Structural and Computational Characterization of the SHV-1 β-Lactamase-β-Lactamase Inhibitor Protein Interface

Kimberly A Reynolds,Jodi M Thomson,Kevin D Corbett,Christopher R Bethel,James M Berger,Jack F Kirsch,Robert A Bonomo,Tracy M Handel

doi:10.1074/jbc.m603878200

Abstract

Beta-lactamase inhibitor protein (BLIP) binds a variety of class A beta-lactamases with affinities ranging from micromolar to picomolar. Whereas the TEM-1 and SHV-1 beta-lactamases are almost structurally identical, BLIP binds TEM-1 approximately 1000-fold tighter than SHV-1. Determining the underlying source of this affinity difference is important for understanding the molecular basis of beta-lactamase inhibition and mechanisms of protein-protein interface specificity and affinity. Here we present the 1.6A resolution crystal structure of SHV-1.BLIP. In addition, a point mutation was identified, SHV D104E, that increases SHV.BLIP binding affinity from micromolar to nanomolar. Comparison of the SHV-1.BLIP structure with the published TEM-1.BLIP structure suggests that the increased volume of Glu-104 stabilizes a key binding loop in the interface. Solution of the 1.8A SHV D104K.BLIP crystal structure identifies a novel conformation in which this binding loop is removed from the interface. Using these structural data, we evaluated the ability of EGAD, a program developed for computational protein design, to calculate changes in the stability of mutant beta-lactamase.BLIP complexes. Changes in binding affinity were calculated within an error of 1.6 kcal/mol of the experimental values for 112 mutations at the TEM-1.BLIP interface and within an error of 2.2 kcal/mol for 24 mutations at the SHV-1.BLIP interface. The reasonable success of EGAD in predicting changes in interface stability is a promising step toward understanding the stability of the beta-lactamase.BLIP complexes and computationally assisted design of tight binding BLIP variants.

Highlights

The extensive contacts removed between position 104 and the Phe-142 loop are consistent with previous mutational data indicating that Phe-142TEM is a hot spot at the TEM11⁄7BLIP interface, whereas Phe-142SHV appears to have a reduced role in the SHV-11⁄7BLIP interface [2]
We further explored the role of position 104 at the SHV-11⁄7BLIP interface through mutagenesis
Ested in using computational methods to guide future Four SHV-1 residues that differ in identity from TEM-1 result mutagenesis studies of the interface, we conducted an extensive in decreased van der Waals (vdW) and electrostatic interactions at the interface examination of the ability of EGAD to predict stability changes according to our calculations

Summary

Introduction

Comparison of the hydrogen bond, salt bridge, and vdW interactions of these residues in the TEM-11⁄7BLIP and SHV-11⁄7BLIP complexes provides some structural insight. The extensive contacts removed between position 104 and the Phe-142 loop are consistent with previous mutational data indicating that Phe-142TEM is a hot spot at the TEM11⁄7BLIP interface, whereas Phe-142SHV appears to have a reduced role in the SHV-11⁄7BLIP interface [2].

Results

Conclusion