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Explore protein quaternary structures, interfaces, and assemblies with PDBe-PISA. Understand biological units, formation of stable complexes, and significant interfaces for protein studies.
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PDBe-PISA a web based service for understandingProtein Interfaces, Surfaces and Assemblies Sanchayita Sen, PhD PDB Depositions
Protein Quaternary Structures (PQS) Assembly of protein chains, stable in native environment • PQS is often a Biological Unit, performing a certain physiological function • PQS is a difficult subject for experimental studies • 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.
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 Asymmetric Unit vs. Biological Unit Crystal = translated Unit Cell Unit Cell = all space symmetry group mates of ASU • a single ASU • part of ASU • several ASU • several parts of neighbouring ASUs PDB file
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
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. • PITA software @ Thornton group EBI (Hannes Ponstingl) J. Appl. Cryst. (2003) 36, 1116 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. Making assemblies from significant interfaces
What is a significant interface? Depends on the problem. • 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.) • and properties that may be important for reaction pathway and dynamics: • Aminoacid composition • Geometrical complementarity • Overall shape, compactness • Charge distribution • etc.
Formation of stable complexes involves interplay between affinity and entropy change and therefore may be less dependent on the interface characteristic features. Real and superficial interfaces • “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. • Few databases available which analyses protein-protein interactions and interfaces derived from PDB - systematic view on factors (macromolecular binding • “…the type of complexes need to be taken into account when characterizing interfaces between them.” Jones, S. & Thornton, J.M., ibid.
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 Factors to consider
Chemical stability of protein complexes • 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: 9 31.10.07
Choice of dissociation subunits: Dissociation into stable subunits with minimum Binding energy Solvation energies of dissociated subunits Free energy of H-bond formation Free energy of salt bridge formation Solvation energy of protein complex Number of H-bonds between dissociated subunits Number of salt bridges between dissociated subunits Binding energy may be viewed as a function of individual interfaces.
Entropy of macromolecules in solutions Translational entropy Rotational entropy Sidechain entropy Solvent-accessible surface area Mass Tensor of inertia Symmetry number Murray C.W. and Verdonik M.L. (2002) J. Comput.-Aided Mol. Design 16, 741-753. ct, cr and F are semiempirical parameters
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
Enumerating assemblies in crystal • 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 13 31.10.07
Detection of Biological Units in Crystals: MethodSummary • Build periodic graph of the crystal • Enumerate all possibly stable assemblies • Evaluate assemblies for chemical stability • 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 14 31.10.07 Macromolecular Structure Database
If you have to ask…. • 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 !!
