The Atomic Structure of Protein-Protein Recognition Sites Loredana Lo Conte 1 , Cyrus Chothia 1 and Joe È l Janin 1,2 * 1 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB1 1JX, England 2 Laboratoire d'Enzymologie et de Biochimie Structurales CNRS UPR9063 91198 Gif-sur-Yvette, France The non-covalent assembly of proteins that fold separately is central to many biological processes, and differs from the permanent macromolecu- lar assembly of protein subunits in oligomeric proteins. We performed an analysis of the atomic structure of the recognition sites seen in 75 pro- tein-protein complexes of known three-dimensional structure: 24 pro- tease-inhibitor, 19 antibody-antigen and 32 other complexes, including nine enzyme-inhibitor and 11 that are involved in signal transduction. The size of the recognition site is related to the conformational changes that occur upon association. Of the 75 complexes, 52 have ``standard-size'' interfaces in which the total area buried by the components in the recog- nition site is 1600 (400) A Ê 2 . In these complexes, association involves only small changes of conformation. Twenty complexes have ``large'' interfaces burying 2000 to 4660 A Ê 2 , and large conformational changes are seen to occur in those cases where we can compare the structure of complexed and free components. The average interface has approximately the same non-polar character as the protein surface as a whole, and carries some- what fewer charged groups. However, some interfaces are signi®cantly more polar and others more non-polar than the average. Of the atoms that lose accessibility upon association, half make contacts across the interface and one-third become fully inaccessible to the solvent. In the latter case, the Voronoi volume was calculated and compared with that of atoms buried inside proteins. The ratio of the two volumes was 1.01 (0.03) in all but 11 complexes, which shows that atoms buried at protein-protein interfaces are close-packed like the protein interior. This conclusion could be extended to the majority of interface atoms by includ- ing solvent positions determined in high-resolution X-ray structures in the calculation of Voronoi volumes. Thus, water molecules contribute to the close-packing of atoms that insure complementarity between the two pro- tein surfaces, as well as providing polar interactions between the two pro- teins. # 1999 Academic Press Keywords: protein-protein complexes; interface area; polar interactions; packing density; conformation changes *Corresponding author Introduction Many biological processes are carried out, or regulated, through the interactions between pre- formed proteins. The importance of such inter- actions in biology has made the protein recognition process an area of considerable interest. Here, we describe aspects of the atomic structure of the rec- ognition sites seen in protein-protein complexes of known structure. The protein-protein complexes whose structures we analyse here are non-covalent assemblies of proteins that fold separately to carry out independent functions before they associate, as opposed to permanent macromolecular assemblies, such as oligomeric proteins, virus capsids or muscle ®bres. Though the two types of complexes have features in common, they also have many that differ, and are best treated separately. General analyses of structural aspects of protein- protein interaction have been carried out (Chothia & Janin, 1975; Argos, 1988; Janin & Chothia, 1990; Janin, 1995, 1996; Jones & Thornton, 1995, 1996, E-mail address of the corresponding author: janin@lebs.cnrs-gif.fr Abbreviations used: ASA, solvent-accessible surface area; PDB, Protein Data Bank; PTI, pancreatic trypsin inhibitor. Article No. jmbi.1998.2439 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 285, 2177±2198 0022-2836/99/052177±22 $30.00/0 # 1999 Academic Press