Lawrence, R. J. | Search bioRxiv

bioRxiv2025

EnvZ signal output correlates with capacity for transmembrane water encapsulation

In Gram-negative bacteria, porins span the outer membrane and control the influx of several prominent groups of antibiotics. Expression of these porins is often altered in clinical isolates exhibiting multidrug resistance. The major regulator of porin expression in Escherichia coli is EnvZ, a canonical sensor histidine kinase. It allosterically processes periplasmic interactions with MzrA and cytoplasmic osmosensing into a single unified change in the ratio of its kinase and phosphatase activities. To better understand how transmembrane communication is achieved by EnvZ, we began by performing disulfide crosslinking to monitor the position and dynamics of helices within the transmembrane domain in vivo and correlated these with modulation of signal output. Subsequently, we employed atomistic molecular dynamic simulations on full-length EnvZ embedded in a model Gram-negative membrane and observed that this in silico analysis accurately predicted the structure of the transmembrane domain embedded in the bacterial membrane. We conclude by proposing that the signal output of EnvZ correlates with its potential for water encapsulation within the transmembrane helical bundle and with unwinding of a helix adjacent to the periplasmic end of the second transmembrane helix. When combined with results from other bacterial receptors, it remains plausible that water encapsulation occurs in other topologically similar sensor kinases.

microbiology↗

bioRxiv2017

In vivo cross-linking and transmembrane helix dynamics support a non-piston model of signaling within E. coli EnvZ

In Gram-negative bacteria, porins span the outer membrane and control the influx of several prominent groups of antibiotics. Thus, it should not be surprising that expression of these porins is often altered in clinical isolates exhibiting multidrug resistance (MDR). The major regulator of porin expression in Escherichia coli is EnvZ, a canonical sensor histidine kinase (SHK). It allosterically processes periplasmic interactions with MzrA and cytoplasmic osmosensing into a single unified change in the ratio of its kinase and phosphatase activities. Unfortunately, the role of the EnvZ transmembrane domain (TMD) in bidirectional communication of these signals remains not well understood. Here, we employed in vivo sulfhydryl-reactivity to probe the dynamics of the TM2 helices and demonstrate that upon stimulus perception, only the region proximal to the periplasm undergoes conformational rearrangement. Furthermore, in silico coarse-grained molecular dynamics (CG-MD) simulations with aromatically tuned variants of EnvZ TM2 demonstrate the existence of both tilting and azimuthal rotational components to transmembrane communication while ruling out piston-type repositioning of TM2. Finally, in contrast to a similar analysis of TM1, we identified position-specific mutants possessing a \"flipped\" phenotype by dual-color fluorescent reporter analysis suggesting that both the periplasmic and cytoplasmic ends of TM2 are critical for maintenance of EnvZ signal output. Taken together, these data strongly support that EnvZ employs a non-piston-type mechanism during transmembrane communication. We conclude by discussing these results within the context of allosteric processing by EnvZ and propose that these results can be used to predict and classify transmembrane communication by various SHKs.\n\nImportanceThe EnvZ sensor histidine kinase serves as the major regulator of porin expression within Escherichia coli. A long-standing question is how stimulus perception by a bacterial receptor on one side of a biological membrane is transmitted to the opposite side of the membrane. To address this question, we monitored the dynamics of the transmembrane domain of EnvZ in vivo and coupled these results with in silico simulations of membrane-embedded EnvZ transmembrane domains. Taken together, these results demonstrate that detection of osmotic stress by the cytoplasmic domain of EnvZ results in non-piston communication across the inner membrane of E. coli. Thus, in addition to understanding how EnvZ regulates porin balance and antibiotic influx, these results contribute to answering the long-standing question of how transmembrane communication is performed by bacterial receptors. Our work concludes with a framework that correlates receptor domain composition and signal transduction mechanisms that could be employed by other research groups on their particular receptors of interest.

biochemistry↗