Crystal structures of the NO sensor NsrR reveal how its iron-sulfur cluster modulates DNA binding

Abstract

NsrR from Streptomyces coelicolor (Sc) regulates the expression of three genes through the progressive degradation of its [4Fe–4S] cluster on nitric oxide (NO) exposure. We report the 1.95 Å resolution crystal structure of dimeric holo-ScNsrR and show that the cluster is coordinated by the three invariant Cys residues from one monomer and, unexpectedly, Asp8 from the other. A cavity map suggests that NO displaces Asp8 as a cluster ligand and, while D8A and D8C variants remain NO sensitive, DNA binding is affected. A structural comparison of holo-ScNsrR with an apo-IscR-DNA complex shows that the [4Fe–4S] cluster stabilizes a turn between ScNsrR Cys93 and Cys99 properly oriented to interact with the DNA backbone. In addition, an apo ScNsrR structure suggests that Asn97 from this turn, along with Arg12, which forms a salt-bridge with Asp8, are instrumental in modulating the position of the DNA recognition helix region relative to its major groove.

Mass Spectrometric Identification of [4Fe–4S](NO)x Intermediates of Nitric Oxide Sensing by Regulatory Iron–Sulfur Cluster Proteins

“…The intensity of the peaks due to these dimeric forms was relatively low, possibly because the protein–protein interactions that mediate dimerization do not efficiently survive the ionization process. Higher intensity and well‐resolved peaks were observed in the monomer region, as previously reported (Figure A). This contained a major peak due to apo‐protein at 17 472 Da (predicted mass 17 474 Da, see Table ), along with small amounts of NsrR with sulfur adducts at +32 and +64 Da.…”

“…ESI‐MS under non‐denaturing conditions was used to study the reaction of [4Fe–4S] NsrR with NO. The m / z spectrum (Figure S2) comprised two regions corresponding to monomeric and dimeric forms of NsrR, as previously reported . The deconvoluted MS spectrum of [4Fe–4S] NsrR revealed broad peaks at 34 942, 35 294, and 35 646 Da corresponding to dimeric apo‐NsrR (see Table for a comparison between observed and predicted mass) and dimeric NsrR containing one (35 294 Da) and two clusters (35 646 Da), respectively, see Figure S3.…”

“…For both regulators, a mono‐nitrosyl complex of the [4Fe–4S] cluster is the first intermediate. For NsrR, the NO is most likely bound at the site‐differentiated iron coordinated by Asp8 . For the all‐Cys coordinated [4Fe–4S] cluster of WhiD, which iron binds the first NO is not clear.…”

“… Structures of S. coelicolor [4Fe–4S] NsrR and M. tuberculosis [4Fe–4S] WhiB1. (A) Cartoon diagram showing the X‐ray structure of one subunit of the NsrR homodimer . (B) Closer view of the [4Fe–4S] cluster showing how Asp8 from the second monomer coordinates the cluster of the first.…”

Metallochaperones and metalloregulation in bacteria

“…We direct the reader to a paper that appeared during the preparation of this manuscript that highlights structural work on a specific member of the Rrf2 superfamily not discussed here [211]. Rrf2 family repressors regulate iron-sulfur cluster biogenesis (IscR), cysteine metabolism (CymR), iron homeostasis (RirA) and the sensing of reactive nitrogen species via a [4Fe-4S) cluster (NsrR).…”