The Tp0684 (MglB-2) Lipoprotein of Treponema pallidum: A Glucose-Binding Protein with Divergent Topology

Abstract

Treponema pallidum, the bacterium that causes syphilis, is an obligate human parasite. As such, it must acquire energy, in the form of carbon sources, from the host. There is ample evidence that the principal source of energy for this spirochete is D-glucose acquired from its environment, likely via an ABC transporter. Further, there is genetic evidence of a D-glucose chemotaxis system in T. pallidum. Both of these processes may be dependent on a single lipidated chemoreceptor: Tp0684, also called TpMglB-2 for its sequence homology to MglB of Escherichia coli. To broaden our understanding of this potentially vital protein, we determined a 2.05-Å X-ray crystal structure of a soluble form of the recombinant protein. Like its namesake, TpMglB-2 adopts a bilobed fold that is similar to that of the ligand-binding proteins (LBPs) of other ABC transporters. However, the protein has an unusual, circularly permuted topology. This feature prompted a series of biophysical studies that examined whether the protein’s topological distinctiveness affected its putative chemoreceptor functions. Differential scanning fluorimetry and isothermal titration calorimetry were used to confirm that the protein bound D-glucose in a cleft between its two lobes. Additionally, analytical ultracentrifugation was employed to reveal that D-glucose binding is accompanied by a significant conformational change. TpMglB-2 thus appears to be fully functional in vitro, and given the probable central importance of the protein to T. pallidum’s physiology, our results have implications for the viability and pathogenicity of this obligate human pathogen.

Crystal stuctures of MglB‐2 (TP0684), a topologically variant d‐glucose‐binding protein from Treponema pallidum, reveal a ligand‐induced conformational change

“…The monosaccharide was essentially solvent‐inaccessible: the calculated solvent accessibilities of the two copies of d ‐glucose in the asymmetric unit were 0.5 and 0.1 Å 2 . As predicted, numerous hydrogen bonds were made between cleft‐lining side chains and hydroxyl groups on the sugar. Also, the phenyl ring of F313 stacked on one face of the d ‐glucose, as observed in other glucose‐specific LBP structures .…”

“…In our previous work, we used isothermal titration calorimetry (ITC) to characterize the interaction of d ‐glucose and other hexoses with a wild‐type, recombinant form of TpMglB‐2 (TpMglB‐2 R ). We found that the protein bound glucose with a K D of 1.1 μ M .…”

“…Hereafter, we refer to this as the “apo” form of the protein. Apo‐TpMglB‐2 WA crystallized in the same space group as apo‐TpMglB‐2 R , with nearly identical unit‐cell dimensions . The apo‐TpMglB‐2 WA crystal structure [Fig.…”

“…However, in TpMglB‐2, the water that makes contact to the anomeric OH group is rigidly held in place by hydrogen bonds to a carboxylate oxygen atom of D91 and the main‐chain oxygen atom of N19; this position favors binding of the β anomer, as the α‐anomeric OH group would have a poor interaction with the water (specifically, a hydrogen‐bonding angle of about 79°). The preferences of both TpMglB‐2 R and EcMglB for d ‐glucose over the epimeric sugar d ‐galactose have been noted; whether the mechanisms of these preferences are the same is unknown, as a structure of d ‐galactose bound to TpMglB‐2 is not available, and the discrimination appears to be subtly distributed around the monosaccharide‐binding site in EcMglB . The observed differences in the binding sites could account for the apparently lower affinity of wild‐type TpMglB‐2 R for d ‐glucose compared to EcMglB (1.1 vs. 0.2 μ M , respectively).…”

“…Earlier, we determined the crystal structure of apo‐TpMglB‐2, a protein predicted to bind to d ‐glucose and d ‐galactose. The ligandless protein was in an open conformation, as would be expected.…”