Wag31, or DivIVA, is an essential protein and a drug target in the 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 enzymes at the pole for exclusive polar growth. The crystal structure of the N-terminal lipid-binding domain of mycobacterial Wag31 was determined at 2.3 Å resolution. The structure revealed a highly polar surface lined with several conserved charged residues that suggest probable sites for interactions with membrane lipids. Crystal-packing analysis revealed a previously unseen `dimer-of-dimers' assembly state of N-terminal Wag31, which is formed by antiparallel stacking of two coiled-coil dimers. Size-exclusion column-chromatography-coupled small-angle solution X-ray scattering data revealed a tetrameric form as a major assembly state of N-terminal Wag31 in solution, further supporting the crystal structure. The results suggest that, in addition to lipid binding, the N-terminal Wag31 can participate in self-assembly to form filamentous structures. Plausible models of linear self-assembly and branching of Wag31 filaments consistent with available data are suggested.

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How proteins localize at the pole remains an enigma. DivIVA/Wag31, which is an essential pole organizing protein in mycobacteria, can assemble at the negatively curved side of the membrane at the growing pole to form a higher order structural scaffold for maintaining cellular morphology and localizing various target proteins for cell-wall biogenesis. A single-site phosphorylation in Wag31 is linked to the regulation of peptidoglycan biosynthesis for optimal mycobacterial growth. The structural organization of polar scaffold formed by coiled-coil rich Wag31, which is a target for anti-tubercular agent amino-pyrimidine sulfonamide, remains to be determined. Here, we report biophysical characterizations of a phospho-mimetic (T73E) and a phosphoablative (T73A) form of mycobacterial Wag31 using circular dichroism, small angle solution X-ray scattering and atomic force microscopy. While data obtained from both variants of Wag31 in solution states suggested formation of alpha-helical, large, elongated particles, their structural organizations were different. Atomic force microscopic images of Wag31 indicate polymer formation, with occasional curving, sharp bending and branching. Observed structural features in this first view of the polymeric forms of Wag31 suggest a basis for higher order network scaffold formation for polar protein localization.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

K-edge anomalous SAXS intensity was measured from a small, dimeric, partly unstructured protein segment of myosin X by using cupric ions bound to its C-terminal polyhistidine tags. Energy-dependent anomalous SAXS can provide key location-specific information about metal-labeled protein structures in solution that cannot be obtained from routine SAXS analysis. However, anomalous SAXS is seldom used for protein research due to practical difficulties, such as a lack of generic multivalent metal-binding tags and the challenges of measuring weak anomalous signal at the metal absorption edge. This pilot feasibility study suggests that weak K-edge anomalous SAXS signal can be obtained from transition metals bound to terminally located histidine tags of small proteins. The measured anomalous signal can provide information about the distribution of all metal–protein distances in the complex. Such an anomalous SAXS signal can assist in the modeling and validation of structured or unstructured proteins in solution and may potentially become a new addition to the repertoire of techniques in integrative structural biology.

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.