Conformational studies of the manganese transport regulator (MntR) from Bacillus subtilis using deuterium exchange mass spectrometry.
The manganese transport regulator (MntR) of Bacillus subtilis is a metalloregulatory protein responsible for regulation of genes involved in manganese uptake by this organism. MntR belongs to the iron-responsive DtxR family, but is allosterically regulated by manganese and cadmium ions. Having previously characterized the metal binding affinities of this protein as well as the DNA-binding activation profiles for the relevant metal ions, we have focused the current study on investigating the structural changes of MntR in solution upon binding divalent transition metal ions. Deuterium exchange mass spectrometry was utilized to investigate the deuterium exchange dynamics between apo-MntR, Co(2+)-MntR, Cd(2+)-MntR, and Mn(2+)-MntR. Comparing the rates of deuteration of each metal-bound form of MntR reveals that the N-terminal DNA-binding motif is more mobile in solution than the C-terminal dimerization domain. Furthermore, significant protection from deuterium exchange is observed in the helices that contribute metal-chelating amino acids to form the metal binding site of MntR. In contrast, the bulk of the DNA-binding winged helix-turn-helix motif shows no difference in deuterium exchange upon metal binding. Mapping of the deuteration patterns onto the crystal structures of MntR yields insight into how metal binding affects the protein structure and complements earlier studies on the mechanism of MntR. Metal binding acts to rigidify MntR, thereby limiting the mobility of the protein and reducing the entropic cost of DNA binding.
Golynskiy
Misha
M
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0358, USA.
Li
Sheng
S
Woods
Virgil L
VL
Cohen
Seth M
SM
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry; D001412:Bacillus subtilis / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D002104:Cadmium / Q000737:chemistry; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003035:Cobalt / Q000737:chemistry; D003903:Deuterium; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry*
D001059:Apoproteins; D001426:Bacterial Proteins; C433792:MntR protein, bacteria; D012097:Repressor Proteins; D002104:Cadmium; D003035:Cobalt; D003903:Deuterium; D010447:Peptide Hydrolases