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J. Biol. Chem., Vol. 281, Issue 36, 26745-26753, September 8, 2006

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

Kimberly A. Reynolds¹, Jodi M. Thomson^¶2, Kevin D. Corbett^||, Christopher R. Bethel^**, James M. Berger^||, Jack F. Kirsch^||3, Robert A. Bonomo^¶**4, and Tracy M. Handel⁵

From the Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California 92093-0684, the ^¶Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106, the Biophysics Group and ^||Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, and ^**Research Service, Louis Stokes Veterans Affairs Medical Center, Cleveland, Ohio 44106

-Lactamase inhibitor protein (BLIP) binds a variety of class A -lactamases with affinities ranging from micromolar to picomolar. Whereas the TEM-1 and SHV-1 -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 -lactamase inhibition and mechanisms of protein-protein interface specificity and affinity. Here we present the 1.6Å 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 1.8Å 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 -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 -lactamase ·BLIP complexes and computationally assisted design of tight binding BLIP variants.

Received for publication, April 24, 2006 , and in revised form, June 21, 2006.

The atomic coordinates and structure factors (code 2G2U and 2G2W) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1 and 2.

¹ Supported by a National Science Foundation (NSF) graduate research fellowship.

² Supported in part by National Institutes of Health (NIH) Grant T32 GM07250 and by the Case Medical Scientist Training Program.

³ Supported by NIH Grant GM35393.

⁴ Supported by NIH Grant 5R01AI635172 and the Veterans Affairs Medical Center Merit Review Program.

⁵ Supported by NSF Grant 0344749. To whom correspondence should be addressed: CMM East Rm. 2057, 9500 Gilman Dr. Mail Code 0684, La Jolla, CA 92093-0684. Tel.: 858-822-6656; Fax: 858-822-6655; E-mail: thandel{at}ucsd.edu.

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