The Subunit Interfaces of Weakly Associated Homodimeric Proteins Sucharita Dey 1 , Arumay Pal 2 , Pinak Chakrabarti 1,2 and Joël Janin 3 ⁎ 1 Bioinformatics Centre, Bose Institute, P-1/12 CIT Scheme VIIM, Calcutta 700 054, India 2 Department of Biochemistry, Bose Institute, P-1/12 CIT Scheme VIIM, Calcutta 700 054, India 3 Yeast Structural Genomics, IBBMC UMR 8619 CNRS, Université Paris-Sud, 91405 Orsay, France Received 16 September 2009; received in revised form 10 February 2010; accepted 10 February 2010 Available online 13 February 2010 We analyzed subunit interfaces in 315 homodimers with an X-ray structure in the Protein Data Bank, validated by checking the literature for data that indicate that the proteins are dimeric in solution and that, in the case of the “weak” dimers, the homodimer is in equilibrium with the monomer. The interfaces of the 42 weak dimers, which are smaller by a factor of 2.4 on average than in the remainder of the set, are comparable in size with antibody–antigen or protease–inhibitor interfaces. Nevertheless, they are more hydrophobic than in the average transient protein–protein complex and similar in amino acid composition to the other homodimer interfaces. The mean numbers of interface hydrogen bonds and hydration water molecules per unit area are also similar in homodimers and transient complexes. Parameters related to the atomic packing suggest that many of the weak dimer interfaces are loosely packed, and we suggest that this contributes to their low stability. To evaluate the evolutionary selection pressure on interface residues, we calculated the Shannon entropy of homologous amino acid sequences at 60% sequence identity. In 93% of the homodimers, the interface residues are better conserved than the residues on the protein surface. The weak dimers display the same high degree of interface conservation as other homodimers, but their homologs may be heterodimers as well as homodimers. Their interfaces may be good models in terms of their size, composition, and evolutionary conservation for the labile subunit contacts that allow protein assemblies to share and exchange components, allosteric proteins to undergo quaternary structure transitions, and molecular machines to operate in the cell. © 2010 Elsevier Ltd. All rights reserved. Edited by M. Sternberg Keywords: protein–protein interaction; monomer–dimer equilibrium; inter- face area; amino acid propensity; atomic packing density Introduction Two major aspects of the interaction between biological (macro)molecules are the timescale over which it occurs and the stability of the assembly as a function of the concentration of its components. The timescale and the concentration scale are broad, and they reflect the diversity of the biological processes that depend on molecular recognition. A macromolecular assembly may be considered as permanent if it is stable over times longer than the life of a cell or as transient if it dissociates or exchanges components on that timescale. Most oligomeric proteins are permanent in that sense: their subunits assemble tightly as soon as they are synthesized, and they stay together afterwards. Many protein–protein com- plexes are also stable once they form, but their components behave as individual units until they come into contact. The Protein Data Bank (PDB) 1 is a rich source of information on the interactions that stabilize oligomeric proteins and protein– protein complexes, and a number of generic studies have analyzed the properties of their subunit interfaces. 2–16 However, permanent struc- tures are not the rule in biology, and genome-wide studies performed in recent years have shown that most macromolecular assemblies contain both a stable core and weakly bound exchangeable components. 17,18 The permanent interactions seen in most oligomeric proteins may be good models *Corresponding author. E-mail address: joel.janin@u-psud.fr. Abbreviations used: BSA, buried surface area; ASA, accessible surface area; NF-κB, nuclear factor κ B; BPTI, bovine pancreatic trypsin inhibitor. doi:10.1016/j.jmb.2010.02.020 J. Mol. Biol. (2010) 398, 146–160 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.