Functional analysis of Rex, a sensor of the NADH/NAD+ redox poise in Streptomyces coelicolor

Maintenance of the intracellular NADH/NAD+ redox poise is vital for energy generation in cells. Gram-positive bacteria, including the antibiotic-producing organism, Streptomyces coelicolor, have evolved a regulatory protein Rex that both senses this ratio and mediates an adaptive response to changes in it. Rex is a dimeric redox-sensitive transcriptional repressor. It is capable of binding to both NAD+ and NADH, although only NADH is an effector, causing dissociation of the protein from operator (ROP) sites. As NADH levels rise during oxygen limitation Rex dissociates from its target genes allowing expression, which helps to restore the NADH/NAD+ ratio. Microarray-based expression studies had suggested that Rex regulated only a small number of genes. In this work, however, ChIP-on-chip analyses revealed 38 genes that are potential regulon members. Analysis of the Rex binding sites in S. coelicolor revealed new insights into the mode of binding and show that Rex can bind with low affinity to incomplete half sites. This work also focused on characterising two key Rex targets, ndh and nuoA-N, that encode non-proton-translocating and proton translocating NADH dehydrogenases, respectively. Whereas nuoAN is not essential and was not expressed in liquid media, ndh was essential for growth. Depletion of NDH from growing cells led to the induction of Rex target genes confirming that ndh and Rex play key roles in maintaining redox homeostasis. Structure-based dissection of Rex, via a close homologue in Thermus aquaticus, identified a key interaction between the NADH- and DNAbinding domains of Rex. An R29-D203’ salt-bridge, that traverses the NADH binding and DNA binding domains of Rex, appeared to stabilise the DNA-bound form of Rex, but is ‘broken’ in the presence of NADH. In the NADH-bound form of Rex, D203 alternatively interacts with Y111, which in turn interacts with the nicotinamide ring of NADH. In order to assess the importance of individual subunits in the dimeric Rex, a single-chain derivative was constructed and the NADH binding and DNA binding domains individually disrupted

Assessing cellular efficacy of bromodomain inhibitors using fluorescence recovery after photobleaching

BACKGROUND: Acetylation of lysine residues in histone tails plays an important role in the regulation of gene transcription. Bromdomains are the readers of acetylated histone marks, and, consequently, bromodomain-containing proteins have a variety of chromatin-related functions. Moreover, they are increasingly being recognised as important mediators of a wide range of diseases. The first potent and selective bromodomain inhibitors are beginning to be described, but the diverse or unknown functions of bromodomain-containing proteins present challenges to systematically demonstrating cellular efficacy and selectivity for these inhibitors. Here we assess the viability of fluorescence recovery after photobleaching (FRAP) assays as a target agnostic method for the direct visualisation of an on-target effect of bromodomain inhibitors in living cells. RESULTS: Mutation of a conserved asparagine crucial for binding to acetylated lysines in the bromodomains of BRD3, BRD4 and TRIM24 all resulted in reduction of FRAP recovery times, indicating loss of or significantly reduced binding to acetylated chromatin, as did the addition of known inhibitors. Significant differences between wild type and bromodomain mutants for ATAD2, BAZ2A, BRD1, BRD7, GCN5L2, SMARCA2 and ZMYND11 required the addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) to amplify the binding contribution of the bromodomain. Under these conditions, known inhibitors decreased FRAP recovery times back to mutant control levels. Mutation of the bromodomain did not alter FRAP recovery times for full-length CREBBP, even in the presence of SAHA, indicating that other domains are primarily responsible for anchoring CREBBP to chromatin. However, FRAP assays with multimerised CREBBP bromodomains resulted in a good assay to assess the efficacy of bromodomain inhibitors to this target. The bromodomain and extraterminal protein inhibitor PFI-1 was inactive against other bromodomain targets, demonstrating the specificity of the method. CONCLUSIONS: Viable FRAP assays were established for 11 representative bromodomain-containing proteins that broadly cover the bromodomain phylogenetic tree. Addition of SAHA can overcome weak binding to chromatin, and the use of tandem bromodomain constructs can eliminate masking effects of other chromatin binding domains. Together, these results demonstrate that FRAP assays offer a potentially pan-bromodomain method for generating cell-based assays, allowing the testing of compounds with respect to cell permeability, on-target efficacy and selectivity

High-Throughput Mass Spectrometry Applied to Structural Genomics

Mass spectrometry (MS) remains under-utilized for the analysis of expressed proteins because it is inaccessible to the non-specialist, and sample-turnaround from service labs is slow. Here, we describe 3.5 min Liquid-Chromatography (LC)-MS and 16 min LC-MSMS methods which are tailored to validation and characterization of recombinant proteins in a high throughput structural biology pipeline. We illustrate the type and scope of MS data typically obtained from a 96-well expression and purification test for both soluble and integral membrane proteins (IMPs), and describe their utility in the selection of constructs for scale-up structural work, leading to cost and efficiency savings. We propose that value of MS data lies in how quickly it becomes available and that this can fundamentally change the way in which it is used

The Zinc-Responsive Regulator Zur Controls Expression of the Coelibactin Gene Cluster in Streptomyces coelicolor▿ †

Streptomyces coelicolor mutants lacking the zinc-responsive Zur repressor are conditionally defective in sporulation, presumably due to the overexpression of one or more Zur target genes. Gene disruption analyses revealed that deregulation of previously known Zur targets was not responsible for the sporulation phenotype. We used microarrays to identify further Zur targets and discovered that Zur controls a cluster of genes predicted to direct synthesis of an uncharacterized siderophore-related non-ribosomally encoded peptide designated coelibactin. Disruption of a key coelibactin biosynthetic gene suppressed the Zur sporulation phenotype, suggesting that deregulation of coelibactin synthesis inhibits sporulation

Structural basis for NADH/NAD+ redox sensing by a Rex family repressor

Nicotinamide adenine dinucleotides have emerged as key signals of the cellular redox state. Yet the structural basis for allosteric gene regulation by the ratio of reduced NADH to oxidized NAD+ is poorly understood. A key sensor among Gram-positive bacteria, Rex represses alternative respiratory gene expression until a limited oxygen supply elevates the intracellular NADH:NAD+ ratio. Here we investigate the molecular mechanism for NADH/NAD+ sensing among Rex family members by determining structures of Thermus aquaticus Rex bound to (1) NAD+, (2) DNA operator, and (3) without ligand. Comparison with the Rex/NADH complex reveals that NADH releases Rex from the DNA site following a 40° closure between the dimeric subunits. Complementary site-directed mutagenesis experiments implicate highly conserved residues in NAD-responsive DNA-binding activity. These rare views of a redox sensor in action establish a means for slight differences in the nicotinamide charge, pucker, and orientation to signal the redox state of the cell

Assessing cellular efficacy of bromodomain inhibitors using fluorescence recovery after photobleaching

Abstract Background Acetylation of lysine residues in histone tails plays an important role in the regulation of gene transcription. Bromdomains are the readers of acetylated histone marks, and, consequently, bromodomain-containing proteins have a variety of chromatin-related functions. Moreover, they are increasingly being recognised as important mediators of a wide range of diseases. The first potent and selective bromodomain inhibitors are beginning to be described, but the diverse or unknown functions of bromodomain-containing proteins present challenges to systematically demonstrating cellular efficacy and selectivity for these inhibitors. Here we assess the viability of fluorescence recovery after photobleaching (FRAP) assays as a target agnostic method for the direct visualisation of an on-target effect of bromodomain inhibitors in living cells. Results Mutation of a conserved asparagine crucial for binding to acetylated lysines in the bromodomains of BRD3, BRD4 and TRIM24 all resulted in reduction of FRAP recovery times, indicating loss of or significantly reduced binding to acetylated chromatin, as did the addition of known inhibitors. Significant differences between wild type and bromodomain mutants for ATAD2, BAZ2A, BRD1, BRD7, GCN5L2, SMARCA2 and ZMYND11 required the addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) to amplify the binding contribution of the bromodomain. Under these conditions, known inhibitors decreased FRAP recovery times back to mutant control levels. Mutation of the bromodomain did not alter FRAP recovery times for full-length CREBBP, even in the presence of SAHA, indicating that other domains are primarily responsible for anchoring CREBBP to chromatin. However, FRAP assays with multimerised CREBBP bromodomains resulted in a good assay to assess the efficacy of bromodomain inhibitors to this target. The bromodomain and extraterminal protein inhibitor PFI-1 was inactive against other bromodomain targets, demonstrating the specificity of the method. Conclusions Viable FRAP assays were established for 11 representative bromodomain-containing proteins that broadly cover the bromodomain phylogenetic tree. Addition of SAHA can overcome weak binding to chromatin, and the use of tandem bromodomain constructs can eliminate masking effects of other chromatin binding domains. Together, these results demonstrate that FRAP assays offer a potentially pan-bromodomain method for generating cell-based assays, allowing the testing of compounds with respect to cell permeability, on-target efficacy and selectivity

Penicillin derivatives inhibit the SARS-CoV-2 main protease by reaction with its nucleophilic cysteine

The SARS-CoV-2 main protease (Mpro) is a medicinal chemistry target for COVID-19 treatment. Given the clinical efficacy of β-lactams as inhibitors of bacterial nucleophilic enzymes, they are of interest as inhibitors of viral nucleophilic serine and cysteine proteases. We describe the synthesis of penicillin derivatives which are potent Mpro inhibitors and investigate their mechanism of inhibition using mass spectrometric and crystallographic analyses. The results suggest that β-lactams have considerable potential as Mpro inhibitors via a mechanism involving reaction with the nucleophilic cysteine to form a stable acyl–enzyme complex as shown by crystallographic analysis. The results highlight the potential for inhibition of viral proteases employing nucleophilic catalysis by β-lactams and related acylating agents

In vitro selection of macrocyclic peptide inhibitors containing cyclic γ2,4-amino acids targeting the SARS-CoV-2 main protease

γ-Amino acids can play important roles in the biological activities of natural products; however, ribosomal incorporation of γ-amino acids into peptides is challenging. Here we report how a selection campaign employing a non-canonical peptide library containing cyclic γ2,4-amino acids (cγAAs) resulted in the discovery of very potent inhibitors of the SARS-CoV-2 main protease (Mpro). Two kinds of cγAAs, cis-3-aminocyclobutane carboxylic acid (γ1) and (1R,3S)-3-aminocyclopentane carboxylic acid (γ2), were ribosomally introduced into a library of thioether-macrocyclic peptides. One resultant potent Mpro inhibitor (IC50 = 50 nM), GM4, comprising 13 residues with γ1 at the 4th position, manifests a 5.2 nM dissociation constant. An Mpro:GM4 complex crystal structure reveals the intact inhibitor spans the substrate binding cleft. γ1 interacts with the S1′ catalytic subsite and contributes to a 12-fold increase in proteolytic stability compared to its alanine-substituted variant. Knowledge of interactions between GM4 and Mpro enabled production of a variant with a 5-fold increase in potency