Highlights d A library of isomeric complex Asn-linked glycans was chemoenzymatically synthesized d Free reducing N-glycans can be generated by treatment with sodium hypochlorite d Unique N-glycan interactions identified with a suite of human proteins and viruses d Differential N-glycan recognition provides insights into biomedical applications

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KPC β-lactamases hydrolyze the “last resort” β-lactam antibiotics (carbapenems) used to treat multi-drug resistant infections, and are compromising efforts to combat life-threatening Gram-negative bacterial infections in hospitals worldwide. Consequently, the development of novel inhibitors is essential for restoring the effectiveness of existing antibiotics. The β-lactamase inhibitor protein (BLIP) is a competitive inhibitor of a number of class A β-lactamases. In this study, we characterize the previously unreported interaction between the KPC-2 β-lactamase and BLIP. Biochemical results show that BLIP is an extremely potent inhibitor of KPC enzymes, binding KPC-2 and KPC-3 with subnanomolar affinity. To understand the basis of affinity and specificity in the β-lactamase/BLIP system, the crystallographic structure of the KPC-2/BLIP complex was solved to 1.9 Å resolution. Computational alanine scanning was also conducted to identify putative hot spots in the KPC-2/BLIP interface. Interestingly, the two complexes making up the KPC-2/BLIP asymmetric unit are distinct, and in one structure the BLIP F142 loop is absent, in contrast to homologous structures where it occupies the active site. This finding and other sources of structural plasticity appear to contribute to BLIP’S promiscuity, enabling it to respond to mutations at the β-lactamase interface. Given the continuing emergence of antibiotic resistance, the high-resolution KPC-2/BLIP structure will facilitate its use as a template for the rational design of new inhibitors of this problematic enzyme.

Summaryβ-lactamase/β-lactamase Inhibitor Protein (BLIP) complexes are emerging as a well characterized experimental model system for studying protein-protein interactions. β-lactamases are enzymes that catalyze the hydrolysis of β-lactam antibiotics. BLIP is a 165 amino acid protein that inhibits several class A β-lactamases with a wide range of affinities: pM affinity for K1; nM affinity for TEM-1, SME-1, and BlaI but only µM affinity for SHV-1 β-lactamase. The large differences in affinity coupled with the availability of extensive mutagenesis data and high resolution crystal structures for the TEM-1/BLIP and SHV-1/BLIP complexes make them attractive systems for the further development of protein engineering and computational design methodologies. We used EGAD, a physics-based computational design program, to redesign BLIP with the goal of increasing affinity for SHV-1. The resulting designed sequences are highly similar to wildtype, with the exception of BLIP residues surrounding β-lactamase position 104. Interestingly, this residue is a known specificity determinant between TEM-1 and SHV-1. Characterization of several of the designs and point mutants revealed that in all cases, the mutations stabilize the interface by 10 to 1000 fold relative to wildtype BLIP. The calculated changes in binding affinity for the mutants were within a mean absolute error of 0.87 kcal/mol from the experimental values, and comparison of calculated and experimental values for a set of 30 SHV-1/BLIP complexes yielded a correlation coefficient of 0.77. Although binding specificity for SHV-1 versus TEM-1 was not explicitly considered in the design process, two of the BLIP variants exhibit a small specificity switch. Structures of the two highest affinity complexes, SHV-1/BLIP (E73M) and SHV-1/BLIP (E73M, S130K, S146M), are presented at 1.7 Å resolution. While the predicted structures have much in common with the experimentally determined structures, they do not coincide perfectly; in particular a salt bridge between SHV-1 D104 and BLIP K74 is observed in