Binding Hot Spots in the TEM1-BLIP Interface in Light of its Modular Architecture

D. Reichmann, M. Cohen, R. Abramovich, O. Dym, D. Lim, N. C.J. Strynadka, G. Schreiber*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

80 Scopus citations


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.

Original languageAmerican English
Pages (from-to)663-679
Number of pages17
JournalJournal of Molecular Biology
Issue number3
StatePublished - 19 Jan 2007
Externally publishedYes

Bibliographical note

Funding Information:
We thank Professor Amnon Horovitz and Professor Igal Burstaein for their comments and stimulating discussions, Eyal Kalie for critical reading of the manuscript, the Israel Structural Proteomics Center (ISPC) for crystallization and structure analysis and BioRad for their collaboration. This research was funded by MINERVA grant 8525 and the Ministry of Science and Technology (MOST) grant 0263.


  • X-ray
  • energetics
  • hot spots
  • protein-protein interaction
  • structure-function


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