title: Identification of transmembrane helix 1 (TM1) surfaces important for EnvZ dimerisation and signal output.
abstract: The Escherichia coli sensor kinase EnvZ modulates porin expression in response to various stimuli, including extracellular osmolarity, the presence of procaine and interaction with an accessory protein, MzrA. Two major outer membrane porins, OmpF and OmpC, act as passive diffusion-limited pores that allow compounds, including certain classes of antibiotics such as β-lactams and fluoroquinolones, to enter the bacterial cell. Even though the mechanisms by which EnvZ detects and processes the presence of various stimuli are a fundamental component of microbial physiology, they are not yet fully understood. Here, we assess the role of TM1 during signal transduction in response to the presence of extracellular osmolarity. Various mechanisms of transmembrane communication have been proposed including rotation of individual helices within the transmembrane domain, dynamic movement of the membrane-distal portion of the cytoplasmic domain and regulated intra-protein unfolding. To assess these possibilities, we have created a library of single-Cys-containing EnvZ proteins in order to facilitate sulfhydryl-reactivity experimentation. Our results demonstrate that the major TM1-TM1' interface falls along a single surface consisting of residue positions 19, 23, 26, 30 and 34. In addition, we show that Cys substitutions within the N- and C-terminal regions of TM1 result in drastic changes to EnvZ signal output. Finally, we demonstrate that core residues within TM1 are responsible for both TM1 dimerisation and maintenance of steady-state signal output. Overall, our results suggest that no major rearrangement of the TM1-TM1' interface occurs during transmembrane communication in response to extracellular osmolarity. We conclude by discussing these results within the frameworks of several proposed models for transmembrane communication.
authors.affiliation: Institute of Biochemistry, Biocenter, Goethe University Frankfurt, D-60438 Frankfurt, Germany. Electronic address: heininger.annika@t-online.de.
authors.forename: Annika
authors.initials: A
authors.lastname: Heininger
authors.affiliation: School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DT, England, UK. Electronic address: rahmi.yusuf@port.ac.uk.
authors.forename: Rahmi
authors.initials: R
authors.lastname: Yusuf
authors.affiliation: School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DT, England, UK. Electronic address: robert.lawrence@port.ac.uk.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Lawrence
authors.affiliation: School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DT, England, UK; Institute of Biomedical and Biomolecular Science, University of Portsmouth, Portsmouth PO1 2DT, England, UK. Electronic address: roger.draheim@port.ac.uk.
authors.forename: Roger R
authors.initials: RR
authors.lastname: Draheim
mesh_terms: D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction / Q000502:physiology
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C057843:envZ protein, E coli; D003545:Cysteine
keywords: EnvZ signalling; Osmosensing; Porin regulation; Sulfhydryl-reactivity; Transmembrane communication