Table 1.
Data collection and refinement statistics for Mycobacterium tuberculosis H37Rv FecB (Rv3044) and FecB2 (Rv0265c).
Fig 1.
Overview of Mtb FecB and FecB2 structures.
A. FecB (i, green) and FecB2 (ii, orange) structures are shown in cartoon form. Both are multi-domain proteins with an N-terminal lobe and a C-terminal lobe connected by a rigid ‘backbone’ helix. These features are labelled, as are the N- and C-termini. Notably, FecB has an extra N-terminal sequence that associates with the C-terminal lobe but has an unstructured connection between the N- and C-terminal lobes (dashed black line). B. FecB and FecB2 structures are superimposed to highlight the differences in secondary structure. Secondary structure elements that overlay well are colored in white (FecB) and grey (FecB2), while divergent secondary structure is colored as in (A) and labelled.
Fig 2.
Comparison of the ligand-binding sites for FecB and FecB2.
Putative ligand-binding site of FecB (left panels) and FecB2 (right panels) show that the FecB2 ligand binding pocket is in a more open form compared to that of FecB. A. FecB and FecB2 are represented in cartoon form and colored as in Fig 1A with potential ligand-binding residues shown as white sticks. B. The electrostatic molecular surface for FecB and FecB2 was generated by APBS (Adaptive Poisson-Boltzmann Solver), where white, red and blue represent hydrophobic, negative and positively charged surfaces, respectively.
Fig 3.
Structural comparison of Mtb FecB and FecB2 with highly similar structural homologs of siderophore-bound PBPs.
Ligand-binding sites for A. Mtb FecB (green, PDB ID 7UQ0), B. Mtb FecB2 (orange, PDB ID 4PM4), C. S. aureus HtsA complexed with ferric-staphyloferrin A (pink, PDB ID 3LI2), D. S. aureus SirA complexed with ferric-staphyloferrin B (white, PDB ID 3MWF), and E. B. subtilis YfiY complexed with ferric-schizokinen (wheat, PDB ID 3TNY) are shown. HtsA, SirA and YfiY are in complex with siderophores (stick representation in yellow with iron as an orange sphere), and coordinating residues are shown as stick representations. Polar interactions are highlighted between the siderophore and coordinating residues (dashed black lines). For FecB and FecB2, conserved or similar residues to HtsA, SirA and YfiY PBPs are shown. Notably, HtsA, SirA and YfiY all have a conserved arginine residue (R126, R125 and R91, respectively) that interacts with the bound siderophore, this arginine is notably absent in FecB and FecB2 with Leu163 and Ala106, respectively, in a similar location.
Table 2.
FecB and FecB2 Kd’s for heme or Fe-cMB.
Fig 4.
Both FecB and FecB2 bind heme, while FecB preferentially binds Fe-cMB compared to FecB2.
Representative fluorescent emission intensities at 335 nm after excitation at 280 nm of 100 nM FecB (left panels) and 100 nM FecB2 (right panels) with increasing concentrations of A. heme and B. Fe-cMB. Notably the experiments in (A) were performed with tagless proteins. Curves were fit using Eq 1 and affinities (Kd) are included for each titration. In (A) FecB binds heme with a higher affinity than FecB2, in contrast to (B) showing that FecB binds Fe-cMB whereas FecB2 does not.
Fig 5.
The affinity of Fe-cMB is attenuated for the FecB-E339S variant compared to WT-FecB.
A. CD shows that both WT-FecB (dashed black line) and FecB-E339S (solid blue line) have the same secondary structural elements. B & C. Comparison of representative fluorescent emission intensities at 335 nm after excitation at 280 nm with increasing concentrations of (B) Fe-cMB and (C) apo-cMB binding to WT-FecB (100 nM, black line) and FecB-E339S (100 nM, blue line). Curves were fit using Eq 1 and cMB affinities (Kd) are included for each titration for WT FecB.
Table 3.
Top periplasmic and membrane protein hits of interest from Msm co-IPs with (A) FecB-FLAG, (B) MmpS4-FLAG, (C) MmpS5-FLAG and (D) FecB2-FLAG.
Fig 6.
Western Blot analysis of Mtb co-IPs suggest that FecB interacts with MmpS5.
Western Blot analysis of protein co-IPs with MtbΔfecB with vectors expressing FecB-FLAG and MmpS5-HA, and the negative control whereby Mtb contains vectors that express the tags alone. Western Blot analysis of both the boiled beads and eluate of the (A) co-IP using the anti-FLAG beads to pull down FecB-FLAG, probed with an anti-HA antibody that recognizes MmpS5-HA, (B) co-IP using the anti-HA beads to pull down MmpS5-HA, probed with an anti-FLAG antibody that recognizes FecB-FLAG, and (C) co-IP using the anti-HA beads to pull down MmpS5-HA, probed with an anti-FecB antiserum that recognizes FecB.