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. 2016 Aug 18;11(8):e0161022.
doi: 10.1371/journal.pone.0161022. eCollection 2016.

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

Chad A Brautigam  1 , Ranjit K Deka  2 , Wei Z Liu  2 , Michael V Norgard  2
Affiliations

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

Chad A Brautigam et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Structural aspects of TpMglB-2.
(A) The overall structure. Shown in ribbons representation is the overall crystal structure of TpMglB-2. Helices (either α- or 310) are shown in green, β-strands are purple arrows, and regions without regular secondary structure are light blue. The three canonical hinge regions are colored orange. The adventitiously bound ligand BIS-TRIS is shown as a group of spheres between the protein’s two lobes. Oxygen atoms are colored red, nitrogen atoms blue, and carbon atoms are gray. The Ca2+ ion bound to the C Lobe is shown as a pink sphere. The span of the protein that forms the fourth hinge region and also comprises the connecting motif not observed in EcMglB (see text) is colored gold. (B) The Ca2+-binding site. Residues 71–79 are shown, with carbon atoms in light blue, and other atoms colored as described in (A). Water molecules are shown as red spheres. Inner-sphere contacts to the Ca2+ ion are depicted as black dashes. (C) The BIS-TRIS binding site. A kicked omit mFo-DFc map [43,44] is shown contoured at the 3-σ level and superposed on the refined coordinates of BIS-TRIS. Hydrogen bonds between the protein and the buffer molecule are drawn as black dashes. H89 is shown because it is in van der Waals contact with the molecule. Its ε N atom is about 3.3 Å from the proximal hydroxyl group on the BIS-TRIS, but this distance coupled with the poor geometry make it unlikely that these two atoms are hydrogen-bonded.
Fig 2
Fig 2. Superposition of TpMglB-2 and EcMglB.
For both structures, a smoothed trace through the main chain is drawn. TpMglB-2 is shown in blue, while EcMglB is red. The bound ligands are depicted as spheres and are colored according to their respective protein chains, and the respective N- and C-termini are noted, with the letters color-coded to the respective structures. The gold coloration for the fourth hinge/connection motif from Fig 1 is retained here.
Fig 3
Fig 3. Comparison of the topologies of the central β-sheets in the N-lobes of TpMglB-2 and EcMglB.
The β-strands for the respective sheets are drawn as arrows, with those from TpMglB-2 shown in blue and those from EcMglB in red. The strands are numbered according to the orders in which they appear in the primary structures of their respective proteins. The positions and sizes of the strands are approximately to scale. The antiparallel strand in TpMglB-2 was not assigned its own number in this scheme because it does not correspond to any strand in EcMglB. Its designation, “pre-3”, indicates that it immediately precedes strand 3 in the primary structure of TpMglB-2.
Fig 4
Fig 4. Relationships between proteins containing sequence and topology homology to TpMglB-2.
(A) A schematic representation of the full cladogram. There are two main clades, which are shown as colored rectangles. The pink “Trep./Spir.” rectangle is occupied by treponemal and spirochetal organisms, while the “Gram Positive/Gut” blue rectangle is inhabited mainly by gram-positive organisms, many of which are mammalian gut commensal organisms. The red rectangle details the area of the figure that is blown up in (B). The full cladogram is available as a text file in the data supplied in S1 File. (B) A close-up of a branch point in the cladogram. The area of the cladogram highlighted in red in (A) is shown. TpMglB-2 is represented twice here (“Tp0684_ref” and “T. pallidum”) because it was included both as a structural template (in.pdb form) and as its native sequence. There is a slight difference between the two because of the 2 missing amino acids in the.pdb file.
Fig 5
Fig 5. DSF data for a panel of carbon sources in the presence of TpMglB-2.
The change in the apparent melting temperature is plotted vs. compound index (ranging from 1 to 96; only results from BIOLOG plate PM1 are shown here). Bars for compounds that caused Tm,app shifts of more than 1.5° C from that observed in the presence of the negative control (water, no compound) are colored red and labeled with the compound’s identity.
Fig 6
Fig 6. D-glucose and D-galactose binding TpMglB-2.
Baseline-subtracted thermograms reconstructed from the raw data using singular-value decomposition are shown in the top panel. In the middle panel are the integrated heats of injection (markers) with error bars depicted estimated measurement errors [54,55]. The lines are fits to these data points. The bottom panel shows the residuals between the data and the fit lines. All lines and markers are colored respectively according to the inset legend. Only one of the three analyzed experiments is shown for each monosaccharide.
Fig 7
Fig 7. D-glucose binding to the W145A variant of TpMglB-2.
The organization of the figure is the same as in Fig 6, but the thermogram peaks and their respective integrated data points are colored according to the color map shown inset in the top panel.
Fig 8
Fig 8. Difference sedimentation velocity of Tp0684 in the presence and absence of sugars.
(A) Example difference SV traces. Three traces are shown after noise and baseline subtraction. The traces are colored according to the time of the scan, with red, blue, and black representing 92, 132, and 172 min after the start of centrifugation. The three traces are from the D-mannose/D-glucose experiment. (B) Three difference sedimentation velocity analysis plots. Data similar to those shown in (A) were treated as described in Materials and Methods; each trace resulted in one of the data points shown here, which are colored according to their respective experiments. Each experiment is labeled; “D-Man/D-Glc” stands for D-mannose vs. D-glucose, “D-Rib/D-Glc” is the D-ribose vs. D-glucose experiment, and “0/0” is the negative control in which no sugar was added to either sector. The gray lines show the linear fits to each data set. Eighty-six traces were analyzed per experiment. The D-Rib/D-Glc data are displaced upward because of the larger difference in the reference/sample sector menisci in this experiment (see Materials & Methods).
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