Insights into Positive and Negative Requirements for Protein–Protein Interactions by Crystallographic Analysis of the β-Lactamase Inhibitory Proteins BLIP, BLIP-I, and BLP Michael Gretes 1 , Daniel C. Lim 1 , Liza de Castro 1 , Susan E. Jensen 2 , Sung Gyun Kang 3 , Kye Joon Lee 4 and Natalie C. J. Strynadka 1 ⁎ 1 Department of Biochemistry and Molecular Biology and the Center for Blood Research, University of British Columbia, Vancouver, British Columbia, Canada 2 Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada 3 Marine Biotechnology Research Center, Korean Ocean Research & Development, Ansan, South Korea 4 School of Biological Sciences, Seoul National University, Seoul 151-742, Korea Received 12 December 2008; received in revised form 15 March 2009; accepted 20 March 2009 Available online 28 March 2009 β-Lactamase inhibitory protein (BLIP) binds a variety of β-lactamase enzymes with wide-ranging specificity. Its binding mechanism and interface interactions are a well-established model system for the characterization of protein–protein interactions. Published studies have examined the binding of BLIP to diverse target β-lactamases (e.g., TEM-1, SME-1, and SHV-1). However, apart from point mutations of amino acid residues, variability on the inhibitor side of this enzyme–inhibitor interface has remained unex- plored. Thus, we present crystal structures of two likely BLIP relatives: (1) BLIP-I (solved alone and in complex with TEM-1), which has β-lactamase inhibitory activity very similar to that of BLIP; and (2) β-lactamase- inhibitory-protein-like protein (BLP) (in two apo forms, including an ultra- high-resolution structure), which is unable to inhibit any tested β-lactamase. Despite categorical differences in species of origin and function, BLIP-I and BLP share nearly identical backbone conformations, even at loop regions differing in BLIP. We describe interacting residues and provide a comparative structural analysis of the interactions formed at the interface of BLIP-I·TEM-1 versus those formed at the interface of BLIP·TEM-1. Along with initial attempts to functionally characterize BLP, we examine its amino acid residues that structurally correspond to BLIP/BLIP-I binding hotspots to explain its inability to bind and inhibit TEM-1. We conclude that the BLIP family fold is a robust and flexible scaffold that permits the formation of high-affinity protein–protein interactions while remaining highly selective. Comparison of the two naturally occurring, distinct binding interfaces built upon this scaffold (BLIP and BLIP-I) shows that there is substantial variation pos- sible in the subnanomolar binding interaction with TEM-1. The cor- responding (non-TEM-1-binding) BLP surface shows that numerous favorable backbone–backbone/backbone–side-chain interactions with a protein partner can be negated by the presence of a few, strongly unfavorable interactions, especially electrostatic repulsions. © 2009 Elsevier Ltd. All rights reserved. Edited by G. Schulz Keywords: protein–protein interactions; interaction hotspots; interaction specificity; β-lactamase inhibitory proteins; crystal structure *Corresponding author. E-mail address: natalie@byron.biochem.ubc.ca. Current address: D.C. Lim, Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Abbreviations used: BLIP, β-lactamase inhibitory protein; BLP, β-lactamase-inhibitory-protein-like protein; PDB, Protein Data Bank; SeMet, selenomethionine; 3D, three-dimensional; RMSD, root-mean-square difference; sc, protein side chain; mc, protein main chain; ITC, isothermal calorimetry; apr, apramycin; PEG, polyethylene glycol; thio, thiostrepton; ALS, Advanced Light Source; CCD, charge-coupled device; APBS, Adaptive Poisson–Boltzmann Solver. doi:10.1016/j.jmb.2009.03.058 J. Mol. Biol. (2009) 389, 289–305 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.