Structural Basis of a Rationally Rewired Protein-Protein Interface Critical to Bacterial Signaling

Podgornaia, Anna I.; Casino, Patricia; Marina, Alberto; Laub, Michael T.

doi:10.1016/j.str.2013.07.005

Cited by 65 publications

(102 citation statements)

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“…Although the ␣3 helices of Actinobacteria, Firmicutes, and chloroflexi display substantial sequence similarity, the highly divergent ␣3 helix sequences of the Xanthomonadaceae/Pseudomonadaceae, the Cyanobacteria, and the Gallionellales cluster target a structurally similar motif that has been shown be bound by several noncognate LexA proteins (50,51). Recent work on the PhoQ-PhoP two-component signal transduction system has shown that there is a remarkable degree of sequence plasticity in the PhoQ-PhoP interface and that evolution has explored only a small fraction of the space of the PhoP-specific PhoQ motifs, due to both physiological and biochemical constraints on available mutational pathways (70)(71)(72). The results presented here suggest that the same is true for the LexA protein-DNA interface, which displays a varied palette of ␣3 helix and LexA-binding motif pairs.…”

Section: Resultsmentioning

confidence: 99%

“…The results presented here suggest that the same is true for the LexA protein-DNA interface, which displays a varied palette of ␣3 helix and LexA-binding motif pairs. Importantly, however, the available data indicate that the coevolution of the LexA recognition domain and its binding motif may face more restrictions than those displayed by two-component systems (70,71,73). In particular, the abundance of highly divergent ␣3 helix sequences targeting a similar, B. subtilis-type LexA-binding motif yields two nonexclusive scenarios.…”

Section: Resultsmentioning

confidence: 99%

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An SOS Regulon under Control of a Noncanonical LexA-Binding Motif in the Betaproteobacteria

et al. 2015

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The SOS response is a transcriptional regulatory network governed by the LexA repressor that activates in response to DNA damage. In the Betaproteobacteria, LexA is known to target a palindromic sequence with the consensus sequence CTGT-N 8 -ACAG. We report the characterization of a LexA regulon in the iron-oxidizing betaproteobacterium Sideroxydans lithotrophicus. In silico and in vitro analyses show that LexA targets six genes by recognizing a binding motif with the consensus sequence GAACGaaCGTTC, which is strongly reminiscent of the Bacillus subtilis LexA-binding motif. We confirm that the closely related Gallionella capsiferriformans shares the same LexA-binding motif, and in silico analyses indicate that this motif is also conserved in the Nitrosomonadales and the Methylophilales. Phylogenetic analysis of LexA and the alpha subunit of DNA polymerase III (DnaE) reveal that the organisms harboring this noncanonical LexA form a compact taxonomic cluster within the Betaproteobacteria. However, their lexA gene is unrelated to the standard Betaproteobacteria lexA, and there is evidence of its spread through lateral gene transfer. In contrast to other reported cases of noncanonical LexA-binding motifs, the regulon of S. lithotrophicus is comparable in size and function to that of many other Betaproteobacteria, suggesting that a convergent SOS regulon has reevolved under the control of a new LexA protein. Analysis of the DNA-binding domain of S. lithotrophicus LexA reveals little sequence similarity with that of other LexA proteins targeting similar binding motifs, suggesting that network structure may limit site evolution or that structural constrains make the B. subtilis-type motif an optimal interface for multiple LexA sequences. IMPORTANCEUnderstanding the evolution of transcriptional systems enables us to address important questions in microbiology, such as the emergence and transfer potential of different regulatory systems to regulate virulence or mediate responses to stress. The results reported here constitute the first characterization of a noncanonical LexA protein regulating a standard SOS regulon. This is significant because it illustrates how a complex transcriptional program can be put under the control of a novel transcriptional regulator. Our results also reveal a substantial degree of plasticity in the LexA recognition domain, raising intriguing questions about the space of protein-DNA interfaces and the specific evolutionary constrains faced by these elements.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

An SOS Regulon under Control of a Noncanonical LexA-Binding Motif in the Betaproteobacteria

et al. 2015

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“…Cross talk at the level of phosphoryl transfer has been described in other TCSs and can be predicted based on the identity of specific coevolving residues found in HKs and RRs (18)(19)(20).…”

Section: Analysis Of Gene Expression By Quantitative Reverse Transcrimentioning

confidence: 99%

“…The intervention of one TCS with the signal transduction of another, termed cross talk between TCSs, can be minimized through the phosphatase activity of HKs, through temporal/spatial regulation, and through specific molecular recognition between cognate HKs and RRs (17). The latter has been extensively explored, with specificity between cognate HK/RR pairs attributed to specific coevolving residues that dictate if a phosphoryl transfer event can occur efficiently (18)(19)(20). With this molecular specificity, an HK can use its kinase and phosphatase functions to control the level of phosphorylated cognate RR without interfering with the signaling of other HK/RR pairs.…”

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confidence: 99%

Functional Dissection of the CroRS Two-Component System Required for Resistance to Cell Wall Stressors in Enterococcus faecalis

Kellogg

Kristich

2016

J Bacteriol

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Bacteria use two-component signal transduction systems (TCSs) to sense and respond to environmental changes via a conserved phosphorelay between a sensor histidine kinase and its cognate response regulator. The opportunistic pathogen Enterococcus faecalis utilizes a TCS comprised of the histidine kinase CroS and the response regulator CroR to mediate resistance to cell wall stresses such as cephalosporin antibiotics, but the molecular details by which CroRS promotes cephalosporin resistance have not been elucidated. Here, we analyzed mutants of E. faecalis carrying substitutions in CroR and CroS to demonstrate that phosphorylated CroR drives resistance to cephalosporins, and that CroS exhibits kinase and phosphatase activities to control the level of CroR phosphorylation in vivo. Deletion of croS in various lineages of E. faecalis revealed a CroS-independent mechanism for CroR phosphorylation and led to the identification of a noncognate histidine kinase capable of influencing CroR (encoded by OG1RF_12162; here called cisS). Further analysis of this TCS network revealed that both systems respond to cell wall stress. IMPORTANCETCSs allow bacteria to sense and respond to many different environmental conditions. The opportunistic pathogen Enterococcus faecalis utilizes the CroRS TCS to mediate resistance to cell wall stresses, including clinically relevant antibiotics such as cephalosporins and glycopeptides. In this study, we use genetic and biochemical means to investigate the relationship between CroRS signaling and cephalosporin resistance in E. faecalis cells. Through this, we uncovered a signaling network formed between the CroRS TCS and a previously uncharacterized TCS that also responds to cell wall stress. This study provides mechanistic insights into CroRS signaling and cephalosporin resistance in E. faecalis. Although Enterococcus faecalis is a normal commensal of the gut microbiome, it poses a serious risk to humans as an opportunistic pathogen. A major risk factor for the pathogenic transition of enterococci is therapy with broad-spectrum cephalosporin antibiotics. Cephalosporins are commonly used to treat bacterial infections; however, nearly all enterococcal isolates are resistant to these compounds. This intrinsic resistance allows for the expansion of resident enterococcal populations during cephalosporin treatment, which is thought to promote dissemination from the gastrointestinal tract, enabling enterococci to establish infections (1, 2). With the emergence of multidrug-resistant isolates of E. faecalis, few options for treating infections are available. Therefore, understanding the mechanisms that support cephalosporin resistance in E. faecalis is crucial for designing new therapies that limit growth during cephalosporin treatment or that can be used as adjuvants with cephalosporins to treat E. faecalis infections. An essential determinant of cephalosporin resistance in E. faecalis is the CroRS two-component system (TCS), consisting of CroS (a transmembrane sensor-histidine kinase) and ...

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“…1B). For histidine kinases, mutating just three or four interfacial residues to match those found in another kinase is often sufficient to reprogram partner specificity Podgornaia et al, 2013).…”

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confidence: 99%

Pervasive degeneracy and epistasis in a protein-protein interface

Podgornaia

Laub

2015

Science

184

218

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Signal transduction pathways rely on transient yet specific protein-protein interactions. How a limited set of amino acids can enforce cognate protein interactions while excluding undesired pairings remains poorly understood, even in cases where the contacting residues have been identified on both protein partners. To tackle this challenge, I performed structure-guided and library-based mutagenesis studies of bacterial two-component signaling pathways. These pathways, typically consisting of a histidine kinase and a response regulator, are an ideal model system for studying protein-protein interactions as they rely almost exclusively on molecular recognition for specificity. The kinase uses a limited set of residues to recognize the regulator in both phosphorylation and dephosphorylation reactions, and to prevent docking with all noncognate regulators.In this thesis I characterized the extent to which interface residues in two-component signaling proteins can be modified without changing the overall behavior of the pathway. In collaboration with another research group I have performed a mutagenesis study of a two-component system from Thermotoga maritima that has proven amenable to structural analysis. By solving the cocrystal structure of a histidine kinase and response regulator containing interface residues from a different interacting pair, we learned the biophysical basis for accommodating these new residues. To understand how many different residue combinations can support a functional interaction, I comprehensively mapped the sequence space of the interface formed by Escherichia coli histidine kinase PhoQ and its partner PhoP. I used a robust high-throughput assay to screen a library of 204 (160,000) PhoQ variants in which I had completely randomized the four key specificity-determining residues. Using deep sequencing, I identified -1,600 (1 %) variants that can phosphorylate and dephosphorylate PhoP as well as the wild-type PhoQ. Strikingly, PhoQ can interact with PhoP via many sets of interfacial residues that are completely different from the wild type. This combinatorial approach to mapping sequence space revealed interdependencies between individual amino acids, illustrating its power relative to screens that only examine substitutions at individual sites. This thesis provides a framework for mapping the sequence space of histidine kinases and has broad implications for understanding protein-protein interaction specificity and the evolution of bacterial signaling pathways.

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Structural Basis of a Rationally Rewired Protein-Protein Interface Critical to Bacterial Signaling

Cited by 65 publications

References 35 publications

An SOS Regulon under Control of a Noncanonical LexA-Binding Motif in the Betaproteobacteria

An SOS Regulon under Control of a Noncanonical LexA-Binding Motif in the Betaproteobacteria

Functional Dissection of the CroRS Two-Component System Required for Resistance to Cell Wall Stressors in Enterococcus faecalis

Pervasive degeneracy and epistasis in a protein-protein interface

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