2007). However, it has also been shown that many

receptors, including TNFa-receptor (Chan, 2007; Chan et al.,

2000), interleukin-receptor (Kramer et al., 2006), EPO receptor

(Livnah et al., 1999), and EGF receptor (Stuttfeld and Ballmer-

Hofer, 2009; Yang et al., 2010), preassemble in the absence of

a ligand on the cell surface. In these cases, it is proposed that

the clustering of receptor molecules increases the affinity for

ligands and is a requirement for effective receptor signaling. It

is notable that the distance of two adjacent VC1 molecules in

the crystal structure is 20–25 A, close to the distance between

subunits in the receptor tyrosine kinase complex (Yuzawa

et al., 2007) and predicted in the active EGF receptor complex

(Burgess et al., 2003). Consistent with a recent FRET study (Xie

et al., 2008), our data suggest a similar preassembly model for

RAGE.

Preassembly has substantial implications for the mechanism

of RAGE activation by its diverse ligands. The molecular basis

for RAGE activation by its diverse set of ligands has remained

enigmatic; RAGE ligands exhibit different structure, size, and

symmetry or even no symmetry, as in the case of glycated

proteins or amyloid-b. Clearly, single copies of dissimilar ligands

cannot induce a regular arrangement of the intracellular receptor

domains as required for initiation of a signal cascade. The

common factor linking these ligands is their tendency to oligo-

merize. The structural and computational data provided here

strongly imply a critical role for the positive electrostatic potential

in ligand recognition and binding.

If RAGE assembles on the cell surface in the absence of a

ligand, ligand binding can be viewed as shifting the equilibrium

distribution between monomer and higher order oligomerization

states (Figure 6A). Thus, receptor assemblies would be stabi-

lized by the binding of multimeric ligands such as S100B,

S100A12, or polymeric glycated proteins. This explains why

RAGE activation by such ligands leads to a positive feedback

cycle to sustain elevated RAGE expression. Moreover, because

increased receptor levels at the cell surface will promote


Figure 5. Structural Model of the S100B-VC1 Complex

(A) Surface representation of VC1 with the electrostatic potential mapped on the surface. Blue represents positively charged areas, and red represents negative

charge.

(B) Overlay of the 600 MHz 15N-1H HSQC NMR spectrum of 15N-enriched V domain in the absence (black) and presence (red) of Ca2+-S100B.

(C) VC1 residues identified in (B) mapped on the VC1 structure. The V domain is colored in green, the C1 domain in light blue, and residues perturbed in the NMR

experiments are colored yellow.

(D) Docking of S100B on VC1 based on the chemical shift perturbations identified in (B).

(E) Surface representations of the structural model of the S100B-VC1 complex demonstrating the contributions of charge complementarity to binding. The

left-hand panel shows S100B engaged with VC1. The right-hand panel pulls apart the complex to reveal the intense negative potential of the binding face of

S100B and the intense positive potential of the binding face of VC1.

(F) Structural model of the S100B-VC1 complex with the S100B ribbon and VC1 displayed with the electrostatic potential surface.

See also Figure S3.

Structure


preassembly, this would explain the hyperactivation of the RAGE

pathway in the development of chronic inflammatory and neuro-

degenerative disorders.

The importance of RAGE oligomerization is underscored by

a number of functional studies. A fusion of the RAGE cytoplasmic

domains to the C terminus of the homodimeric glutathione

S-transferase (GST) was able to bind the cytoplasmic RAGE

mediators Diaphanous-1 (Hudson et al., 2008b) and ERK kinase

(Ishihara et al., 2003) in vitro, implying that theGST-fusion protein

mimics the active receptor. Because the C termini in the GST


homodimer are located in the same orientation and at a distance

of approximately 25 A, these observations imply the cytoplasmic

domains in the fusion are separated by the same distance as in

the activated receptor.

Although preassembly of RAGE might facilitate ligand binding

and increase its efficiency in signaling, preassembled receptors

could promote nonspecific activation with adverse conse-

quences. Therefore, preassembly of RAGE must be tightly regu-

lated. The major alternative splice product of RAGE comprises

the extracellular region and occurs as soluble protein (sRAGE)

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Figure 6. Models of RAGE Activation and Inhibition by

sRAGE

(A) Model in which RAGE preassembles in the plasma

membrane. Ligand binding to RAGE stabilizes oligomers,

which then can bind a signaling adaptor protein (gray sphere)

to the cytoplasmic region of RAGE.

(B) Diagram showing the action of sRAGE, in which interaction

with intact RAGE to form a hetero-oligomer limits binding of

intracellular adaptors and blocks signal transduction.

Structure


(Hudson et al., 2008a), which represents an adaptable regulator

of RAGE activation. It has been proposed that soluble alternative

splice products of RAGE can act as decoy receptors, decreasing

the concentration of available ligands. Our data suggest a

different explanation for how sRAGE might interfere with RAGE

activation: both sRAGE and the splice variant lacking only the

intracellular domain (DN-RAGE) can form heterocomplexes

with full-length RAGE, resulting in nonfunctional assemblies

(Figure 6B). This notion is largely supported by the observation

that coexpression of a RAGE isoform devoid of its intracellular

domain has a dominant negative effect on RAGE signaling (Hut-

tunen et al., 1999; Taguchi et al., 2000). Dominant inhibition of

signaling by heterocomplex formation has also been observed

for tumor necrosis factor–related apoptosis-inducing ligand

receptor (Clancy et al., 2005) and Fas death receptor (Siegel

et al., 2000), where coexpression of truncated forms of the

receptors blocked signaling of full-length receptors by ligand-

independent association.

RAGE is a critically important mediator of the inflammatory

response and plays an important role in innate immunity. Thera-

peutic approaches to target RAGE will be valuable for the

treatment of diabetic complications, chronic inflammations,

and neurodegenerative disorders. The structure of VC1 and

NMR-based models of the VC1-S100B complex provide

detailed insights into the mechanism of ligand recognition and

binding, as well as how RAGE can be activated by the diverse

Structure 18, 1342–1352, October

range of ligands. Both structural and biochemical

data support the proposal that RAGE assembles

in the cytoplasmic membrane without the presence

of a ligand. These results define approaches to

block ligand binding or reduce self-association,

which represent a promising avenue for the pursuit

of therapeutic strategies based on interfering with

RAGE activation.

EXPERIMENTAL PROCEDURES

Protein Expression and Purification

Protein encompassing the V and C1 domains of RAGE and

S100 proteins were expressed in Escherichia coli and purified

as described previously (Allmen et al., 2008; Dattilo et al.,

2007; Ostendorp et al., 2005).

Crystallization, Data Collection, and Structure

Determination

Crystals were grown by hanging drop vapor diffusion at 298 K

bymixing 2 ml of protein with 2 ml of 0.1MNa cacodylate, 0.2M

Zn acetate (pH 6.5), and 11% PEG 8000. Crystals of a size

of 200 mm 3 100 mm 3 200 mm appeared after 10 to

20 days. The crystals were soaked for 1 min in three consec-

utive steps in mother liquor containing 4%, 8%, and 12% glycerol and were

flash-frozen in liquid nitrogen. Crystals diffracted to a resolution of 1.85 A.

Because Zn2+ turned out to be essential for crystallization, we assumed that

Zn2+ specifically interacts with RAGE VC1 and could, therefore, be used for

experimental phasing. Multiple anomalous dispersion data were collected at

a wavelength of 1.28 A at the Swiss Light Source (Villigen, Switzerland) beam-

line X06SA at 100 K. An additional data set collected at a wavelength of 1.0 A

was used for refinement. The data sets were reduced and scaled with the XDS

package (Kabsch, 1993). Crystals belonged to the space group P21212 with

unit cell parameters of 74.85, 119.96, and 28.89, as determined by XPREP

(2005 version, Bruker-AXS). The zinc substructure was determined using

SHELXD (Schneider and Sheldrick, 2002). Refinement of the zinc sites and

phase calculation was done with SHARP (Fortelle and Bricogne, 1997)

followed by solvent flattening and initial fragment building using RESOLVE

(Terwilliger, 2000). The final model was built in COOT (Emsley and Cowtan,

2004) and was refined using REFMAC5 (Murshudov et al., 1997; Winn et al.,

2001) with final Rwork and Rfree of 20.9% and 24.0%, respectively (Table 1).

Ninety-nine percent of the residues were located in the allowed regions, and

1% were located in the generously allowed regions of the Ramachandran

plot (Table 1).

Solution NMR Mapping Studies with Ca2+-Loaded S100B

Substoichiometric additions of Ca2+-loaded S100B were made into uniformly

labeled 15N-V in 10 mM Imidazole-d4 (pH 6.5), 100 mMNaCl, and 5mMCaCl2.

Similar additions of VC1 were made into a uniform solution of 15N-labeled

S100B in 20 mM KCl (pH 7.1), 2.5 mM Ca2+, and 5% D2O. Perturbations in

the NMR signals were monitored by 2D 15N-1H HSQC spectra using a Bruker

DRX500 spectrometer. Data were processed using Topspin 2.0b (Bruker) and

analyzed using NMRViewJ. The intensities (I) were extrapolated from peak

heights and plotted as a ratio (I/Io) versus the initial intensity (Io).

13, 2010 ª2010 Elsevier Ltd All rights reserved 1349

Structure


Protein Oligomerization Assay

Protein oligomerization assays of VC1 were performed at 298 K. A stock

solution of VC1 with 10 mg ml�1 in 10 mM sodium acetate buffer (pH 5.0)

was diluted into 20 mM Tris and 150 mM NaCl (pH 7.6). Large oligomers

formed at increasing VC1 concentrations and in the presence of Zn2+. The

oligomers were pelleted by centrifugation (10 min at 12,000 g). The amount

of oligomer formed was calculated by subtracting the amount of protein

remaining in the supernatant from total protein. VC1 concentration in the

supernatant was determined by the UV absorbance using an extinction coef-

ficient of 3278nm = 32500 M�1 cm�1. In experiments monitoring the effect of

Zn2+ ions, 200 mM ZnCl2 was present in the buffer.

Docking Calculations

Residues exhibiting significant NMR chemical shift perturbations were used

as input for HADDOCK calculations (Dominguez et al., 2003) to generate an

experimentally derived model of the S100B-VC1 complex. Specifically, those

residues in S100B and in the V-domain of RAGE whose intensities were

reduced by more than one standard deviation from the mean reduction in

intensity were selected. The active and passive categories of restraint were

assigned based on criteria outlined in HADDOCK2 (de Vries et al., 2007).

The calculation proceeded in a similar manner to that reported by Nordquist

et al. (2010). To improve the conformation sampling, twenty structures each

were generated from the X-ray crystal structures of S100B (PDB accession

number 2H61) and VC1 using room temperature MD simulations in AMBER 9

(Pearlman and Connelly, 1995). Using these starting structures, 1000 complexes

were generated from rigid-body docking, of which 200 were selected for further

refinement in explicit water. One hundred and four of the 200 structures fell into

11 clusters, of which the twowith the lowest target energieswere themost popu-

lated (cluster 1,25; cluster2,16).Cluster1had the lowestaverageHaddockscore

and highest average buried surface area, so the 20 structures with the lowest

energies were selected as the representative ensemble for further analysis.

Free docking calculations of Ca2+-loaded S100B with VC1 were performed

using HEX 5.0 (Mustard and Ritchie, 2005; Ritchie and Kemp, 2000) on amulti-

processor Linux workstation. HEX uses 3D parametric functions, which are

used to encode both surface shape and electrostatic charge and potential

distributions for docking calculations.

Modeling and Analysis

A homology model of the activated leukocyte adhesion molecule (ALCAM) Ig

domains 1 and 2 was constructed using VC1 as a template in MODELLER

(Eswar et al., 2007; Sali et al., 1995). Electrostatic potentials were calculated

with APBS (Baker et al., 2001). All molecular figures were generated using

PyMol (DeLano Scientific).

ACCESSION NUMBERS

Coordinates and structure-factor amplitudes have been deposited in the

Protein Data Bank with accession number 3CJJ.

SUPPLEMENTAL INFORMATION

Supplemental Information includes three figures and can be found with this

article online at doi:10.1016/j.str.2010.05.017.

ACKNOWLEDGMENTS

We thank thestaff at theSwissLightSourcebeamlineX06SA for excellent assis-

tance with data collection and Benjamin Chagot for technical expertise with

HADDOCK2.0.Financial supportwasprovidedby theDeutscheForschungsge-

meinschaft (grants FR 1488/3-1 and FR 1488/5-1 to G.F.) and the National

Institutes of Health (grant RO1 GM62112 to W.J.C.). S.C. was supported by

a Canadian Institutes of Health and Research postdoctoral fellowship.

Received: September 25, 2009

Revised: May 6, 2010

Accepted: May 15, 2010

Published: October 12, 2010


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Structural Basis for Ligand Recognition and Activation of RAGE