Dissecting Subunit Interfaces in Homodimeric Proteins Ranjit Prasad Bahadur, 1 Pinak Chakrabarti, 1 Francis Rodier, 2 and Joe ¨ l Janin 2 * 1 Department of Biochemistry, Bose Institute, Calcutta, India 2 Laboratoire d’Enzymologie et de Biochimie Structurales, CNRS UPR 9063, Gif-sur-Yvette, France ABSTRACT The subunit interfaces of 122 ho- modimers of known three-dimensional structure are analyzed and dissected into sets of surface patches by clustering atoms at the interface; 70 interfaces are single-patch, the others have up to six patches, often contributed by different structural domains. The average interface buries 1,940 Å 2 of the surface of each monomer, contains one or two patches burying 600 –1,600 Å 2 , is 65% nonpolar and includes 18 hydrogen bonds. However, the range of size and of hydrophobicity is wide among the 122 interfaces. Each interface has a core made of resi- dues with atoms buried in the dimer, surrounded by a rim of residues with atoms that remain accessible to solvent. The core, which constitutes 77% of the interface on average, has an amino acid composi- tion that resembles the protein interior except for the presence of arginine residues, whereas the rim is more like the protein surface. These properties of the interfaces in homodimers, which are permanent assemblies, are compared to those of protein– protein complexes where the components associate after they have independently folded. On average, subunit interfaces in homodimers are twice larger than in complexes, and much less polar due to the large fraction belonging to the core, although the amino acid compositions of the cores are similar in the two types of interfaces. Proteins 2003;53:708 –719. © 2003 Wiley-Liss, Inc. Key words: interface area; protein–protein recogni- tion; protein–protein interaction; inter- faces; hydrophobicity; polar interac- tions; oligomeric proteins INTRODUCTION Homodimers are the simplest example of the non- covalent self-assembly of proteins. This class of proteins is abundantly represented in the Protein Data Bank (PDB). 1 Their subunit interfaces, defined as the regions of the protein surface that are involved in subunit contacts, display geometric and chemical properties that give the assembly its stability and specificity. The interfaces formed by the components of protein–protein complexes could be expected to share the same properties. However, most homodimeric proteins are permanent assemblies, and their polypeptide chains assemble at the time they fold. In contrast, complexes involve proteins that fold separately and remain monomers until they meet and associate. Several studies have been devoted to subunit interfaces in oligomeric proteins 2,3 and in protein–protein complexes. 3,4 As the number of new X-ray structures increases rapidly, these studies must be updated regularly. Here, we exam- ine a sample of 122 homodimer interfaces with tools that we already applied to interfaces in complexes. 5 We show that subunit interfaces in homodimers are very diverse both in their size and in their chemical composition. On average, they are more hydrophobic and bury twice as much protein surfaces as in complexes. The larger inter- faces involve several patches of the subunit surface, often contributed by separate structural domains. Like in com- plexes, the subunit interfaces have a core of residues containing atoms that are fully buried in the dimer. The core has an amino acid composition similar to that of the protein interior, and is surrounded by a rim that remains solvent accessible and has an amino acid composition similar to that of the protein surface. METHODS One hundred twenty-two entries representing X-ray structures of homodimeric proteins were taken from the Protein Data Bank 1 at the Research Collaboratory for Structural Bioinformatics or the Protein Quaternary Struc- ture Server (PQS) 6 at the European Bioinformatics Insti- tute. When more than one entry was available for a particular protein, the structure with the highest resolu- tion was retained; 84 out of 122 structures have resolu- tions better than 2.0 Å; 32 have resolution in the range 2–2.5 Å, and 6 in the range 2.5–3.0 Å. The sequence identity between any two proteins in the set was less than 25%. In cases where the dimer did not constitute the crystal asymmetric unit, it was generated with the informa- tion of record BIOMT if it was present. Alternatively, we used the program CRISPACK 7 to generate neighbors, and selected the pair of twofold related subunits that had the largest interface area. The procedure was implemented in program ASPIC. The solvent accessible surface area (ASA) was computed using the program ACCESS, 8 which is an implementation of the algorithm of Lee and Richards. 9 The subunit inter- face area was estimated as B/2 where B was: Grant sponsor: Indo-French Centre for the Promotion of Advanced Research (CEFIPRA); Grant number: 2203-2. *Correspondence to: J. Janin, LEBS-CNRS, 91198-Gif-sur-Yvette, France. E-mail: janin@lebs.cnrs-gif.fr Received 10 December 2002; Accepted 20 February 2003 PROTEINS: Structure, Function, and Genetics 53:708 –719 (2003) © 2003 WILEY-LISS, INC.