Structure-Function Analysis 117 Jan 2006 DNA/Protein structure-function analysis and prediction Protein-protein Interaction (PPI) and Docking: Protein-protein Interaction –Interfaces –Solvation –Energetics –Conformational change –Allostery Examples –Arfaptin – Rac –Ribosome Docking –Search space –Docking methods

Structure-Function Analysis 217 Jan 2006 PPI Characteristics Universal –Cell functionality based on protein-protein interactions Cyto-skeleton Ribosome RNA polymerase Numerous –Yeast: ~6.000 proteins at least 3 interactions each  ~ interactions –Human: estimated ~ interactions Network –simplest: homodimer (two) –common: hetero-oligomer (more) –holistic: protein network (all)

Structure-Function Analysis 317 Jan 2006 Interface Area Contact area –usually >1100 Å 2 –each partner >550 Å 2 each partner loses ~800 Å 2 of solvent accessible surface area –~20 amino acids lose ~40 Å 2 –~ J per Å 2 Average buried accessible surface area: –12% for dimers –17% for trimers –21% for tetramers 83-84% of all interfaces are flat Secondary structure: –50%  -helix –20%  -sheet –20% coil –10% mixed Less hydrophobic than core, more hydrophobic than exterior

Structure-Function Analysis 417 Jan 2006 Complexation Reaction A + B  AB –K a = [AB]/[A][B]  association –K d = [A][B]/[AB]  dissociation

Structure-Function Analysis 517 Jan 2006 Experimental Methods 2D (poly-acrylamide) gel electrophoresis  mass spectrometry Liquid chromatography –e.g. gel permeation chromatography Binding study with one immobilized partner –e.g. surface plasmon resonance In vivo by two-hybrid systems or FRET Binding constants by ultra-centrifugation, micro-calorimetry or competition experiments with labelled ligand –e.g. fluorescence, radioactivity Role of individual amino acids by site directed mutagenesis Structural studies –e.g. NMR or X-ray

Structure-Function Analysis 617 Jan 2006 Surface Plasmon Resonance 1 Evanescent field

Structure-Function Analysis 717 Jan 2006 Surface Plasmon Resonance 2 sensitivity limit for layer thickness ~ nm.

Structure-Function Analysis 817 Jan 2006 Surface Plasmon Resonance 3 plasmon curve –Reflexion intensity as function of angle of incidence  dotted line: after addition of adsorbing molecules. kinetics during adsorption process –Reflexion intensity as function of time at fixed angle .

Structure-Function Analysis 917 Jan 2006 PPI Network

Structure-Function Analysis 1017 Jan 2006 Protein-protein interactions Complexity: –Multibody interaction Diversity: –Various interaction types Specificity: –Complementarity in shape and binding properties

Structure-Function Analysis 1117 Jan 2006 Binding vs. Localization Obligate oligomers Non-obligate weak transient Non-obligate triggered transient e.g. GTPPO 4 - Non-obligate co-localised e.g. in membrane Non-obligate permanent e.g. antibody-antigen strong weak co-expresseddifferent places

Structure-Function Analysis 1217 Jan 2006 Some terminology Transient interactions: –Associate and dissociate in vivo Weak transient: –dynamic oligomeric equilibrium Strong transient: –require a molecular trigger to shift the equilibrium Obligate PPI: –protomers not stable structures on their own –(functionally obligate)

Structure-Function Analysis 1317 Jan 2006 Strong – medium – weak Nanomolar to sub-nanomolar  K d < Micromolar to nanomolar  > K d > Micromolar  > K d > A + B  AB  K d = [A][B]/[AB]  dissociation

Structure-Function Analysis 1417 Jan 2006 Analysis of 122 Homodimers 70 interfaces single patched 35 have two patches 17 have three or more

Structure-Function Analysis 1517 Jan 2006 Patches Cluster in different domains –(structurally defined units often with specific function) two domains anticodon-binding catalytic

Structure-Function Analysis 1617 Jan 2006 Interfaces ~30% polar ~70% non-polar

Structure-Function Analysis 1717 Jan 2006 Interface Rim is water accessible rim core

Structure-Function Analysis 1817 Jan 2006 Interface composition Composition of interface essentially the same as core But % surface area can be quite different!

Structure-Function Analysis 1917 Jan 2006 Propensities Interface vs. surface propensities –as ln(f int /f surf )

Structure-Function Analysis 2017 Jan 2006 Conformational Change Chaperones –extreme conformational changes upon complexation  ligand unfolds within the chaperone GroEL/GroES Allosteric proteins –conformational change at 'active' site –ligand binds to 'regulating' site Peptides –often adopt 'bound' conformation –different from the 'free' conformation

Structure-Function Analysis 2117 Jan 2006 Allostery 1 Regulation by 'remote' modulation of binding affinity (complex strength)

Structure-Function Analysis 2217 Jan 2006 Allostery 2 Substrate binding is cooperative Binding of first substrate at first active site –stimulates active shape –promotes binding of second substrate

Structure-Function Analysis 2317 Jan 2006 Allostery 3 Committed step of metabolic pathway –regulated by an allosteric enzyme Pathway end product –can regulate the allosteric enzyme for the first committed step Inhibitor binding favors inactive form

Structure-Function Analysis 2417 Jan 2006 DNA/Protein structure-function analysis and prediction Protein-protein Interaction (PPI) and Docking: Protein-protein Interaction –Interfaces –Solvation –Energetics –Conformational change –Allostery Examples –Arfaptin – Rac –Ribosome Docking –Search space –Docking methods

Structure-Function Analysis 2517 Jan 2006 Small G-proteins cross-talk Mediated by Arfaptin

Structure-Function Analysis 2617 Jan 2006 Arfaptin and Rac Micromolar K d But specific

Structure-Function Analysis 2717 Jan 2006 Arfaptin Interacting surface

Structure-Function Analysis 2817 Jan 2006 Arfaptin Interacting patches

Structure-Function Analysis 2917 Jan 2006 Arfaptin Exposed surface

Structure-Function Analysis 3017 Jan 2006 Rac Interacting patch

Structure-Function Analysis 3117 Jan 2006 Arfaptin + Rac Exposed surface

Structure-Function Analysis 3217 Jan S structure at 5.5 Å (Noller et al. Science 2001)

Structure-Function Analysis 3317 Jan S structure

Structure-Function Analysis 3417 Jan S-50S interface Overall buried surface area ~8500 Å 2 < 37.5 Å Å 2 – 75 Å 2 > 75 Å 2

Structure-Function Analysis 3517 Jan 2006 Protein-nucleic acid Interactions

Structure-Function Analysis 3617 Jan 2006 Interactions in the Ribosome

Structure-Function Analysis 3717 Jan 2006 DNA/Protein structure-function analysis and prediction Protein-protein Interaction (PPI) and Docking: Protein-protein Interaction –Interfaces –Solvation –Energetics –Conformational change –Allostery Examples –Arfaptin – Rac –Ribosome Docking –Search space –Docking methods

Structure-Function Analysis 3817 Jan 2006 Docking - ZDOCK Protein-protein docking –3-dimensional (3D) structure of protein complex –starting from 3D structures of receptor and ligand Rigid-body docking algorithm (ZDOCK) –pairwise shape complementarity function –all possible binding modes –using Fast Fourier Transform algorithm Refinement algorithm (RDOCK) –top 2000 predicted structures –three-stage energy minimization –electrostatic and desolvation energies molecular mechanical software (CHARMM) statistical energy method (Atomic Contact Energy) 49 non-redundant unbound test cases: –near-native structure (<2.5Å) for 37% test cases for 49% within top 4

Structure-Function Analysis 3917 Jan 2006 Protein-protein docking Finding correct surface match Systematic search: –2 times 3D space! Define functions: –‘1’ on surface –‘  ’ or ‘  ’ inside –‘0’ outside  

Structure-Function Analysis 4017 Jan 2006 Protein-protein docking Correlation function: C  = 1/N 3  o  p  q exp[2  i(o  + p  + q  )/N] C o,p,q

Structure-Function Analysis 4117 Jan 2006 Docking Programs ZDOCK, RDOCK AutoDock Bielefeld Protein Docking DOCK DOT FTDock, RPScore and MultiDock GRAMM Hex 3.0 ICM Protein-Protein docking KORDO MolFit MPI Protein Docking Nussinov-Wolfson Structural Bioinformatics Group …