Protein Structure Modelling

Many sequences - few structures

Homology Modelling - Based on Sequence Similarity with Sequences of Known Structures.

COMPLEXES

Protein:DNAComplex

Initiation of

Transcription.

Protein1

ComplexFormation

ConformationalChange

Function

Protein2

MoreProteins

Structures of Macromolecular Complexes

• Very few experimentally determined– e.g. antibodies:antigens

• ~1000 antibody sequences known• ~100 antibody structures known• ~10 antibody:antigen complex structures known

• Can we use calculation?

Structures of Isolated Components?

•- crystallography

•- NMR

•- Homology Modelling

Structure of Complex?•Rigid-Body Shape Complementarity(based on hydrophobic effect and van der Waals packing)•Conformational Change on Complexation?•Electrostatic Complementarity?•Solvation Effects?•Experiment?

Complementarity Determining Region Loops (CDRs):(i) Uniform Conformational Searching(ii) Canonical Loop Modelling(iii) Data-Base Searching of Loop Conformations(iv) Molecular Dynamics in vacuo and with solvated CDRs.

> 90 models.

Clustering and Screening for Consistency with Experimental Antibody Structures.

4 Dynamically Interconvertible Models.

Homology Model of Framework Residues.

Modelling of Isolated Antibody

Clustering and Screening for:

(i) Buried Surface Area.(ii) Electrostatic Complementarity. (iii) Consistency with existing Ab:Ag complex structures.

> 18 models. Refinement of Atomic-Detail Models with Molecular Dynamics

in Explicit Solvent.

6 Models.

Initial Generation

Low -Resolution Shape Complementarity.

> 41,585 models

Modelling of Ab:Ag Complex

Which Experiments can help Model the

Structures of Complexes?

• Site-Directed Mutagenesis– Mutate then measure affinity.– Swap Mutations e.g. Glu:Lys for Lys:Glu

• Optical Spectroscopy.– e.g. Fluorescence energy transfer gives direct

information on distances between sites.

Functional Binding Site on Toxin

Red: Affinity Lowered >100-fold

Yellow: Affinity Lowered 10-100 fold

Toxin and M 23 Functional Binding Sites

Red - >100 fold affinity loss on mutation

Yellow - 10-100 fold affinity loss on mutation

Three Models of Calculated M23 Paratope

Red: Residues contacting antigen energy coreYellow: Residues contacting functional epitope

Orientation of toxin onM23 combining site in

the two remaining models.