Influence of substrates and inhibitors on the structure of Klebsiella pneumoniae carbapenemase-2

Abstract

The hydrolysis of last resort carbapenem antibiotics by Klebsiella pneumoniae carbapenemase-2 (KPC-2) presents a significant danger to global health. Combined with horizontal gene transfer, the emergence KPC-2 threatens to quickly expand carbapenemase activity to ever increasing numbers of pathogens. Our understanding of KPC-2 has greatly increased over the past decade thanks, in great part, to 20 crystal structures solved by groups around the world. These include apo KPC-2 structures, along with structures featuring a library of 10 different inhibitors representing diverse structural and functional classes. Herein we focus on cataloging the available KPC-2 structures and presenting a discussion of key aspects of each structure and important relationships between structures. Although the available structures do not provide information on dynamic motions with KPC-2, and the family of structures indicates small conformational changes across a wide array of bound inhibitors, substrates, and products, the structures provide a strong foundation for additional studies in the coming years to discover new KPC-2 inhibitors.

Impact statement: The work herein is important to the field as it provides a clear and succinct accounting of available KPC-2 structures. The work advances the field by collecting and analyzing differences and similarities across the available structures. This work features new analyses and interpretations of the existing structures which will impact the field in a positive way by making structural insights more widely available among the beta-lactamase community.

Keywords: Beta-lactamase; KPC-2; inhibitors.

Figure 1.

Panel A shows the two sub-domains of KPC-2, the α+β sandwich (blue) and the α-helical cluster (red). Panel B highlights the three loop regions near the active site. The sidechain of W105 has been rendered with stick representation to highlight the “gating” role it plays for the active site. Panel C is a rendering of the active site with the surface of the active site colored by element and select active site residues shown as sticks. The KPC-2 structure with PDB code 2OV5 was used as the basis for all representations in this figure. (A color version of this figure is available in the online journal.)

Figure 2.

Panel A is a rendering of the active site of the KPC-2 3NPBA complex (PDB ID 3RXX). Select active site residues (green) are shown as sticks. The boron of 3NPBA is covalently bonded to the side chain oxygen atom of catalytic S70, and the oxyanion hole is occupied. Panel B is a rendering of the active site of KPC-2 with S02030 (PDB ID 5EEC), though for clarity, only one conformation of S02030 is shown. The carboxyl moiety does not interact with T235 or T237 in the second conformation (not shown). Panel C is a rendering of the active site of KPC-2 in complex with a phenyl boronic acid with a prop-2-enoic substituent (PDB ID 5LL7). Like in PDB ID 3RXX, the side chain of W105 is positioned such that the active site cleft is open compared to the positioning in PDB ID 5EEC. Panel D is an overlay of the three structures from panels A–C. The most dramatic changes are in the positioning of W105. (A color version of this figure is available in the online journal.)

Figure 3.

Panel A shows the active site of the PSR3–226 complex (PDB ID 3RXW). For clarity, the citrate molecules are not shown, and for W105 only the side chain rotamer corresponding to bound PSR3-226 is depicted. Panel B is a rendering of the hydrolyzed faropenem product (PDB ID 5UJ4). Two conformers for S130 are shown and the side chain of S70 is rotated into the oxyanion hole. Panel C is a rendering of the cefotaxime hydrolyzed product (PDB ID 5UJ3). The side chain of S70 is rotated into the oxyanion hole, which is thought to assist in expulsion of product from the active site. The wide and shallow active site of KPC-2 is readily accommodating of the comparatively bulky substituents within cefotaxime. Panel D is an overlay of the three structures from panels A–C. (A color version of this figure is available in the online journal.)

Figure 4.

Panel A shows the active site of the KPC-2 Avibactam complex (PDB ID 4ZBE). The sulfate group of avibactam interacts with residues from the KTG motif and T216. Panel B shows the active site in complex with WCK 4234 (PDB ID 6B1H). The positioning of W105 is consistent for the WCK DABCO compounds, though each are different than the W105 rotamer observed in the avibactam bound KPC-2 structure (PDB ID 4ZBE). Panel C shows the active site of the WCK 5107 complex (PDB ID 6B1H). Notable amongst the WCK compounds, the piperidine ring interacts with the carbonyl of C238. Further functionalization might yield other stabilizing interactions. Panel D shows the active site of the WCK 5153 complex (PDB ID 6B1X). The pyrrolidine ring does not have the same interaction with the carbonyl of C238 as seen in 6B1H. Panel E overlays all four of DABCO-bound structures in panels A–D. Consistent with the structures displayed in Figures 2 and 3, the most dramatic changes in the active site are seen for the side chain of W105. (A color version of this figure is available in the online journal.)