TY - JOUR
T1 - Structural and computational characterization of the SHV-1 β-lactamase-β-lactamase inhibitor protein interface
AU - Reynolds, Kimberly A.
AU - Thomson, Jodi M.
AU - Corbett, Kevin D.
AU - Bethel, Christopher R.
AU - Berger, James M.
AU - Kirsch, Jack F.
AU - Bonomo, Robert A.
AU - Handel, Tracy M.
PY - 2006/9/8
Y1 - 2006/9/8
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M603878200
DO - 10.1074/jbc.M603878200
M3 - Article
C2 - 16809340
AN - SCOPUS:33748753961
SN - 0021-9258
VL - 281
SP - 26745
EP - 26753
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 36
ER -