EBI is an Outstation of the European Molecular Biology Laboratory. PDBe-PISA a web based service for understanding Protein Interfaces, Surfaces and Assemblies

EBI is an Outstation of the European Molecular Biology Laboratory.

PDBe-PISA

a web based service for understandingProtein Interfaces, Surfaces and

Assemblies

Sanchayita Sen, PhDPDB Depositions

Protein Data Bank Europe http://www.ebi.ac.uk/pdbe

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Protein Quaternary Structures (PQS)

PQS is often a Biological Unit, performing a certain physiological function

PQS is a difficult subject for experimental studies

Assembly of protein chains, stable in native environment

Light/Neutron/X-ray scattering: mainly composition and multimeric state may be found. 3D shape may be guessed from mobility measurements.

Electron microscopy: not a fantastic resolution and not applicable to all objects

NMR is not good for big chains, even less so for protein assemblies.

In PDB, very few quaternary structures have been identified experimentally.


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More than 80% of the structures are solved by x-ray diffraction methods

An X-ray diffraction experiment produces atomic coordinates of the crystal’s Asymmetric Unit (ASU).

In general, neither ASU nor Unit Cell has any relation to Biological Unit, or stable protein complex which acts as a unit in physiological processes.

Biological Unit may be made of

Unit Cell = all space symmetry group mates of ASU

Crystal = translated Unit Cell

PDB file

• a single ASU• part of ASU

• several ASU• several parts of

neighbouring ASUs

Asymmetric Unit vs. Biological Unit


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Significant interfaces?

PQS server @ EBI (Kim Henrick) Trends in Biochem. Sci. (1998) 23, 358 PITA server @ EBI (Hannes Ponstingl) J. Appl. Cryst. (2003) 36, 1116


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PQS server @ PDBe-EBI (Kim Henrick) Trends in Biochem. Sci. (1998) 23, 358

http://pqs.ebi.ac.uk Method: progressive build-up by addition of monomeric chains that suit the selection criteria. The results are partly curated.

http://www.ebi.ac.uk/thornton-srv/databases/pita/ Method: recursive splitting of the largest complexes as allowed by crystal symmetry. Termination criteria is derived from the individual statistical scores of crystal contacts. The results are not curated.

PITA software @ Thornton group EBI (Hannes Ponstingl) J. Appl. Cryst. (2003) 36, 1116

Making assemblies from significant interfaces


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Protein functionality: the interface should be engaged in any sort of interaction, including transient short-living protein-ligand and protein-protein etc. associations. Obviously important properties:

• Affinity (comes from area, hydrophobicity, electrostatics, H-bonding etc.)

Depends on the problem.

• Aminoacid composition• Geometrical complementarity• Overall shape, compactness• Charge distribution• etc.

and properties that may be important for reaction pathway and dynamics:

What is a significant interface?


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“No single parameter absolutely differentiates the interfaces from all other surface patches”Jones, S. & Thornton, J.M. (1996) Principles of protein-protein interactions, Proc. Natl. Acad. Sci. USA, 93, 13-20.

Formation of stable complexes involves interplay between affinity and entropy change and therefore may be less dependent on the interface characteristic features.

“…the type of complexes need to be taken into account when characterizing interfaces between them.”

Jones, S. & Thornton, J.M., ibid.

Real and superficial interfaces

•Few databases available which analyses protein-protein interactions and interfaces derived from PDB - systematic view on factors (macromolecular binding


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Factors to consider

• Stability of a macromolecular complex is governed by the following physicochemical properties:

• free energy of formation • solvation energy gain• interface area (buried surface area > 10% ASA)• hydrogen bonds and saltbridges across the

interface• Hydrophobic specificity


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It is not properties of individual interfaces but rather chemical stability of protein complex in general that really matters

Protein chains will most likely associate into largest complexes that are still stable

A protein complex is stable if its free energy of dissociation is positive:

0int0 STGGdiss

Chemical stability of protein complexes

RTG

ni i

ddiss

A

AK

0

exp


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Binding energy

sbsbhbhb

n

iisolnsol NENEAGAAAGG

121 ,int

Solvation energy of protein complex

Solvation energies of dissociated

subunits

Free energy of H-bond formation

Number of H-bonds between

dissociated subunits

Free energy of salt bridge

formation

Number of salt bridges between

dissociated subunits

321 AAA 321 AAA

Dissociation into stable subunits with minimum

STGGdiss int

Choice of dissociation subunits:

Binding energy may be viewed as a function of

individual interfaces.


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Entropy of macromolecules in solutions

aSISmSS surfSrottrans ,ˆ

Translational entropy Rotational entropy Sidechain entropy

MassSolvent-accessible

surface areaTensor of inertia

mRcmS ttrans log23

2321log2,ˆ SrSrot IIIRcIS

FaaSsurf

Murray C.W. and Verdonik M.L. (2002)J. Comput.-Aided Mol. Design 16, 741-753.

Symmetry number

ct , cr and F are semiempirical parameters


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Which assembly?Which assembly?

We now know (or we think that we know) how to evaluate chemical stability of protein complexes.

Given a 3D-arrangement of protein chains, we can now say whether there are chances that this arrangement is a stable assembly, or a biological unit.

How one can get potential assemblies in first place?

- find all assemblies that are allowed by crystal symmetry


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• crystal is represented as a periodic graph with monomeric chains as nodes and interfaces as edges

• each set of assemblies is identified by engaged interface types

• Due to crystal symmetry engaged

interfaces must satisfy 2 conditions. 1)If an interface of a particular type is engaged, all other interfaces of the same type are also engaged 2)An interface cannot be engaged if doing

so results in assembly that contains equivalent monomeric units in parallel orientations

• all assemblies may be enumerated by a backtracking scheme engaging all possible combinations of different interface types

Enumerating assemblies in crystal


14 Macromolecular Structure Database31.10.0714

Method Summary

1. Build periodic graph of the crystal

2. Enumerate all possibly stable assemblies

3. Evaluate assemblies for chemical stability

4. Leave only sets of stable assemblies in the list and range them by chances to be a biological unit :

• Larger assemblies take preference• Single-assembly solutions take preference• Otherwise, assemblies with higher Gdiss take preference

Detection of Biological Units in Crystals:


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• What quaternary structure can my crystal structure have?

• What are the crystal contacts and interfaces in my structure ?

• What are the energetics that keep my quaternary structure together ?

• Are there any other structures in the PDB that have similar interfaces ?

USE Pisa

Upload your own PDB file for analysis !!

If you have to ask….


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http://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver


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Assembly Information


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Interfaces


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Details about the interface…

Documents

EBI is an Outstation of the European Molecular Biology Laboratory. PDBe-PISA a web based service for understanding Protein Interfaces, Surfaces and Assemblies