Abstract
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.
Highlights
The sensing of membrane curvature plays a critical role in diverse physiological processes such as the maintenance of cellular morphology, polar or hyphal growth in bacteria and endocytosis in eukaryotes (Cannon et al, 2017)
Mycobacterial Wag31 (P9WMU1, Rv2145c) is a 260-residue long filament-forming protein containing two domains: an Nterminal lipid- or membrane-binding domain and a C-terminal domain that participates in polar protein localization
The crystal structure of N-terminal lipidbinding domain of tbWag31 (N-Wag31) reported here reveals a tetrameric form of N-Wag31, which is further supported by solution SAXS data
Summary
The sensing of membrane curvature plays a critical role in diverse physiological processes such as the maintenance of cellular morphology, polar or hyphal growth in bacteria and endocytosis in eukaryotes (Cannon et al, 2017). Wag plays a critical role in regulating peptidoglycan biosynthesis and localizing many cell-wall synthesizing enzymes at the pole to support polar growth (Kang et al, 2008; Jani et al, 2010; Meniche et al, 2014; Xu et al, 2014). While depletion of Wag leads to a ‘rod to spherical cell’ transition (Nguyen et al, 2007; Kang et al, 2008; Meniche et al, 2014), Wag is shown to contribute to restoration of rod shape in spherical cells (Melzer et al, 2018)
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