ABSTRACT
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.
Subject(s)
Proteins , Proteins/chemistry , Scattering, Small Angle , X-Ray DiffractionABSTRACT
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 2.3â 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.
ABSTRACT
DivIVA or Wag31, which is an essential pole organizing protein in mycobacteria, can self-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. The structural organization of polar scaffold formed by polymerization of coiled-coil rich Wag31, which is implicated in the anti-tubercular activities of amino-pyrimidine sulfonamides, remains to be determined. A single-site phosphorylation in Wag31 regulates peptidoglycan biosynthesis in mycobacteria. We report biophysical characterizations of filaments formed by mycobacterial Wag31 using circular dichroism, atomic force microscopy and small angle solution X-ray scattering. Atomic force microscopic images of the wild-type, a phospho-mimetic (T73E) and a phospho-ablative (T73A) form of Wag31 show mostly linear filament formation with occasional curving, kinking and apparent branching. Solution X-ray scattering data indicates that the phospho-mimetic forms of the Wag31 polymers are on average more compact than their phospho-ablative counterparts, which is likely due to the extent of bending/branching. Observed structural features in this first view of Wag31 filaments suggest a basis for higher order Wag31 scaffold formation at the pole.
