Novel structural features drive DNA binding properties of Cmr, a CRP family protein in TB complex mycobacteria

Abstract

Mycobacterium tuberculosis (Mtb) encodes two CRP/FNR family transcription factors (TF) that contribute to virulence, Cmr (Rv1675c) and CRPMt (Rv3676). Prior studies identified distinct chromosomal binding profiles for each TF despite their recognizing overlapping DNA motifs. The present study shows that Cmr binding specificity is determined by discriminator nucleotides at motif positions 4 and 13. X-ray crystallography and targeted mutational analyses identified an arginine-rich loop that expands Cmr’s DNA interactions beyond the classical helix-turn-helix contacts common to all CRP/FNR family members and facilitates binding to imperfect DNA sequences. Cmr binding to DNA results in a pronounced asymmetric bending of the DNA and its high level of cooperativity is consistent with DNA-facilitated dimerization. A unique N-terminal extension inserts between the DNA binding and dimerization domains, partially occluding the site where the canonical cAMP binding pocket is found. However, an unstructured region of this N-terminus may help modulate Cmr activity in response to cellular signals. Cmr’s multiple levels of DNA interaction likely enhance its ability to integrate diverse gene regulatory signals, while its novel structural features establish Cmr as an atypical CRP/FNR family member.

Cyclic nucleotide signaling in Mycobacterium tuberculosis: an expanding repertoire

“…A crystal structure of Cmr also reveals several novel structural features not found in CRP or other CRP/FNR family proteins (Ranganathan et al 2017). One of these is an arginine loop (Arg loop ) consisting of Arg93-96 that likely makes additional contacts with DNA.…”

“…Additional DNA contact regions outside of the DNA-binding helices and a cooperative DNA-binding mechanism also differentiate Cmr's DNA-binding interactions from those of CRP MT . The nature of this cooperativity is such that Cmr is proposed to dimerize on the DNA rather than prior to DNA binding, which differs from other CRP/FNR family proteins (Ranganathan et al 2017).…”

“…Cmr and CRP MT recognize very similar DNA motifs, but their genomic DNA-binding profiles are distinct, with Cmr binding far fewer sites than CRP MT (Knapp et al 2015;Ranganathan et al 2016Ranganathan et al , 2017. A recent study found that nucleotides at positions 4 and 13 of the binding motif are discriminatory bases that distinguish between Cmr and CRP MT binding.…”

“…A recent study found that nucleotides at positions 4 and 13 of the binding motif are discriminatory bases that distinguish between Cmr and CRP MT binding. Differences in the aa's that contact DNA in the DNA-binding helices of each protein, with substitution of Glu189 in CRP MT for Pro209 in Cmr, likely drive this binding specificity (Ranganathan et al 2017). Additional DNA contact regions outside of the DNA-binding helices and a cooperative DNA-binding mechanism also differentiate Cmr's DNA-binding interactions from those of CRP MT .…”

C1188D mutation abolishes specific recognition between MLL1‐CXXC domain and CpG site by inducing conformational switch of flexible N‐terminal

“…The inhibition of recognition between MLL1‐CXXC domain and CpG site was then attributed to a decrease of electrostatic attraction due to C1188D mutation 8,9 . Recently, the importance of conformational changes of protein in the binding process was also highlighted by molecular dynamic simulations 27‐29 . Molecular dynamic simulations on binding between TRF1 and its target DNA revealed the binding process in atomic resolution for the first time and showed that conformational changes of protein have the ability to affect the outcome of protein‐DNA binding 30 …”