Specificity and cooperativity at β-lactamase position 104 in TEM-1/BLIP and SHV-1/BLIP interactions
Article first published online: 3 FEB 2011
Copyright © 2011 Wiley-Liss, Inc.
Proteins: Structure, Function, and Bioinformatics
Volume 79, Issue 4, pages 1267–1276, April 2011
How to Cite
Hanes, M. S., Reynolds, K. A., McNamara, C., Ghosh, P., Bonomo, R. A., Kirsch, J. F. and Handel, T. M. (2011), Specificity and cooperativity at β-lactamase position 104 in TEM-1/BLIP and SHV-1/BLIP interactions. Proteins, 79: 1267–1276. doi: 10.1002/prot.22961
- Issue published online: 8 MAR 2011
- Article first published online: 3 FEB 2011
- Accepted manuscript online: 13 DEC 2010 04:39PM EST
- Manuscript Accepted: 2 DEC 2010
- Manuscript Revised: 30 NOV 2010
- Manuscript Received: 9 AUG 2010
- NSF. Grant Number: 0344749
- U.S. Department of Veterans Affairs Merit Review Program. Grant Number: VISN 10 GRECC
- National Institutes of Health (NIH). Grant Number: 1R01 A1063517-01
- NIGMS. Grant Number: GM35393.
- double mutant cycle;
- salt bridge;
- protein–protein interaction
Establishing a quantitative understanding of the determinants of affinity in protein–protein interactions remains challenging. For example, TEM-1/β-lactamase inhibitor protein (BLIP) and SHV-1/BLIP are homologous β-lactamase/β-lactamase inhibitor protein complexes with disparate Kd values (3 nM and 2 μM, respectively), and a single substitution, D104E in SHV-1, results in a 1000-fold enhancement in binding affinity. In TEM-1, E104 participates in a salt bridge with BLIP K74, whereas the corresponding SHV-1 D104 does not in the wild type SHV-1/BLIP co-structure. Here, we present a 1.6 Å crystal structure of the SHV-1 D104E/BLIP complex that demonstrates that this point mutation restores this salt bridge. Additionally, mutation of a neighboring residue, BLIP E73M, results in salt bridge formation between SHV-1 D104 and BLIP K74 and a 400-fold increase in binding affinity. To understand how this salt bridge contributes to complex affinity, the cooperativity between the E/K or D/K salt bridge pair and a neighboring hot spot residue (BLIP F142) was investigated using double mutant cycle analyses in the background of the E73M mutation. We find that BLIP F142 cooperatively stabilizes both interactions, illustrating how a single mutation at a hot spot position can drive large perturbations in interface stability and specificity through a cooperative interaction network. Proteins 2011. © 2011 Wiley-Liss, Inc.