PNNL

Abstract

Due to the evolution of multi-drug and extremely drug-resistant M. tuberculosis strains there is an urgent need to develop new anti-tuberculosis strategies to prevent TB epidemics in the industrial world. Among the potential new drug targets are two small non-heme iron-binding proteins, rubredoxin A (Rv3251c) and B (Rv3250c), believed to play a role in electron transfer processes. Here we report the solution structure and biophysical properties for one of these two proteins, rubredoxin B (Mt-RubB), determined in the zinc-substituted form. The zinc-substituted protein was prepared by expressing Mt-RubB in minimal media containing excess zinc acetate. Size exclusion chromatography and NMR spectroscopy indicated Mt-RubB was a monomer in solution. The structure (PDB ID 2KN9) was generally similar to other rubredoxins, containing one three-stand anti-parallel ß-sheet (ß2:ß1:ß3) and a metal tetrahedrally coordinated to the sulfur atoms of four cysteine residues (C9, C12, C42, and C45). The first cysteine is at the C-terminal end of the first ß-strand and the second pair of cysteines are towards the C-terminal end of the loop between ß2 and and ß3. The structure shows the metal buried deeply within the protein, an observation supported by the inability to remove the metal with excess EDTA at room temperature. Circular dichroism spectroscopy shows that this stability extends to high temperature, with essentially no change observed in the CD spectrum of Mt-RubB upon heating to ~80 ºC.