WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus . WalKR regulates peptidoglycan synthesis, but this function alone does not explain its essentiality. Here, to further understand WalKR function, we investigate a suppressor mutant that arose when WalKR activity was impaired; a histidine to tyrosine substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PAS CYT ) domain of the histidine kinase WalK. Introducing the WalK H271Y mutation into wild-type S. aureus activates the WalKR regulon. Structural analyses of the WalK PAS CYT domain reveal a metal-binding site, in which a zinc ion (Zn 2+ ) is tetrahedrally-coordinated by four amino acids including H271. The WalK H271Y mutation abrogates metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn 2+ -binding negatively regulates WalKR. Promoter-reporter experiments using S. aureus confirm Zn 2+ sensing by this system. Identification of a metal ligand recognized by the WalKR system broadens our understanding of this critical S. aureus regulon.

The human norepinephrine transporter (NET) is implicated in many neurological disorders and is a target of tricyclic antidepressants and nisoxetine (NX). We used molecular docking simulations to guide the identification of residues likely to affect substrate transport and ligand interactions at NET. Mutations to alanine identified a hydrophobic pocket in the extracellular cavity of NET, comprising residues Thr80, Phe317, and Tyr317, which was critical for efficient norepinephrine (NE) transport. This secondary NE substrate site (NESS-2) overlapped the NX binding site, comprising Tyr84, Phe317, and Tyr317, and was positioned ϳ11 Å extracellular to the primary site for NE (NESS-1). Thr80 in NESS-2 appeared to be critical in positioning NE for efficient translocation to NESS-1. Three residues identified as being involved in gating the reverse transport of NE (Arg81, Gln314, and Asp473) did not affect NE affinity for NESS-1. Mutating residues adjacent to NESS-2 abolished NET expression (D75A and L76A) or appeared to affect NET folding (S419A), suggesting important roles in stabilizing NET structure, whereas W308A and F388A at the top of NESS-2 abolished both NE transport and NX binding. Our findings are consistent with a multistep model of substrate transport by NET, for which a second, shallow extracellular NE substrate site (NESS-2) is required for efficient NE transport by NET.

Evolution of a nano-machine consisting of multiple parts, each with a specific function, is a complex process. A change in one part should eventually result in changes in other parts, if the overall function is to be conserved. In bacterial flagella, the filament and the hook have distinct functions and their respective proteins, FliC and FlgE, have different three-dimensional structures. The filament functions as a helical propeller and the hook as a flexible universal joint. Two proteins, FlgK and FlgL, assure a smooth connectivity between the hook and the filament. Here we show that, in Campylobacter, the 3D structure of FlgK differs from that of its orthologs in Salmonella and Burkholderia, whose structures have previously been solved. Docking the model of the FlgK junction onto the structure of the Campylobacter hook provides some clues about its divergence. These data suggest how evolutionary pressure to adapt to structural constraints, due to the structure of Campylobacter hook, causes divergence of one element of a supra-molecular complex in order to maintain the function of the entire flagellar assembly.

WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus. WalKR regulates peptidoglycan synthesis, but this function alone appears not to explain its essentiality. To understand WalKR function we investigated a suppressor mutant that arose when WalKR activity was impaired; a single histidine to tryptophan substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PASCYT) domain of the histidine kinase WalK. Introduction of the WalKH271Y mutation into wild-type S. aureus activated the WalKR regulon. Structural analyses of the WalK PASCYT domain revealed a hitherto unknown metal binding site, in which a zinc ion (Zn2+) was tetrahedrally-coordinated by four amino acid residues including H271. The WallkH271Y mutation abrogated metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn2+-binding negatively regulates WalKR activity. Identification of a metal ligand sensed by the WalKR system substantially expands our understanding of this critical S. aureus regulon.