LbGlcK and the HsHxKIV d glucose complex PDB entry IDH

LbGlcK and the HsHxKIV-d-glucose complex (PDB entry 3IDH) [17] were superimposed from individual subunits. The active site regions revealed the key Closantel Sodium mg binding residues to be present for LbGlcK; also, the superposition showed that HsHxKIV had residues that were absent in LbGlcK, such as K169 and T168 (Fig. 1c). A structure-based sequence alignment was also established between the same crystal structures (Fig. 2). The overall sequence identity based on that alignment was 15%, which is quite low but desirable from a drug discovery perspective, as an overall goal is to find inhibitor selectivity toward the parasite homologue. A noteworthy structural difference is with two distinct loops at the outer part of the active site. In HsHxKIV, there exists two large loop regions [(L165 – G178); (S281 – Q287)] that interact with each other through van der Waals contact. Such loops were evaluated from our previous work and hypothesized to cause steric hindrance to glucosamine analogue inhibitors in accessing the active site. The S281 – Q287 loop is oriented adjacent to the glucose binding site and N283, Q286, and Q287 have potential to block this region due to the proximity of their side chains [16]. In TcGlcK, these two loop regions [(G102 – I110); (K230 – N234)] also exist but they are substantially shorter in residue count. As a result, they do not interact with each other and they also do not preclude inhibitor access to the active site. LbGlcK also has these two loop regions [G114 – I122); (A248 – I260)]. The first loop region contains 9 residues and is very similar to the loop segment found in TcGlcK; however, the second loop region is a 13-residue loop and is longer than both the loop segments of TcGlcK and HsHxKIV. Through our crystallography, we observed a 4-residue segment [K247 – A250] in both the A- and B- chains that had disorder and could not be modeled in. As this segment is part of the 13-residue loop, although being a longer loop, it appears that it still would not cause deterrence to inhibitor access to the active site even if the 4-residue segment could be modeled in, from a visual observation.
Conclusions With glucose kinases being critical enzymes for glycolysis and the PPP due to the requirement of d-glucose as a major carbon source for Leishmania spp., the crystal structure of LbGlcK will provide important guidance regarding the design of new inhibitors. Competitive inhibitor designs for LbGlcK can follow a structure-activity relationship method because HPOP-GlcN was shown to have competitive inhibition, as such, variations in the structure of HPOP-GlcN will provide a starting point for new inhibitor designs. Thus, different variants of glucosamine analogue inhibitors are expected to strongly inhibit LbGlcK. Such inhibitors may also be biologically active against Leishmania parasites. Future studies in this regard will be reported in the course of time.
Accession code The atomic coordinates and structure factors for apo-LbGlcK were deposited in the Protein Data Bank (www.rcsb.org) having the accession code of 6EDI.
Acknowledgments The work was supported by an Advanced Support for Innovative Research Excellence: Track I (ASPIRE-I) grant to E.L.D. and undergraduate research grants through the Magellan program to G.S.B. and M.E.M. that were provided by the University of South Carolina, Office of the Vice President for Research. We thank the beamlines of the Northeastern Collaborative Access Team (NE-CAT), which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P41 GM103403). The Pilatus 6M detector on beamline 24-ID-C is funded by a NIH-ORIP HEI grant (S10 RR029205). This research used resources from the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.