Classification of Protein Complexes based on Biophysics of Association Sandor Vajda

Classification of Protein Complexes based on Biophysics of Association

Sandor Vajda

Boston University

“Tell me with whom you go,and I'll tell you what you are.” Italian Proverb

“FYI” filtered yeast interactome (Vidal 2004):• involves ~1500 proteins,• making ~2500 physical interactions

H. Jeong et al, Nature 2001

Structure: Nature of Intreractions

PDB: ~ 25’000 solved crystal structures; ~ 10% complexes

computational prediction of structure and specificity of

protein – protein complexes

“Tell me how you contact your partners,and I'll tell you who you are.”

List of Interactions

Protein-protein docking How proteins interact with each other? Docking problem

Predict docking configuration from the structures of component proteins

Bound vs. unbound docking Conformational change

Bound vs.unbound: at least side chain conformations change

Coarse details

Fine details

Trypsin/APPI

Receptor

Ligand

Talk outline

1.What is the current state of docking?

2.What docking calculations tell us about the nature of protein - protein complexes?

3.How to deal with side chain flexibility?

Proteins: Basics

ADEFFGKLSTKK……. Sequence

CASP

Structure

N

O

OO

N

O

N

O

N

N

O

......

Building Blocks:backbone & side chains

CAPRI

+

Complex

Monomers

Rigid body degrees of freedom 3 translation3 rotation

de novo docking

Structure Prediction

Benchmark set of protein complexes: Chen, R. et al. (2003) A protein-protein docking benchmark. Proteins,

52, 88-91. 22 enzyme-inhibitor 19 antigen-antibody 11 “other” types 7 “difficult” cases

Comeau, S. et al. (2003) ClusPro: An automated docking and discrimination method for the prediction of protein complexes. Bioinformatics, 20, 45-50.

Chen, R. et al. (2003) ZDOCK: An initial-stage protein-docking algorithm Proteins, 52, 80-87

Li, L. et al. (2003) RDOCK: Refinement of rigid-body protein docking predictions. Proteins, 53, 693-707.

Gray, J.J. et al. (2003) Protein–protein docking with simultaneous optimization of rigid-body displacement and side-chain conformations. J. Molec. Biol. 331, 281-299

How current protein docking programs work?

Submit 10 models to

CAPRI

Rigid Body Search

Select docked structures with low energy

Cluster retained conformations

Refine structures Flexible side

chains

Filter 1: 20,000

Filter 2: 2,000

Filter 3: 30

Filter 4: 1?

Algorithms of the 3 docking methods

Method (Investigator)

Step 1: Rigid body search Step 2: Rescoring, ranking, filtering, and refinement

ClusPro (Camacho and Vajda)

Fast Fourier Transform (FFT) correlation approach using ZDOCK or DOT

Re-scoring with empirical potentials and clustering

Gray and Baker

Monte-Carlo search using simplified protein geometry and scoring function

Iterative repacking of side chains and rigid-body docking repeated until convergence. Final selection by clustering.

ZDOCK (Weng)

FFT correlation with shape complementarity, electrostatics, and desolvation

Clustering of conformations to avoid redundancies

RDOCK (Weng)

FFT correlation with shape complementarity

Re-scoring with empirical potentials

Effect of the interface area

difficult

uncertain

easy very difficult

GOOD

Effect of hydrophobicity

easy

uncertain

-4

Type IVdifficult

Type IIIuncertain

Type IIeasy

Type Ieasy

Size vs. Hydrophobicity

Type Vdifficult

difficultType IVdifficult

Type Ieasy

Type III uncertain

Benchmark by type

Type II easy

Type Vdifficult

Interface Area

Des

olva

tion

free

ene

rgy

1400 2000 3400

-4

Type IEasy

Enzymes

Type IIEasy

Largemultienzymecomplexes

Type IIIUncertain

Antibody/Antigen

Type IVDifficult

Type VHopeless

Transitionalcomplexes with

substantial conformational

change

Small signallingcomplexes

Type II Or

Type V?

Table I. Major differences between enzyme-inhibitor and antibody-antigen complexes

Property Enzyme-inhibitor complexes Antibody-antigen complexes

Interface area ASA 1400 Å2 < ASA < 2000 Å2, Possibly < 1400 Å2

Interface connectedness Single patch Frequently multiple patches

Interface shape Convex-concave Mostly planar

Binding free energy G, kcal/mol

-17.5 kcal/mol < G < -13.0 kcal/mol


% Nonpolar residues in interface

61% nonpolar (can reach 71%)

51% nonpolar (can be as low as 44%)

Desolvation free energy Negative (favorable) Positive (unfavorable)

Conformational change Generally moderateCan be substantial; loop and/or hinge motion

Crystallographic water positions

Around perimeter of interface Within the interface

Conformati-onal

Interface

Type change ASAa Hydrophobicity Docking outcome

Example

I Small (rigid interface)

Standardb Strong; the convex-concave interface provides good shape complementarity

Successful, unless key side chains are in wrong conformations

Trypsinogen and trypsin inhibitor (1cgi): KD = 0.2 pM, ASA = 1950 Å2, and Gdes = -18.3 kcal/mol. Most complexes of enzymes with their protein inhibitors are in this category

II Small ASA > 2000 Å2

Unimportant Successful Ribonuclease a and ribonuclease inhibitor (1dfj): ASA = 2580 Å2, Gdes = 18.6 kcal/mol, Eelec=-63.9 kcal/mol KD = 0.15 nM

III Moderate, but larger than for Type I

Standard Variable, but generally weak. Charge-charge interactions can be strong

Unpredictable; can be very difficult, even with know hypervariable regions of antibody

Hyhel-5 Fab with lysozyme (1mlc): KD = 126M, ASA = 1390 Å2, and Gdes = -3.84 kcal/mol. Most antibody – antigen complexes are in this category

IV Restricted to side chains

ASA <1400 Å2

Weak; mostly polar and charge-charge interactions

Hits are found, but are generally lost in scoring and ranking

Ras and Ras interacting domain (1lfd) KD = 2M, ASA = 1130 Å2, and Gdes = 3.6 kcal/mol. A number of weak complexes are in this category

V Substantial backbone change, C RMSD > 2 Å

ASA > 2000 Å2

Generally moderate

Rigid body methods seem to always fail for these complexes

Cyclin A and cyclin-dependent kinase 2 (1fin): KD = 47.6 nM, ASA = 3390 Å2, and Gdes = 4.7 kcal/mol

a ASA – Acessible Surface Area, bStandard interface: 1400 Å2 < ASA < 2000 Å2, c C RMSD - carbon Root Mean Square Deviation

Classification of complexes

alpha-chymotrypsinogen

trypsin inhibitor variant 3

Type I:Enzyme-Inhibitor Complexes

Interface in the complex of alpha-chymotrypsinogen with trypsin inhibitor

Table I. Major differences between enzyme-inhibitor and antibody-antigen complexes

Property Enzyme-inhibitor complexes Antibody-antigen complexes

Interface area ASA 1400 Å2 < ASA < 2000 Å2, Possibly < 1400 Å2

Interface connectedness Single patch Frequently multiple patch

Interface shape Convex-concave Mostly planar

Binding free energy G, kcal/mol



% Nonpolar residues in interface

61% nonpolar (can reach 71%)

51% nonpolar (can be as low as 44%)

Desolvation free energy Negative (favorable) Positive (unfavorable)

Conformational change Generally moderateCan be substantial; loop and/or hinge motion

Crystallographic water positions

Around perimeter of interface Within the interface

Conformati-onal

Interface


Example










Hyhel-5 Fab with lysozyme (1mlc): KD = 126M, ASA = 1390 Å2, Gdes = -3.84 kcal/mol, Eelec = --21.4 kcal/mol, Most antibody – antigen complexes are in this category


ASA <1400 Å2





ASA > 2000 Å2

Generally moderate





chicken lysozyme

Monoclonal antibody fab d44.1

Type III:Antigen-Antibody Complexes

Interface in the complex of chicken lysozyme with antibody fab d44.1

Interface Area

Des

olva

tion

free

ene

rgy

1400 2000 3400

-4

Type IEasy

Enzymes

Type IIEasy


Type IIIUncertain

Antibody/Antigen

Type IVDifficult

Type VHopeless



change


Type II Or

Type V?

Conformati-onal

Interface


Example












ASA <1400 Å2





ASA > 2000 Å2

Generally moderate





ribonuclease a

Ribonuclease inhibitor

Interface in the complex of ribonuclease a with ribonuclease inhibitor

Interface Area

Des

olva

tion

free

ene

rgy

1400 2000 3400

-4

Type IEasy

Enzymes

Type IIEasy


Type IIIUncertain

Antibody/Antigen

Type IVDifficult

Type VHopeless



change


Type II Or

Type V?

Conformati-onal

Interface


Example












ASA <1400 Å2



Ras and Ras interacting domain (1lfd) KD = 2M, ASA = 1250 Å2, Gdes = 3.6 kcal/mol, and Eelec =-39.5 kcal/mol A number of weak complexes are in this category


ASA > 2000 Å2

Generally moderate


Cyclin A and cyclin-dependent kinase 2 (1fin): KD = 47.6 M, ASA = 3550 Å2,

Gdes = 3.9 kcal/mol, and Eelec= -66.5 kcal/mol.



ras protein

ras-interacting domain of ralgds

GNP (5'-guanosyl-imido-triphosphate

Interface in the complex of ras-interacting domain with ras

Interface Area

Des

olva

tion

free

ene

rgy

1400 2000 3400

-4

Type IEasy

Enzymes

Type IIEasy


Type IIIUncertain

Antibody/Antigen

Type IVDifficult

Type VHopeless



change


Type II Or

Type V?

Conformati-onal

Interface


Example












ASA <1400 Å2





ASA > 2000 Å2

Generally moderate


Cyclin A and cyclin-dependent kinase 2 (1fin): KD = 47.6 nM, ASA = 3550 Å2,

Gdes = 3.9 kcal/mol, Eelec =-66.5 kcal/mol



Type V:Large interface andlarge conformational change

Cyclin-ACyclin-dependent kinase

Li, L. et al. (2003) RDOCK

Gray, J.J. et al. (2003)

Overall Success rates of participants in CAPRI 1-5

2: How the community is doing?

Classification of CAPRI 1-2 Targets

Interface area ASA

1000 1500 2000 2500 3000 3500

Des

olv

atio

n f

ree

ener

gy,

kca

l/mo

l

-10

-5

0

5

10

15

20

T2 T1

T7

T5 T3T4T6

Type IVdifficult

Type IIIuncertain

Type IIeasy

Type Ieasy

Type Vvery difficult


Interface area ASA

1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500

De

solv

ati

on

fre

e e

ne

rgy,

kc

al/m

ol

-15

-10

-5

0

5

10

15

20

25

T13

T18

T8

T10

T12

T19

T14

T9

Type IIIuncertain

Type IIeasy Type V

very difficult

Type Ieasy

Classification of CAPRI 3-5 Targets


Interface Area

Des

olva

tion

free

ene

rgy

1400 2000 3400

-4

Type IEasy

Enzymes

Type IIEasy


Type IIIUncertain

Antibody/Antigen

Type IVDifficult

Type VHopeless



change

Small signalling

complexes

Type II Or

Type V?

Expected Improvements

Much improved

3. How to deal with side chain flexibility?

Coarse details

Fine details

Trypsin/APPI

Receptor

Ligand

Recognition mechanisms:

Lock-and-key vs. Induced fit

Key-and-latch mechanismRajamani, D., Thiel, S. Vajda, S. and C.J. Camacho. Anchor residues in protein-protein

interactions. Proc. Natl. Acad. Sci. USA, 101: 11287-11292, 2004.

Key-Latch model

KEYS which stay close to the bound conformation in solution

LATCHES do not show preference to stay near bound conformation.

key

latch

UnboundBoundSimulated

Solvated protein

Individually crystallized protein Predisposition

0

1

2

3

4

5

6

7

1 2 3 4Time (ns)

RM

SD

BoundUnbound

RMSD of Arg39 of ribonuclease A with respect to the structure found in the complex (bound; PDB code 1DFJ) and in the individually crystallized ribonuclease A (unbound; PDB code 7RSA). The RMSD was computed for 2000 snapshots of a 4ns MD simulation of 7RSA.

3.32.72.2

2.41.8

1.52.4

0

100

200

300

2.3 2.9 2.4 1.8 1.7 3.5 1.7 3.1

Clusters

Clu

ste

r s

ize

Clustering of the conformations of Arg39 in ribonuclease A. The 16 largest clusters were derived from a pairwise RMSD analysis of the MD snapshots, and clustering using a radius of 2Å. The RMSD of the cluster center from the bound conformation is shown on the top/bottom of each bar. The bound conformation is shown in blue, unbound in red, and the dominant conformation from the MD simulations is shown in green.

Complex of trypsin with amyloid β-protein inhibitor (APPI). Key residue Arg-15 is a major contributor to the total binding free energy.

HIV-1 NEF/FYN tyrosine kinase SH3 domain complex. Trp-119 is within 1 and 2 Å of the bound conformation for 36% and 96% of the MD. It is stabilized in this native-like conformation by Tyr-93 (and therefore also native-like) in the free state. Thr-97 buries the second largest SASA (70 Å2).

Tyr93Thr97

Asp100

Trp119

Lys68Arg45

Hyhel-5 Fab/lysozyme complex. The main key residue, Arg-45, has a SASA value of 147 Å2; a second key residue, Lys-68, is found buried with a SASA = 93 Å2. Both side chains show native-like properties, sampling during 50% and 97% of the time conformations that were less than 2 Å rmsd from their corresponding bound rotamer.

The complex of acetylcholinesterase with fasciculin. The main key Met-33 is in a native-like conformation during most of the simulation. The SASA encompassed by Met-33 is comparable with the next largest SASA of 78 Å2 resulting from the burial of Arg-27; this anchor is in a native-like conformer during 95% of the MD.

Met33

Thr8Arg27

Complex Receptor/Ligand Anchora ΔSASAb

ΔGbind Residence time, %c

PDB ID ResID Å2 kcal/mol Rank MD Rotamer

library

Enzyme/Inhibitor

1BRC Trypsin/APPI (1AAP) Arg 15 251.24 -11.9 1 32† 7.4†

2SIC Subtilisin BPN/Inhibitor Met 70 196.33 -7.1 1 51†

2SNI Subtilisin novo/CI2 (2CI2) Ile 56 189.79 -7.6 1 37‡ 96.6‡

1CHO α-Chymotrypsin/OMTKY3 Leu 18 180.33 -7.9 1 73‡*

1CSE Subtilisin C/eglin C (1ACB) Leu 45 165.07 -5.1 1 50‡ 97.4‡

1BRS Barnase/barstar (1A19) Asp 35 125.06 -2.5 3 97‡

1UGH** UDG/UGI Leu 272 180.38 -5.2 1 66‡

1DFJ Ribonuclease inhibitor/ Asn 67 101.18 -1.2 8 41‡ 28.5‡

ribonuclease A (7RSA)

1FSS AchE/FasII (1FSC) Thr 8 96.29 -3.4 4 99‡

Antigen/Antibody

1BQL Hyhel5 Fab/QBL (1DKJ) Arg 45 165.3 -10.1 1 49† 38.3†

1FBI IgG1 Fab/lysozyme Arg 73 132.72 -1.9 4 46†

1DQJ Hyhel63 Fab/HEL (3LZT) Arg 21 131.4 5.4 92† 29.1†

PDB IDa

Receptor/Ligandb

Native-like Predictions

Anchor replacementBoundc UBd Resurf

e

Enzyme/Inhibitor complexes ResiduefRMSDBound Predictions

UBg MDh Bi MDj

2SNI Subtilisin Novo/Chymotrypsin

inhibitor2(2CI2)151 62 124 Met59 2.9 1.5 99 82

151 62 Ile56 0.6 0.9 73 74

1DFJ Ribonuclease inhibitor/ Ribonuclease A

(7RSA)63 22 43 Arg39 3.6 2.7 22 43

1BRC Trypsin/APPI (1AAP) 98 24 45 Arg15 3.8 2.4 70 57

1CHO A-Chymotrypsin/Ovomucoid 3rd domain 182 61 91 Leu/Met18 32 68

1BRS Barnase/Barstar (1A19) 54 23 27 Asp35 0.8 0.6 16 17

1CSE Subtilisin Carlsberg/Eglin C(1ACB) 176 105 116 Leu45 0.6 1 133 119

1FSS Snake venom

acetylcholinesterase/FasciculinII (1FSC)21 3 4 Thr9 0.5 0.6 4 5

2BTF β-actin/Profilin 43 18 15 Arg74* 2.4 1.9 25 28

1WQ1 RAS activating domain/ RAS 29 26 9 Tyr32 0.2 1.5 26 21

Gln61* 2.4 1.5 28 40

Credits

Crystallographers: Please submit to CAPRI

Dr. Carlos Camacho (University of Pittsburgh)

Graduate students at Boston University Stephen ComeauDeepa RajamaniDima KozakovYang ShenRyan Brenke

National Institute of Health

Documents

Classification of Protein Complexes based on Biophysics of Association Sandor Vajda