Binding Hot Spots in the TEM1–BLIP Interface in Light of its Modular Architecture D. Reichmann 1 , M. Cohen 1 , R. Abramovich 1 , O. Dym 2 , D. Lim 3 N. C. J. Strynadka 3 and G. Schreiber 1 ⁎ 1 Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, 76100, Israel 2 Department of Structural Biology, Weizmann Institute of Science, Rehovot, 76100, Israel 3 Department of Biochemistry and Molecular Biology , University of British Columbia, Vancouver, Canada V6T 1Z3 Proteins bind one another in aqua’s solution to form tight and specific complexes. Previously we have shown that this is achieved through the modular architecture of the interaction network formed by the interface residues, where tight cooperative interactions are found within modules but not between them. Here we extend this study to cover the entire interface of TEM1 β-lactamase and its protein inhibitor BLIP using an improved method for deriving interaction maps based on REDUCE to add hydrogen atoms and then by evaluating the interactions using modifications of the programs PROBE, NCI and PARE. An extensive mutagenesis study of the interface residues indeed showed that each module is energetically independent on other modules, and that cooperativity is found only within a module. By solving the X-ray structure of two interface mutations affecting two different modules, we demonstrated that protein–protein binding occur via the structural reorganization of the binding modules, either by a “lock and key” or an induced fit mechanism. To explain the cooperativity within a module, we performed multiple-mutant cycle analysis of cluster 2 resulting in a high- resolution energy map of this module. Mutant studies are usually done in reference to alanine, which can be regarded as a deletion of a side-chain. However, from a biological perspective, there is a major interest to understand non-Ala substitutions, as they are most common. Using X-ray crystallography and multiple-mutant cycle analysis we demonstrated the added complexity in understanding non-Ala mutations. Here, a double mutation replacing the wild-type Glu,Tyr to Tyr,Asn on TEM1 (res id 104,105) caused a major backbone structural rearrangement of BLIP, changing the composition of two modules but not of other modules within the interface. This shows the robustness of the modular approach, yet demonstrates the complexity of in silico protein design. © 2006 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: protein–protein interaction; energetics; X-ray; structure-function; hot spots Introduction The interest in the mechanisms of binding and networking of protein–protein interactions has grown increasingly over the past years stemming from the realization that much of the cellular complexity is related to such interactions. The ability of certain proteins to form specific stable protein–protein complexes is fundamental for biological processes, including events such as signal transduction, cell cycle regulation, immune response etc. This is feasible due to the almost unlimited potential to generate unique binding sites on proteins, characterized by their shape and surface chemistry. Much effort has been invested in understanding the biophysical basis allowing for proteins to interact in aqueous solution. Mutational analysis of protein interfaces has led to the conclusion that only a minority of Abbreviations used: TEM1, TEM1-β-lactamase; BLIP, β- lactamase inhibitor protein; C1 to C6, cluster 1 to cluster 6; SPR, surface plasmon resonance; PDB, Protein Data Bank; WT, wild-type. E-mail address of the corresponding author: gideon.schreiber@weizmann.ac.il doi:10.1016/j.jmb.2006.09.076 J. Mol. Biol. (2007) 365, 663–679 0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.