McsB/YwlE

The CtsR stress response

The CtsR/McsB regulon of Gram-positive bacteria. Under non-stress conditions, CtsR represses expression of indicated heat-shock genes.

All cells have evolved highly efficient signaling pathways that sense the presence of damaged proteins and transmit the signal “folding stress” to dedicated transcription factors, which then adjust the expression of the protein quality control factors. One of the most intensely studied stress-response pathways is the bacterial heat-shock system. In B. subtilis, the transcription factor CtsR is a major component of this system. CtsR represses the clpC heat shock operon (clpC, mcsA, mcsB, ctsR) and the clpE and clpP genes, by binding specifically to a 7-nucleotide direct repeat sequence located upstream of the transcriptional start sites. Stress-induced transcription of the clp genes depends on the inactivation of CtsR by McsB, however the precise mechanism of this process and the subsequent reactivation remain to be discovered.

Our findings: 2009 McsB is a protein arginine kinase targeting CtsR * 2013 The PTP YwlE is a specific arginine phosphatase

2013 YwlE is a specific arginine phosphatase counteracting McsB

Crystallized reaction intermediate representing the arginine:phospho-enzyme complex.

Many cellular pathways are regulated by the competing activity of protein kinases and phosphatases. The recent identification of arginine phosphorylation as a novel protein modification prompted us to analyze the molecular basis of targeting phospho-arginine.

In this work, we characterized the arginine phosphatase YwlE, which was shown to counteract McsB activity. Initially, YwlE was annotated as a tyrosine phosphatase, however our structural studies of YwlE reaction intermediates provide a direct view on a captured arginine residue. Together with biochemical data, the crystal structures depict the evolution towards a highly specific phospho-arginine phosphatase that employs an intricate size-and-polarity filter for distinguishing phosphorylated arginine from other phosphorylated side chains. To confirm the proposed mechanism, we performed bioinformatic searches for phosphatases employing a similar selectivity filter, and identified a protein in Drosophila melanogaster exhibiting robust arginine phosphatase activity. In sum, our findings uncover the molecular framework for specific targeting of phospho-arginine and suggest that protein arginine (de)phosphorylation may be relevant in eukaryotes.

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2009 McsB is a protein arginine kinase that phosphorylates and inhibits the transcriptional repressor Cts

To delineate the molecular basis of the CtsR/McsB interplay in the bacterial stress response, we tried to reconstitute the corresponding system from various Gram-positive bacteria using recombinant proteins. Finally, we succeeded in reconstituting the Bacillus stearothermophilus CtsR/McsB system in vitro.

The crystal structure of the CtsR repressor in complex with DNA revealed how partial asymmetry in a dimeric transcription factor allows high affinity binding to tandem DNA repeats. Moreover, biochemical characterization of McsB highlighted a novel protein kinase activity targeting arginine residues. McsB specifically phosphorylates arginine residues in the DNA-binding domain of CtsR, like for example Arg62 of the beta-wing, thereby impairing its function as a repressor of stress response genes. Identification of the CtsR/McsB arginine phospho-switch reveals the mechanism of transcriptional regulation in bacterial stress response. The first identification of a specific protein arginine kinase, not displaying any homology to previously known protein kinases, opens new perspectives in the understanding of this largely overseen protein modification that may play important roles both in bacteria and higher organisms.

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