β-Lactamase inhibitor protein (BLIP) binds a variety of class A β-lactamases with affinities ranging from micromolar to picomolar. Whereas the TEM-1 and SHV-1 β-lactamases are almost structurally identical, BLIP binds TEM-1 ∼1000-fold tighter than SHV-1. Determining the underlying source of this affinity difference is important for understanding the molecular basis of β-lactamase inhibition and mechanisms of protein-protein interface specificity and affinity. Here we present the 1.6 Å 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 1.8 Å 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 β- 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 β-lactamase·BLIP complexes and computationally assisted design of tight binding BLIP variants.
ASJC Scopus subject areas