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

2. Protein Data Bank Europe http://www.ebi.ac.uk/pdbe 2 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.

3. Protein Data Bank Europe http://www.ebi.ac.uk/pdbe 3  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

4. Protein Data Bank Europe http://www.ebi.ac.uk/pdbe 4 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

5. Protein Data Bank Europe http://www.ebi.ac.uk/pdbe 5  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

6. Protein Data Bank Europe http://www.ebi.ac.uk/pdbe 6 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?

7. Protein Data Bank Europe http://www.ebi.ac.uk/pdbe 7  “ 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

8. Protein Data Bank Europe http://www.ebi.ac.uk/pdbe 8 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

9. Protein Data Bank Europe http://www.ebi.ac.uk/pdbe 9 31.10.07 9  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: Chemical stability of protein complexes

10. Protein Data Bank Europe http://www.ebi.ac.uk/pdbe 10 Binding energy 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 Dissociation into stable subunits with minimum Choice of dissociation subunits: Binding energy may be viewed as a function of individual interfaces.

11. Protein Data Bank Europe http://www.ebi.ac.uk/pdbe 11 Entropy of macromolecules in solutions Translational entropyRotational entropySidechain entropy Mass Solvent-accessible surface area Tensor of inertia Murray C.W. and Verdonik M.L. (2002) J. Comput.-Aided Mol. Design 16, 741-753. Symmetry number c t, c r and F are semiempirical parameters

12. Protein Data Bank Europe http://www.ebi.ac.uk/pdbe 12 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

13. Protein Data Bank Europe http://www.ebi.ac.uk/pdbe 13 31.10.07 13 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. Protein Data Bank Europe http://www.ebi.ac.uk/pdbe 14 Macromolecular Structure Database31.10.07 14 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  G diss take preference Detection of Biological Units in Crystals:

15. Protein Data Bank Europe http://www.ebi.ac.uk/pdbe 15 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….

16. Protein Data Bank Europe http://www.ebi.ac.uk/pdbe 16 http://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver

17. Protein Data Bank Europe http://www.ebi.ac.uk/pdbe 17 Assembly Information

18. Protein Data Bank Europe http://www.ebi.ac.uk/pdbe 18 Interfaces

19. Protein Data Bank Europe http://www.ebi.ac.uk/pdbe 19 Details about the interface…

20. Protein Data Bank Europe http://www.ebi.ac.uk/pdbe 20 Entropy of dissociation Fitted parameter Mass of i-th subunit k-th principal moment of inertia of i-th subunit By its very nature, entropy of dissociation is function of protein complex rather than that of individual interfaces. ∆G diss = ∆G 0 diss + RTlogπ i A i /A