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. 2014 Jan 23;426(2):436-46.
doi: 10.1016/j.jmb.2013.10.014. Epub 2013 Oct 17.

Binding of MgtR, a Salmonella Transmembrane Regulatory Peptide, to MgtC, a Mycobacterium Tuberculosis Virulence Factor: A Structural Study

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Free PMC article

Binding of MgtR, a Salmonella Transmembrane Regulatory Peptide, to MgtC, a Mycobacterium Tuberculosis Virulence Factor: A Structural Study

Frantz L Jean-Francois et al. J Mol Biol. .
Free PMC article

Abstract

MgtR, a highly hydrophobic peptide expressed in Salmonella enterica serovar Typhimurium, inhibits growth in macrophages through binding to the membrane protein MgtC that has been identified as essential for replication in macrophages. While the Mycobacterium tuberculosis MgtC is highly homologous to its S. Typhi analogue, there does not appear to be an Mtb homologue for MgtR, raising significant pharmacological interest in this system. Here, solid-state NMR and EPR spectroscopy in lipid bilayer preparations were used to demonstrate the formation of a heterodimer between S. Typhi MgtR and the transmembrane helix 4 of Mtb MgtC. Based on the experimental restraints, a structural model of this heterodimer was developed using computational techniques. The result is that MgtR appears to be ideally situated in the membrane to influence the functionality of MgtC.

Keywords: MD; PISEMA; distance restraints; electron spin resonance; molecular dynamics; orientational restraints; polarization inversion spin exchange at the magic angle; restrained molecular dynamics; solid-state nuclear magnetic resonance.

Figures

Figure 1
Figure 1
a) PISEMA spectra of MgtR selectively 15N labeled constructs in aligned DMPC bilayers. Labeled residues are: construct 1, Ala10, Leu15, Ile16, and Ala24 (black); construct 2, Ala10, Leu11, Ile12, and Phe13 (blue); construct 3, Ile8, Leu23, Ala24, and Leu25 (red). The simulated 32° PISA wheel (green) is superimposed on the spectra using torsion angles of ϕ = −57° and ψ = −47°. b) Experimental ρ values taken from the PISA wheel compared to theoretical values assuming an ideal helix. The linear correlation demonstrates that a uniform helix without kinks or bends extends from residue 8 to residue 24. c) Calculated structure of MgtR in a DMPC lipid bilayer, with a 32° tilt angle. A helix cross section (yellow) is shown to identify the top, bottom and side of the helix. Backbone nitrogens of the Ala/Ser motif (Ala10, Ser17, and Ala24) are shown as red spheres. Sidechains shown in sticks include: Ala/Ser motif (red); Ile9, Ile16, and Leu23 (black); Trp26 (green); and Lys7 (blue). DMPC phosphorus atoms are shown as light blue spheres.
Figure 2
Figure 2
PISEMA spectra of MgtR peptide in aligned DMPC bilayers with (green, red) and without (black) MgtC TM4 unlabeled peptide. a) Construct with 15N labeling at Ala10, Leu15, Ile16, and Ala24. b) Construct with 15N labeling at Ala10, Leu11, Ile12, and Phe13. Dotted circles are drawn to enclose resonances attributed to the same residue. Spectra were acquired at 310 K with 3 K scans per t1 increment.
Figure 3
Figure 3
One-dimensional anisotropic 15N chemical shift spectra of MgtC TM4 in aligned DMPC bilayers with (red) and without (black) unlabeled MgtR. Spectra were acquired at 310 K with 3 K scans per t1 increment. Black vertical lines reflect an estimate for the limits of the chemical shift dispersion for the four resonance frequencies. Simulated PISA wheels predict a dispersion of chemical shifts for ideal helices having a tilt of 17° (orange), 19° (magenta) and 21° (green). The black dots on the 19° PISA wheel illustrate the distribution of resonances for Ala93, Ala94, Val102, and Ala107 assuming rotational angles of 0°, 100°, 180° and 320° in an ideal helix. Similar dispersion of these resonances on the PISA wheel is obtained if a different helical rotation is assumed.
Figure 4
Figure 4
EPR spectra of MgtR and MgtC TM4 reconstituted in DMPC liposomes. a) MgtR; b) MgtC TM4; and c) MgtR/MgtC TM4 mixture. Red curves display the spectra of fully labeled peptides. Black curves display controls using 30% labeled peptides in a) and b) and the sum of singly labeled peptides in c). Broadening of the red spectra indicates spin-spin coupling within 25 Å.
Figure 5
Figure 5
Structural models of the MgtR/MgtC TM4 dimer generated by RosettaDock. a) Scatter plot of interface scores versus root-mean-square-deviations from a seed model with low interface score. Two low-score clusters, with 300 and 13 poses, respectively, are highlighted in cyan and orange boxes. b) Conformations of the two clusters. The green helix is MgtR, and the cyan and orange helices are MgtC TM4 in the major and minor clusters, respectively. The Cα atoms of the Ala/Ser motifs of MgtR and MgtC TM4 are shown as spheres. Trp97 of MgtC TM4 is shown as sticks in order to assist in locating the position and orientation of this peptide. Models of c) the major cluster and d) the minor cluster. Ala/Ser motif residues are shown as spheres; other residues are shown as sticks.
Figure 6
Figure 6
Structural model of the MgtR/MgtC TM4 dimer refined by MD simulation in the DMPC bilayer. MgtR and MgtC TM4 are represented in green and cyan, respectively. a) Positioning of the dimer in the DMPC bilayer. Each peptide is shown as a helix, with three residues shown as van der Waals spheres. DMPC phosphorus atoms are shown as light blue spheres. b) Representation of the dimer by van der Waals surface, illustrating the snug fit in the interface.

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