Identification of transmembrane helix 1 (TM1) surfaces important for EnvZ dimerisation and signal output

Heininger, Annika; Yusuf, Rahmi; Lawrence, Robert; Draheim, Roger Russell

doi:10.1016/j.bbamem.2016.05.002

Cited by 7 publications

(11 citation statements)

References 71 publications

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“…This argues against large displacement of the two helices relative to one another in response to modulation. Interestingly, similar results have been reported for the sensor kinases DcuS and EnvZ (34,35). Those authors have instead proposed a symmetrical piston movement of the TM helices to transduce the signal.…”

Section: Resultssupporting

confidence: 67%

Conformational Changes of an Interdomain Linker Mediate Mechanical Signal Transmission in Sensor Kinase BvgS

et al. 2017

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The whooping cough agent, Bordetella pertussis, controls the expression of its large virulence regulon in a coordinated manner through the two-component system BvgAS. BvgS is a dimeric, multidomain sensor kinase. Each monomer comprises, in succession, tandem periplasmic Venus flytrap (VFT) domains, a transmembrane segment, a cytoplasmic Per-Arnt-Sim (PAS) domain, a kinase module, and additional phosphorelay domains. BvgS shifts between kinase and phosphatase modes of activity in response to chemical modulators that modify the clamshell motions of the VFT domains. We have shown previously that this regulation involves a shift between distinct states of conformation and dynamics of the two-helix coiled-coil linker preceding the enzymatic module. In this work, we determined the mechanism of signal transduction across the membrane via a first linker, which connects the VFT and PAS domains of BvgS, using extensive cysteine cross-linking analyses and other approaches. Modulator perception by the periplasmic domains appears to trigger a small, symmetrical motion of the transmembrane segments toward the periplasm, causing rearrangements of the noncanonical cytoplasmic coiled coil that follows. As a consequence, the interface of the PAS domains is modified, which affects the second linker and eventually causes the shift of enzymatic activity. The major features of this first linker are well conserved among BvgS homologs, indicating that the mechanism of signal transduction unveiled here is likely to be generally relevant for this family of sensor kinases.IMPORTANCE Bordetella pertussis produces virulence factors coordinately regulated by the two-component system BvgAS. BvgS is a sensor kinase, and BvgA is a response regulator that activates gene transcription when phosphorylated by BvgS. Sensor kinases homologous to BvgS are also found in other pathogens. Our goal is to decipher the mechanisms of BvgS signaling, since these sensor kinases may represent new targets for antibacterial agents. Signal perception by the sensor domains of BvgS triggers small motions of the helical linker region underneath. The protein domain that follows this linker undergoes a large conformational change that amplifies the initial signal, causing a shift of activity from kinase to phosphatase. Because BvgS homologs harbor similar regions, these signaling mechanisms are likely to apply generally to that family of sensor kinases.

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Section: Resultssupporting

confidence: 67%

Conformational Changes of an Interdomain Linker Mediate Mechanical Signal Transmission in Sensor Kinase BvgS

et al. 2017

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“…4). It should be noted that this significant difference at the periplasmic end of the TMD between cells grown under the low‐ and high‐osmolarity regime was not observed during similar analyses of TM1 (25).…”

Section: Resultsmentioning

confidence: 80%

“…We previously found that the Cys-less version of EnvZ had similar steady-state signal output and response to environmental osmolarity as the wild-type version of EnvZ making it suitable for comparisons of in vivo sulfhydryl-reactivity and signal output analysis. We initially determined that no major rearrangements occur along the TM1-TM1’ interface upon stimulus perception (25). As minimal change was observed along this helical interface in response to osmolarity, we continued by examining the TM2-TM2’ interface.…”

Section: Resultsmentioning

confidence: 99%

“…The in vivo nature of this assay facilitated mapping of the TM2-TM2’ interface under different osmotic conditions, which is an important first step toward understanding how EnvZ processes allosteric inputs from periplasmic MzrA binding and cytoplasmic osmosensing into a single uniform modulation of bacterial porin balance (Fig.1). In a similar manner to mapping TM1-TM1’ interactions (25), Cys-containing EnvZ variants were expressed in EPB30/pRD400 cells and upon entering the early exponential phase (OD 600nm ≈ 0.25) they were subjected to 250 μM molecular iodine for 10 minutes analysed by non-reducing SDS-PAGE and immunoblotting (Fig. S4).…”

Section: Resultsmentioning

confidence: 99%

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In vivocross-linking and transmembrane helix dynamics support a bidirectional non-piston model of signaling withinE. coliEnvZ

Yusuf

Heininger

et al. 2017

Preprint

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61 62In Gram-negative bacteria, porins span the outer membrane and control the influx of several prominent 63 groups of antibiotics. Thus, it should not be surprising that expression of these porins is often altered in 64 clinical isolates exhibiting multidrug resistance (MDR). The major regulator of porin expression in 65Escherichia coli is EnvZ, a canonical sensor histidine kinase (SHK). It allosterically processes 66 periplasmic interactions with MzrA and cytoplasmic osmosensing into a single unified change in the 67 ratio of its kinase and phosphatase activities. Unfortunately, the role of the EnvZ transmembrane 68 (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/206888 doi: bioRxiv preprint first posted online EnvZ regulates porin balance and antibiotic influx, these results contribute to answering the long-92 standing question of how transmembrane communication is performed by bacterial receptors. Our work 93 concludes with a framework that correlates receptor domain composition and signal transduction 94 mechanisms that could be employed by other research groups on their particular receptors of interest. 95 96 All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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“…The selection of residues previously assessed to contain TM1 was described in [28]. Briefly, the primary sequence of EnvZ from Escherichia coli K-12 MG1655 (NP_417863.1) was subjected to a full protein scan with DGpred, which calculates the ΔG app for transmembrane insertion throughout the entire length of the submitted sequence [29].…”

Section: Selection Of Residues For Assessmentmentioning

confidence: 99%

Employing scan-SCAM to identify residues that compose transmembrane helix 1 (TM1) of EnvZ

Chavalwan

Hasan

Cheesman

et al. 2020

Preprint

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The Escherichia coli sensor kinase EnvZ modulates porin expression in response to various stimuli including intracellular osmolarity, intracellular pH and periplasmic interaction with MzrA. The expression of two major outer membrane porins, OmpF and OmpC, are regulated by EnvZ, and act as passive diffusion-limited pores allowing compounds, including certain classes of antibiotics such as β-lactams and fluoroquinolones, to enter the bacterial cell. Even though allosteric processing occurs within both the periplasmic and cytoplasmic domains of EnvZ, how the transmembrane domain bi-directionally transmits these signals remains not fully understood. Here, we employ a library of single-Cys-containing EnvZ proteins to perform scan-SCAM in order to map the precise residue composition of TM1. Our results demonstrate that residue positions 19 through 30 reside within the membrane core and compose a tightly packed portion of TM1. We also show that positions 15 through 18 and position 31 are interfacial and slightly splayed apart compared to those tightly packed within the hydrophobic core. Finally, we reveal that residue positions 33 and 34 reside in the periplasm and participate in robust protein-protein interactions, while the periplasmic positions 35 through 41 exhibit helical periodicity. We conclude by synthesizing these new insights with recent high-resolution structural information into a model of membrane-spanning allosteric coupling between the periplasmic and cytoplasmic domains of EnvZ.

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Identification of transmembrane helix 1 (TM1) surfaces important for EnvZ dimerisation and signal output

Cited by 7 publications

References 71 publications

Conformational Changes of an Interdomain Linker Mediate Mechanical Signal Transmission in Sensor Kinase BvgS

Conformational Changes of an Interdomain Linker Mediate Mechanical Signal Transmission in Sensor Kinase BvgS

In vivocross-linking and transmembrane helix dynamics support a bidirectional non-piston model of signaling withinE. coliEnvZ

Employing scan-SCAM to identify residues that compose transmembrane helix 1 (TM1) of EnvZ

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