Cloning, purification, crystallization and preliminary X-ray analysis of ESX-1-secreted protein regulator (EspR) fromMycobacterium tuberculosis

Abstract

ESX-1-secreted protein regulator (EspR; Rv3849) is a key regulator in Mycobacterium tuberculosis that delivers bacterial proteins into the host cell during infection. EspR binds directly to the Rv3616c-Rv3614c promoter and activates transcription and secretes itself from the bacterial cell by the ESX-1 system. The three-dimensional structure of EspR will aid in understanding the mechanisms by which it binds to the Rv3616c-Rv3614c promoter and is involved in transcriptional activation. This study will significantly aid in the development of EspR-based therapeutics against M. tuberculosis. The full-length EspR gene from M. tuberculosis (H37Rv strain) was cloned and overexpressed as a soluble protein in Escherichia coli. The protein was purified by affinity chromatography using His-tagged protein followed by size-exclusion chromatography. EspR was crystallized using polyethylene glycol 3350 as precipitant. The crystals diffracted to 3.2 Å resolution using synchrotron radiation of wavelength 0.97625 Å . The crystal belonged to space group P3 1 21 and contained three monomers in the asymmetric unit. Native and heavy-atom-derivatized data sets were collected from EspR crystals for use in ab initio structure-solution techniques.

EspR, a key regulator of Mycobacterium tuberculosis virulence, adopts a unique dimeric structure among helix-turn-helix proteins

“…Although the solution was complicated by pseudotranslation and perfect merohedral twinning, we were able to refine to an R free of 23% using least-squares, twinned refinement (6). As was the case for another group with a related crystal form, we were unable to solve the structure with molecular replacement (7). Attempts to fit our data with models of SinR, the closest structural homolog discovered by tertiary structure prediction (3,8), and with models of other classic HTH proteins including lambda (9) and phage 434 repressors (10), failed to produce a solution, likely because these were monomeric domains that were only distantly related to EspR sequence (<25% identical).…”

Comparison of four different crystal forms of theMycobacterium tuberculosisESX-1 secreted protein regulator EspR

“…The resulting EspR plasmid was transformed in Escherichia coli BL21(DE3) cells and the protein was expressed in the soluble fraction. The EspR was purified using Ni-NTA and sizeexclusion chromatography as described in Gangwar et al (2011). The recombinant EspR protein contains 152 residues [132 residues from native EspR and 20 residues from the pET28a(+) vector containing a 6ÂHis tag and a thrombin cleavage site].…”

“…30 mg of seleno-dl-methionine (Sigma-Aldrich) was added to 1 l of the selenomethionine medium. The expression and purification of selenomethionine-substituted EspR protein were similar to those of native EspR described in Gangwar et al (2011).…”

“…Initial crystallization screenings were performed on native and selenomethionine-substituted EspR as described in Gangwar et al (2011). The selenomethionine-substituted EspR crystals were obtained in several conditions using the sitting-drop vapour-diffusion technique.…”

“…We recombinantly expressed EspR protein in E. coli and purified it using Ni-NTA affinity and size-exclusion chromatography as described in Gangwar et al (2011). The native EspR eluted as a dimer (molecular mass of $30 kDa) from a Superdex 75 (16/60) column (Fig.…”