Abstract
CmtR from Mycobacterium tuberculosis is a winged helical DNA-binding repressor of the ArsR-SmtB metal-sensing family that senses cadmium and lead. Cadmium-CmtR is a dimer with the metal bound to Cys-102 from the C-terminal region of one subunit and two Cys associated with helix alphaR from the other subunit, forming a symmetrical pair of cadmium-binding sites. This is a significant novelty in the ArsR-SmtB family. The structure of the dimer could be solved at 312 K. The apoprotein at the same temperature is still a dimer, but it experiences a large conformational exchange at the dimer interface and within each monomer. This is monitored by an overall decrease of the number of nuclear Overhauser effects and by an increase of H(2)O-D(2)O exchange rates, especially at the dimeric interface, in the apo form with respect to the cadmium-bound state. The C-terminal tail region is completely unstructured in both apo and cadmium forms but becomes less mobile in the cadmium-bound protein due to the recruitment of Cys-102 as a metal-ligand. DNA binds to the apo dimer with a ratio 1:3 at millimolar concentration. Addition of cadmium to the apo-CmtR-DNA complex causes DNA detachment, restoring the NMR spectrum of free cadmium-CmtR. Cadmium binding across the dimer interface impairs DNA association by excluding the apo-conformers suited to bind DNA.
Highlights
Life depends on multiple metals [1]
IC-mass spectrometry shows that cadmium-CmtR has a mass of 25,211 under native conditions with volatile buffers, matching a dimer plus two cadmium atoms
Binding of cadmium reduces the conformational heterogeneity of regions within the CmtR subunits and locks the two subunits into a better defined reciprocal orientation (Fig. 4)
Summary
Life depends on multiple metals [1]. Iron-, copper-, and zincresponsive transcriptional regulators are known in yeast (AFT1, MAC1, ACE1, and ZAP1) [2] and higher eukaryotes (MTF1) [3, 4]. Multiple families of metal-responsive transcriptional regulators have been described, including ArsR/ SmtB-like DNA-binding repressors [5,6,7,8]. These are a subgroup of winged helix repressors. Mycobacterium tuberculosis contains ten genes encoding ArsR/SmtB sensors making it a useful model organism for studies of these regulators. We report a solution NMR investigation of both the demetallated and metallated proteins We show that the former has a tertiary and quaternary structure that is fluxional, whereas the latter has a more rigid conformation with implications for the metal-sensing mechanism. We visualize cadmium inhibition of CmtR DNA binding by NMR
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
