Protein-Protein Interactions: Stability, Function and ...

1 Protein-Protein Interactions: Stability, Function and LandscapeStructural Aspects of Protein-Protein interactions Agenda Understand the importance of studying Protein-Protein interactions at the structural level Classify the various types of interactions Look at one structure-based method for predicting Protein-Protein interactionsLINKP rotein interaction Definition Specific interactions between two or more proteins. Examples Enzyme-inhibitor complex; antibody-antigen complex; receptor-ligand interactions , multiprotein complexes such as ribosomes or RNA usuallypermanentand optimized ( , thehomodimer cytochrome c9 (1)) (Fig. 1a).Heterocomplexes can also have suchproperties, or they can be non-obligatory,being made and broken according to theenvironment or external factors and involveproteins that must also exist independently[ , the enzyme inhibitorcomplex trypsin with the inhibitor frombitter gourd (2) (Fig.)]

2 1b) and the antibody protein complex HYHEL-5 with lysozyme(3) (Fig. 1c)].It is important to distinguish between the different types of complexes when analyzing the intermolecularinterfaces that occur within : Protein-Protein interactions can be arbitrarily classified based on theproteins involved (structural or functional groups) or based on their physicalproperties (weak and transient, non-obligate vs. strong and permanent). Proteininteractions are usually mediated by defined domains, hence interactions can also beclassified based on the underlying : All of molecular biology is about Protein-Protein interactions (Alberts etal. 2002, Lodish et al. 2000). Protein-Protein interactions affect all processes in a cell:structural proteins need to interact in order to shape organelles and the whole cell,molecular machines such as ribosomes or RNA polymerases are hold together byprotein- protein interactions , and the same is true for multi-subunit channels orreceptors in distinguishes such interactions from random collisions that happen byBrownian motion in the aqeous solutions inside and outside of cells.

3 Note that manyproteins are known to interact although it remains unclear whether certain interactionshave any physiological of interactions : It is estimated that even simple single-celled organisms suchas yeast have their roughly 6000 proteins interact by at least 3 interactions per protein , a total of 20,000 interactions or more. By extrapolation, there may be on the orderof ~100,000 interactions in the human Protein-Protein interaction network in interaction map of the yeast proteome assembled from published interactions . The map contains 1,548 proteins (boxes) and 2,358 interactions (connecting lines).Homo- and hetero-oligomeric complexesProtein- protein interactions (PPIs) occur between identical or non-identical chains ( homo- or hetero-oligomers). (A-B)Oligomers of identical or homologous protein units can be organized in an isologous or heterologous way (Monod et al.)

4 , 1965) with structural symmetry (Goodsell and Olson, 2000). An isologous association involves the same surface on both monomers ( Arc repressor and lysin; Figure 1A and C), related by a 2-fold symmetry axis. In contrast to an isologous association that can only further oligomerize using a different interface ( form a dimer of dimers with three 2-fold axes of symmetry), heterologous assemblies use different interfaces that, without a closed (cyclic) symmetry, can lead to infinite and obligate complexesAs well as composition, two different types of complexescan be distinguished on the basis of whether a complex isobligate or non-obligate. In an obligate PPI, the protomersare not found as stable structures on their own in complexes are generally also functionally obligate;for example, the Arc repressor dimer (Figure 1A) isessential for DNA binding.

5 Many of the hetero-oligomeric structures in the protein Data Bank involve non-obligateinteractions of protomers that exist independently, such asintracellular signalling complexes ( RhoA RhoGAP;Figure 1D) and antibody antigen, receptor ligand andenzyme inhibitor ( thrombin rodniin; Figure 1E) components of such protein protein complexesare often initially not co-localized and thus need tobe independently stable. However, some homo-oligomers,which by definition are co-localized, can also form nonobligateassemblies ( sperm lysin; Figure 1C).Transient and permanent complexesPPIs can also be distinguished based on the lifetime of thecomplex. In contrast to a permanent interaction that isusually very stable and thus only exists in its complexedform, a transient interaction associates and dissociatesin vivo. We distinguish weak transient interactions thatfeature a dynamic oligomeric equilibrium in solution,where the interaction is broken and formed continuously( lysin; Figure 1C), and strong transient associationsthat require a molecular trigger to shift the oligomericequilibrium.

6 For example, the heterotrimeric G protein (Figure 1F) dissociates into the Ga and Gbg subunitsupon guanosine triphosphate (GTP) binding, but forms astable trimer with guanosine diphosphate (GDP) or functionally obligate interactions areusually permanent, whereas non-obligate interactionsmay be transient or of Protein-Protein interactions (PPI)Obligate PPIthe protomers are notfound as stable structures on their own in vivoNon-obligate PPIO bligate homodimerP22 Arc repressor DNA-bindingObligate heterodimerHuman cathepsin D1 LYBNon-obligate homodimerSperm lysinNon-obligate heterodimerRhoAand RhoGAPsignaling complexTypes of Protein-Protein interactions (PPI)Obligate PPIusually permanentthe protomers are notfound as stable structures on their own in vivoNon-obligate PPIO bligate heterodimerHuman cathepsin DNon-obligate transient homodimer, Sperm lysin(interaction is broken and formed continuously)Permanent(many enzyme-inhibitor complexes)dissociation constant Kd=[A][B] / [AB] 10-7-10-13 MTransientWeak(electron transport complexes)KdmM- MNon-obligate permanent heterodimerThrombinand rodniininhibitorIntermediate(antibody-an tigen, TCR-MHC-peptide, signal transduction PPI), Kd M-nMStrong(require a molecular trigger to shift the oligomericequilibrium)KdnM-fMBovine G protein dissociates into G and G subunits upon GTP, but forms a stable trimer upon GDPT ypes of Protein-Protein interactions (PPI)

7 Obligate PPIusually permanentthe protomers are notfound as stable structures on their own in vivoNon-obligate PPIO bligate heterodimerHuman cathepsin DNon-obligate transient homodimer, Sperm lysin(interaction is broken and formed continuously)Permanent(many enzyme-inhibitor complexes)dissociation constant Kd=[A][B] / [AB] 10-7 10-13 MTransientWeak(electron transport complexes)KdmM- MNon-obligate permanent heterodimerThrombinand rodniininhibitorIntermediate(antibody-an tigen, TCR-MHC-peptide, signal transduction PPI), Kd M-nMStrong(require a molecular trigger to shift the oligomericequilibrium)KdnM-fMBovine G protein dissociates into G and G subunits upon GTP, but forms a stable trimer upon GDPS tructural features of protein -interaction sites The contact area between two proteins is almost always bigger than 1100 2 with each of the interacting partners contributing at least 550 2 of complementary surface.

8 On average each partner loses about 800 2 of solvent-accessible surface upon contact, contributed by some 20 amino acid residues of each partner, the average interface residue covers some 40 2. NACCESS The Accessible surface area (ASA) of the complexes is calculated using an implementaion of the Lee and Richards (1971) algorithm devloped by Hubbard (1992). With a probe sphere, of radius angstroms, the ASA was defined as the surface mapped out by the centre of the probe as if it were rolled around the van der Waals surface of the protein . The program is used to calculate the ASA of each protomer in the complex and then the complete ASA shown in the results table is for a single subunit (chain1 as designated by the user on the submission form (this subunit is indiacted at the top of the table and coloured purple)). Forces that mediate Protein-Protein interactions include electrostatic interactions , hydrogen bonds, the van der Waals attraction and hydrophobic effects.

9 The average Protein-Protein interface is not less polar or more hydrophobic than the surface remaining in contact with the solvent. Water is usually excluded from the contact region. Non-obligate complexes tend to be more hydrophilic in comparison, as each component has to exist independently in the cell. It has been proposed that hydrophobic forces drive Protein-Protein interactions and hydrogen bonds and salt bridges confer : Independent studies showed that 83-84% of interfaces are more or less flat. With few exceptions, the interfaces are approximately circular areas on the protein surface in both permenant and non-obligate complexes. Interfaces in permanent associations tend to be larger, less planar, more highly segmented (in terms of sequence), and closer packed than interfaces in non-obligate : can be measured in terms of fitting surface shape.

10 Interfaces in homodimers, enzyme-inhibitor complexes, and permanent heterocomplexes are the most complementary, whilst the antibody-antigen complexes and the non-obligate heterocomplexes are the least structure: In one study the loop interactions contributed, on average, 40% of the interface contacts. In another study (involving 28 homodimers), 53% of the interface residues were a-helical, 22% beta sheets, and 12% ab, with the rest being acid composition: Interfaces have been shown to be more hydrophobic thanthe exterior but less hydrophobic than the interior of a protein . In one study, 47% ofinterface residues were hydrophobic, 31% polar and 22% charged. Permanentcomplexes have interfaces that contain hydrophobic residues, whilst the interfaces in 5 non-obligate complexes favour the more polar residues. Site-directed mutagenesisshowed that in many cases a large majority ( > 50%) of interface residues can bemutated to alanine with little effect on Kd: the functional epitope is a subset of thestructural relevance and applications of Protein-Protein interaction analysisBiologically active proteins such as peptide hormones or antibodies act by interactingwith other proteins such as receptors or antigens, respectively.