Higher order assembling of the mycobacterial polar growth factor DivIVA/Wag31

Choukate, Komal; Gupta, Aanchal; Basu, Brohmomoy; Virk, Karman; Ganguli, Munia; Chaudhuri, Barnali N.

doi:10.1016/j.jsb.2019.107429

Cited by 5 publications

(6 citation statements)

References 58 publications

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“…A combination of the two such N-terminal and C-terminal dimer-of-dimers can be used to build a linear filament (figure 3). Such linear filaments, with sporadic branching, were reported for full-length tbWag31 earlier (Choukate et al, 2019). Furthermore, the two-fold symmetry related interfaces in the N-Wag31 suggest a natural way for lateral or sideway association of dimeric N-Wag31 units to form a hexamer and other higher order oligomers (figure 3).…”

Section: A Suggested Model Of Wag31 Filament Formationsupporting

confidence: 51%

“…The tetrameric form of N-Wag31 naturally explained linear, and branched, assembly formation of tbWag31 ( figure 3). Full-length tbWag31 forms linear filament, which is at times branched (Choukate et al, 2019). Filaments, networks and doublering structures formed by bsDivIVa were reported earlier (Stahlberg et al, 2004;Eswaramoorthy et al, 2011).…”

Section: Discussionmentioning

confidence: 64%

“…Crystal structure of N-Wag31 that comprises of the first 60 residues of tbWag31 raises question about the possible structural role of phosphorylated T73 residue in fulllength tbWag31. The T73 residue is in a putative intrinsically unstructured region adjacent to the N-terminal domain (Choukate et al, 2019), and proximal to several glycine residues, such as G 63 G 64 G 65 X 66 G 67 (figure 2). Small and flexibility-imparting glycine residues are typically rare in coiled coils (Surkont and Pereira-Lal, 2015).…”

Section: Discussionmentioning

confidence: 99%

“…Recently, we showed that the full-length mycobacterial Wag31 (tbWag31, P9WMU1, Rv2145c) form several microns long primarily linear polymers in vitro, with occasional bending or branching (Choukate et al, 2019). Here, we report a crystal structure of the N-terminal membrane binding domain of tbWag31 (N-Wag31) at 2.3 Å resolution.…”

Section: Introductionmentioning

confidence: 92%

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Crystal structure of the membrane anchoring domain of mycobacterial Wag31: a dimer-of-dimer suggests how a Wag31 filament might self-assemble

Choukate

Chaudhuri

2019

Preprint

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Wag31, or DivIVA, is an essential protein and a drug target in human pathogen Mycobacterium tuberculosis that self-assembles at the negatively curved membrane surface to form a higher-order structural scaffold, maintains rod-shaped cellular morphology, and localizes key cell-wall synthesizing proteins at the pole for exclusive polar growth. We determined the crystal structure of N-terminal membrane anchoring domain of mycobacterial Wag31 at 2.3 Å resolution using molecular replacement method. Crystal packing analysis revealed a previously unseen dimer-of-dimer assembly state of N-terminal Wag31 with C 2 point group symmetry, which is formed by antiparallel stacking of two coiled coil dimers. Size-exclusion column chromatography-coupled small angle solution X-ray scattering data showed a tetrameric form as a major assembly state of N-terminal Wag31 in solution, further supporting the crystal structure. Plausible models of linear self-assembling, and branching, of Wag31 filaments consistent with available data are suggested.

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Section: A Suggested Model Of Wag31 Filament Formationsupporting

confidence: 51%

Section: Discussionmentioning

confidence: 64%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 92%

See 2 more Smart Citations

Crystal structure of the membrane anchoring domain of mycobacterial Wag31: a dimer-of-dimer suggests how a Wag31 filament might self-assemble

Choukate

Chaudhuri

2019

Preprint

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“…Rv2145c (Wag31) is a homolog of DivIVA (a cell division and cell shape protein), which regulates cell morphology in gram-positive bacteria ( 32 ) and contains two coiled-coil domains on the N-terminal side (32-59 aa) and C-terminal side (166-193 aa), a DivIVA functional site (3-63 aa), and an N-terminal-membrane binding domain. Therefore, we produced two truncated Rv2145c proteins: the N-terminal part (1-90 aa, domain 1; D1) and the C-terminal part (91-260 aa, domain 2; D2) ( Figure S6A ).…”

Section: Resultsmentioning

confidence: 99%

Mycobacterium tuberculosis Rv2145c Promotes Intracellular Survival by STAT3 and IL-10 Receptor Signaling

et al. 2021

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Although Mycobacterium tuberculosis (Mtb) is an intracellular pathogen in phagocytic cells, the factors and mechanisms by which they invade and persist in host cells are still not well understood. Characterization of the bacterial proteins modulating macrophage function is essential for understanding tuberculosis pathogenesis and bacterial virulence. Here we investigated the pathogenic role of the Rv2145c protein in stimulating IL-10 production. We first found that recombinant Rv2145c stimulated bone marrow-derived macrophages (BMDMs) to secrete IL-10, IL-6 and TNF-α but not IL-12p70 and to increase the expression of surface molecules through the MAPK, NF-κB, and TLR4 pathways and enhanced STAT3 activation and the expression of IL-10 receptor in Mtb-infected BMDMs. Rv2145c significantly enhanced intracellular Mtb growth in BMDMs compared with that in untreated cells, which was abrogated by STAT3 inhibition and IL-10 receptor (IL-10R) blockade. Expression of Rv2145c in Mycobacterium smegmatis (M. smegmatis) led to STAT3-dependent IL-10 production and enhancement of intracellular growth in BMDMs. Furthermore, the clearance of Rv2145c-expressing M. smegmatis in the lungs and spleens of mice was delayed, and these effects were abrogated by administration of anti-IL-10R antibodies. Finally, all mice infected with Rv2145c-expressing M. smegmatis died, but those infected with the vector control strain did not. Our data suggest that Rv2145c plays a role in creating a favorable environment for bacterial survival by modulating host signals.

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Polar growth protein Wag31 undergoes changes in homo-oligomeric network topology, and has distinct functions at both cell poles and the septum

Arejan

Patel

Quintanilla

et al. 2022

Preprint

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Mycobacterial cell elongation occurs at the cell poles; however, it is not clear how cell wall insertion is restricted to the pole and organized. Wag31 is a pole-localized cytoplasmic protein that is essential for polar growth, but its molecular function has not been described. Wag31 homo-oligomerizes in a network at the poles, but it is not known how the structure of this network affects Wag31 function. In this study we used a protein fragment complementation assay to identify Wag31 residues involved in homo-oligomeric interactions, and found that amino acids all along the length of the protein mediate these interactions. We then used both N-terminal and C-terminal splitGFP fusions to probe Wag31 network topology at different sites in the cell, and found that Wag31 C-terminal-C-terminal interactions predominate at the septa, while C-terminal-C-terminal and C-terminal-N-terminal interactions are found equally at the poles. This suggests the Wag31 network is formed through an ordered series of associations. We then dissected Wag31’s functional roles by phenotyping a series of wag31 alanine mutants; these data show that Wag31 has separate functions in not only new and old pole elongation, but also inhibition of both septation and new pole elongation. This work establishes new functions for Wag31, and indicates that changes in Wag31 homo-oligomeric network topology may contribute to cell wall regulation in mycobacteria.

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Higher order assembling of the mycobacterial polar growth factor DivIVA/Wag31

Cited by 5 publications

References 58 publications

Crystal structure of the membrane anchoring domain of mycobacterial Wag31: a dimer-of-dimer suggests how a Wag31 filament might self-assemble

Crystal structure of the membrane anchoring domain of mycobacterial Wag31: a dimer-of-dimer suggests how a Wag31 filament might self-assemble

Mycobacterium tuberculosis Rv2145c Promotes Intracellular Survival by STAT3 and IL-10 Receptor Signaling

Polar growth protein Wag31 undergoes changes in homo-oligomeric network topology, and has distinct functions at both cell poles and the septum

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