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

Abstract

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.