LbGlcK crystallized as a ligandless

LbGlcK crystallized as a ligandless dimer in the asymmetric unit of space group P21 (Fig. 1a) with two identical subunits stabilized by a large contact surface area of 2295 Å2 (13% of the total surface area) as determined by the Protein Interfaces, Surfaces, and Assemblies (PISA) server [18]. Each monomer has a large α + β domain (residues 157–383) and a small α/β domain (residues 1–142 and 384–398) (Fig. 1b). Each subunit of LbGlcK was observed in an open conformation between large and small domains and this comparison was made by having a superposition against the TcGlcK – BENZ-GlcN complex (PDB entry 5BRD) (Figure S2, SI), which has a low bending region angle of 4.9° [16]. Specifically, the large domains of LbGlcK and TcGlcK were superimposed onto each other while the small domains oriented differently. The small domain of LbGlcK (open form) had a bending region angle that was greater than 20° from the small domain of TcGlcK (closed form), as determined by the program DynDom [21]. When the LbGlcK closed form crystal structure becomes available, a better analysis of the bending region angle can be established between the two domains. For a trypanosomatid glucokinase, this structure reveals the first open conformation structure reported. The overall fold of the LbGlcK dimer is almost similar when compared to that of the TcGlcK-BENZ-GlcN complex (PDB entry 5BRD) [16] except with changes noted from the bending angle between large and small domains. When superimposed, this resulted in a median root-mean-square deviation (r.m.s.d.) of 4.10 Å for 733 Cα atoms or an r.m.s.d. of 1.673 Å for 522 Cα atoms (after 5 cycles of Calcium Gluceptate rejections). The differences in fold are attributed to the partially open conformation (TcGlcK) and the full open conformation (LbGlcK) between crystal structures. A superposition of the crystal structures reveals conserved features within the active site (Figure S3, SI). R.m.s.d. values were determined through the MacPyMOL software [19]. Active site residues of the TcGlcK-BENZ-GlcN complex (PDB entry 5BRD) [16] that are indeed responsible in making hydrogen bonding interactions with the glucose moiety of the competitive inhibitor were evaluated with respect to the conserved residues of LbGlcK through a superposition of their crystallographic coordinates (Figure S3, SI). TcGlcK was selected as the comparison model because it had the highest protein sequence identity to LbGlcK across all deposited PDB entries. A structure-based sequence alignment between the two enzymes revealed which active site residues were conserved and indicated a 44% sequence identity (Figure S4, SI). Since LbGlcK is in the apo form (ligandless) in the structure presented in this work, we assumed that its following residues make hydrogen bonding interactions with d-glucose during catalysis: N142, D143, E262, and E220 (note: residues N130, D131, E236, and E207 of TcGlcK correspond respectively as the conserved residues). Furthermore, all residues of the active site and the outer part of the active site were presented in Figure S3, SI. Similar to the TcGlcK active site structural environment, LbGlcK also includes a hydrophobic pocket at the outer part region. Hydrophobic residues F368 and M364 of LbGlcK are in the same superimposed positions as TcGlcK residues F337 and M334, respectively. Interestingly, as some of the glucosamine analogue inhibitors from our previous work were observed to make π-stacking and van der Waals interactions in the hydrophobic pocket, which afforded enhanced competitive inhibitor binding strength [16], we predict that residue F368 in LbGlcK will participate in the same kind of role as F337 of TcGlcK. Structural and enzyme – inhibitor kinetics comparisons were made between LbGlcK, TcGlcK, and HsHxKIV to assess whether glucosamine analogues, that have a linker extended from the C2 position of the glucose moiety (refer to the SI section for example chemical structures of such compounds), could indeed bind in the active site cavity of LbGlcK. Pertaining to structural assessments, they included the analysis of residues within the active site that are subject to cause deterrence for the binding of a glucosamine analogue and the analysis of two protein loops by the outer part of the active site for the possibility of deterrence of inhibitor access into the active site.