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Alternative ?I/anti-?I factors represent a unique form of bacterial ?/anti-? complex.

ABSTRACT: The ?70 family alternative ?I factors and their cognate anti-?I factors are widespread in Clostridia and Bacilli and play a role in heat stress response, virulence, and polysaccharide sensing. Multiple ?I/anti-?I factors exist in some lignocellulolytic clostridial species, specifically for regulation of components of a multienzyme complex, termed the cellulosome. The ?I and anti-?I factors are unique, because the C-terminal domain of ?I (SigIC) and the N-terminal inhibitory domain of anti-?I (RsgIN) lack homology to known proteins. Here, we report structure and interaction studies of a pair of ?I and anti-?I factors, SigI1 and RsgI1, from the cellulosome-producing bacterium, Clostridium thermocellum. In contrast to other known anti-? factors that have N-terminal helical structures, RsgIN has a ?-barrel structure. Unlike other anti-? factors that bind both ?2 and ?4 domains of the ? factors, RsgIN binds SigIC specifically. Structural analysis showed that SigIC contains a positively charged surface region that recognizes the promoter -35 region, and the synergistic interactions among multiple interfacial residues result in the specificity displayed by different ?I/anti-?I pairs. We suggest that the ?I/anti-?I factors represent a distinctive mode of ?/anti-? complex formation, which provides the structural basis for understanding the molecular mechanism of the intricate ?I/anti-?I system.

SUBMITTER: Wei Z

PROVIDER: S-EPMC6582324 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Alternative σI/anti-σI factors represent a unique form of bacterial σ/anti-σ complex.

Wei Zhen Z Chen Chao C Liu Ya-Jun YJ Dong Sheng S Li Jie J Qi Kuan K Liu Shiyue S Ding Xiaoke X Ortiz de Ora Lizett L Muñoz-Gutiérrez Iván I Li Yifei Y Yao Hongwei H Lamed Raphael R Bayer Edward A EA Cui Qiu Q Feng Yingang Y

Nucleic acids research 20190601 11

The σ70 family alternative σI factors and their cognate anti-σI factors are widespread in Clostridia and Bacilli and play a role in heat stress response, virulence, and polysaccharide sensing. Multiple σI/anti-σI factors exist in some lignocellulolytic clostridial species, specifically for regulation of components of a multienzyme complex, termed the cellulosome. The σI and anti-σI factors are unique, because the C-terminal domain of σI (SigIC) and the N-terminal inhibitory domain of anti-σI (Rs ...[more]

PMID: 31106374

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Project description:Extracytoplasmic function σ factors that are stress inducible are often sequestered in an inactive complex with a membrane-associated anti-σ factor. Mycobacterium tuberculosis membrane-associated anti-σ factors have a small, stable RNA gene A (ssrA)-like degron for targeted proteolysis. Interaction between the unfoldase, ClpX, and a substrate with an accessible degron initiates energy-dependent proteolysis. Four anti-σ factors with a mutation in the degron provided a set of natural substrates to evaluate the influence of the degron on degradation strength in ClpX-substrate processivity. We note that a point mutation in the degron (X-Ala-Ala) leads to an order-of-magnitude difference in the dwell time of the substrate on ClpX. Differences in ClpX/anti-σ interactions were correlated with changes in unfoldase activities. Green fluorescent protein (GFP) chimeras or polypeptides with a length identical to that of the anti-σ factor degron also demonstrate degron-dependent variation in ClpX activities. We show that degron-dependent ClpX activity leads to differences in anti-σ degradation, thereby regulating the release of free σ from the σ/anti-σ complex. M. tuberculosis ClpX activity thus influences changes in gene expression by modulating the cellular abundance of ECF σ factors.IMPORTANCE The ability of Mycobacterium tuberculosis to quickly adapt to changing environmental stimuli occurs by maintaining protein homeostasis. Extracytoplasmic function (ECF) σ factors play a significant role in coordinating the transcription profile to changes in environmental conditions. Release of the σ factor from the anti-σ is governed by the ClpXP2P1 assembly. M. tuberculosis ECF anti-σ factors have an ssrA-like degron for targeted degradation. A point mutation in the degron leads to differences in ClpX-mediated proteolysis and affects the cellular abundance of ECF σ factors. ClpX activity thus synchronizes changes in gene expression with environmental stimuli affecting M. tuberculosis physiology.

Project description:Extracytoplasmic function σ factors (ECFs) belong to the most abundant signal transduction mechanisms in bacteria. Among the diverse regulators of ECF activity, class I anti-σ factors are the most important signal transducers in response to internal and external stress conditions. Despite the conserved secondary structure of the class I anti-σ factor domain (ASDI) that binds and inhibits the ECF under noninducing conditions, the binding interface between ECFs and ASDIs is surprisingly variable between the published cocrystal structures. In this work, we provide a comprehensive computational analysis of the ASDI protein family and study the different contact themes between ECFs and ASDIs. To this end, we harness the coevolution of these diverse protein families and predict covarying amino acid residues as likely candidates of an interaction interface. As a result, we find two common binding interfaces linking the first alpha-helix of the ASDI to the DNA-binding region in the σ4 domain of the ECF, and the fourth alpha-helix of the ASDI to the RNA polymerase (RNAP)-binding region of the σ2 domain. The conservation of these two binding interfaces contrasts with the apparent quaternary structure diversity of the ECF/ASDI complexes, partially explaining the high specificity between cognate ECF and ASDI pairs. Furthermore, we suggest that the dual inhibition of RNAP- and DNA-binding interfaces is likely a universal feature of other ECF anti-σ factors, preventing the formation of nonfunctional trimeric complexes between σ/anti-σ factors and RNAP or DNA.IMPORTANCE In the bacterial world, extracytoplasmic function σ factors (ECFs) are the most widespread family of alternative σ factors, mediating many cellular responses to environmental cues, such as stress. This work uses a computational approach to investigate how these σ factors interact with class I anti-σ factors-the most abundant regulators of ECF activity. By comprehensively classifying the anti-σs into phylogenetic groups and by comparing this phylogeny to the one of the cognate ECFs, the study shows how these protein families have coevolved to maintain their interaction over evolutionary time. These results shed light on the common contact residues that link ECFs and anti-σs in different phylogenetic families and set the basis for the rational design of anti-σs to specifically target certain ECFs. This will help to prevent the cross talk between heterologous ECF/anti-σ pairs, allowing their use as orthogonal regulators for the construction of genetic circuits in synthetic biology.

Dataset Information

Alternative ?I/anti-?I factors represent a unique form of bacterial ?/anti-? complex.

Publications

Alternative σI/anti-σI factors represent a unique form of bacterial σ/anti-σ complex.

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