1H, 13C and 15N backbone and side chain resonance assignments of a Myxococcus xanthus anti-repressor with no known sequence homologues

Abstract

The CarS antirepressor activates a photo-inducible promoter in Myxococcus xanthus by physically interacting with the CarA repressor and eliminating the latter's binding to operator DNA. Interestingly, interactions with both CarS and operator are crucially dependent on the DNA recognition helix of the CarA winged-helix DNA-binding domain. The CarA-CarS and the CarA-operator interfaces therefore overlap, and CarS may have structural features that mimic operator DNA. CarS has no known sequence homologues and its Gly and Pro contents are unusually high. Here, we report (1)H, (13)C and (15)N backbone and side chain assignments of CarS1, an 86-residue truncated yet fully functional variant of CarS. Secondary structural elements inferred from these data differ from those predicted from sequence.

A bacterial antirepressor with SH3 domain topology mimics operator DNA in sequestering the repressor DNA recognition helix

“…Given its excellent spectral dispersion and overall quality, NMR studies were carried out with CarS1. 1 H, 15 N and 13 C resonances were assigned using standard triple resonance methods (20). The ensemble of the 20 lowest-energy structures calculated using experimental constraints (Supplementary Table S2) revealed a disordered nine-residue N-terminal segment, a relatively well-defined loop with two arms (residues 10–13 and 17–19) in extended conformations, a 3 10 helix (residues 37–39) and a five-stranded β-sheet (Figure 1A and Supplementary Figure S1).…”

“…CarS variants, generated as described above, were overexpressed using pET15b and purified as described for CarS. 15 N-labeled and 15 N, 13 C-labeled H 6 CarS1 were overexpressed and purified as reported previously (20). Synthetic N-acetylated, C-amidated peptides were purchased from Caslo Laboratory, Denmark.…”

NMR structure note: N-terminal domain of Thermus thermophilus CdnL

“…This peptide, which could be eliminated by gel filtration, has narrow NMR line-widths, chemical shifts very close to reference random coil peptide values, and negative or near-zero 15 N-{ 1 H}-NOEs (Supplementary Material (SM) Figure SF1 and Table ST1), suggesting that it is disordered and does not interact with TtCdnLNt. Based on the analyses of a series of 3D NMR spectra (CBCANH, CBCAcoNH, HBHANH, HBHAcoNH, HNCO, HNCA and HNHA; Sattler et al 1999) H a and/or 1 H bb' identified in the 3D spectra, as we have previously described for another P-rich protein (León et al 2009). Based on the chemical shift difference between 13 C b and 13 C c carbons (Schubert et al 2002), the X-P bonds are in the trans conformation (Dd Cb-Cc = 2.4-4.7 ppm) for all P except P17, where it is cis (Dd Cb-Cc = 9.4 ppm).…”