Membrane depolarization causes voltage-gated ion channels to transition from a resting/closed conformation to an activated/open conformation. We used voltage-clamp fluorometry to measure protein motion at specific regions of the Shaker Kv channel. This enabled us to construct new structural models of the resting/closed and activated/open states based on the Kv1.2 crystal structure using the Rosetta-Membrane method and molecular dynamics simulations. Our models account for the measured gating charge displacement and suggest a molecular mechanism of activation in which the primary voltage sensors, S4s, rotate by approximately 180 degrees as they move "outward" by 6-8 A. A subsequent tilting motion of the S4s and the pore domain helices, S5s, of all four subunits induces a concerted movement of the channel's S4-S5 linkers and S6 helices, allowing ion conduction. Our models are compatible with a wide body of data and resolve apparent contradictions that previously led to several distinct models of voltage sensing.

Voltage-sensing domains (VSDs) confer voltage dependence on effector domains of membrane proteins. Ion channels use four VSDs to control a gate in the pore domain, but in the recently discovered phosphatase Ci-VSP, the number of subunits has been unknown. Using single-molecule microscopy to count subunits and voltage clamp fluorometry to detect structural dynamics, we found Ci-VSP to be a monomer, which operates independently, but nevertheless undergoes multiple voltage-dependent conformational transitions.

SUMMARYIn voltage-gated sodium, potassium, and calcium channels the functions of ion conduction and voltage sensing are performed by two distinct structural units: the pore domain and the voltage-sensing domain (VSD). In the Hv1 voltage-gated proton channel, the VSD has the remarkable property of performing both functions. Hv1 was recently found to dimerize and to form channels made of two pores. However, the channels were also found to function when dimerization was prevented, raising a question about the functional role of dimerization. Here we show that the two subunits of the Hv1 dimer influence one another during gating, with positive cooperativity shaping the response to voltage of the two pores. We also find that the two voltage sensors undergo conformational changes that precede pore opening and that these conformational changes are allosterically coupled between the two subunits. Our results point to a major role of dimerization in the modulation of Hv1 activity.

The chemotaxis pathway of Escherichia coli and Salmonella typhimurium is the paradigm for the ubiquitous class of 2-component signaling pathways in prokaryotic organisms. Chemosensing begins with the binding of a chemical attractant to a transmembrane receptor on the cell surface. The resulting transmembrane signal regulates a cytoplasmic, multiprotein signaling complex that controls cellular swimming behavior by generating a diffusible phosphoprotein. The minimal functional unit of this signaling complex, termed the core complex, consists of the transmembrane receptor, the coupling protein CheW, and the histidine kinase CheA. Though the structures of individual components are largely known and the core complex can be functionally reconstituted, the architecture of the assembled core complex has remained elusive. To probe this architecture, the present study has utilized an enhanced version of the protein-interactions-by-cysteine-modification method (PICM-β) to map out docking surfaces on CheA essential for kinase activity and for core complex assembly.The approach employed a library of 70 single, engineered cysteine residues, scattered uniformly over the surfaces of the five CheA domains in a cysteine-free CheA background. These surface Cys residues were further modified by the sulfhydryl-specific alkylating agent, 5-fluorescein-maleimide (5FM). The functional effects of individual Cys and 5FM-Cys surface modifications were measured by kinase assays of CheA activity in both the free and core complex-associated states, and by direct binding assays of CheA associations with CheW and the receptor. The results define (i) two mutual docking surfaces on the CheA substrate and catalytic domains essential for the association of these domains during autophosphorylation, (ii) a docking surface on the CheA regulatory domain essential for CheW binding, and (iii) a large docking surface encompassing regions of the CheA dimerization, catalytic, and regulatory domains proposed to bind the receptor. To test the generality of these findings, a CheA sequence alignment was analyzed, revealing that the newly identified docking surfaces are highly conserved among CheA homologues. These results strongly suggest that the same docking sites are widely utilized in prokaryotic sensory pathways. Finally, the results provide new structural constraints allowing the development of improved models for core complex architecture.The homodimeric, histidine kinase CheA is the central processing unit of the conserved signaling pathway that controls chemotaxis in Escherichia coli, Salmonella typhimurium, and many other bacteria (for reviews see refs 1-7). This pathway is the prototype for the large, ubiquitous class of two-component pathways that regulate a wide array of cell processes in prokaryotes. CheA and the other pathway components assemble to form large clusters of signaling complexes at the poles of the cell. The minimal signaling unit required for receptorregulated kinase activity, termed the core complex, comprises the oligomeri...

The independently folding C2 domain motif serves as a Ca(2+)-dependent membrane docking trigger in a large number of Ca(2+) signaling pathways. A comparison was initiated between three closely related C2 domains from the conventional protein kinase C subfamily (cPKC, isoforms alpha, beta, and gamma). The results reveal that these C2 domain isoforms exhibit some similarities but are specialized in important ways, including different Ca(2+) stoichiometries. In the absence of membranes, Ca(2+) affinities of the isolated C2 domains are similar (2-fold difference) while Hill coefficients reveal cooperative Ca(2+) binding for the PKC beta C2 domain but not for the PKC alpha or PKC gamma C2 domain (H = 2.3 +/- 0.1 for PKC beta, 0.9 +/- 0.1 for PKC alpha, and 0.9 +/- 0.1 for PKC gamma). When phosphatidylserine-containing membranes are present, Ca(2+) affinities range from the sub-micromolar to the micromolar (7-fold difference) ([Ca(2+)](1/2) = 0.7 +/- 0.1 microM for PKC gamma, 1.4 +/- 0.1 microM for PKC alpha, and 5.0 +/- 0.2 microM for PKC beta), and cooperative Ca(2+) binding is observed for all three C2 domains (Hill coefficients equal 1.8 +/- 0.1 for PKC beta, 1.3 +/- 0.1 for PKC alpha, and 1.4 +/- 0.1 for PKC gamma). The large effects of membranes are consistent with a coupled Ca(2+) and membrane binding equilibrium, and with a direct role of the phospholipid in stabilizing bound Ca(2+). The net negative charge of the phospholipid is more important to membrane affinity than its headgroup structure, although a slight preference for phosphatidylserine is observed over other anionic phospholipids. The Ca(2+) stoichiometries of the membrane-bound C2 domains are detectably different. PKC beta and PKC gamma each bind three Ca(2+) ions in the membrane-associated state; membrane-bound PKC alpha binds two Ca(2+) ions, and a third binds weakly or not at all under physiological conditions. Overall, the results indicate that conventional PKC C2 domains first bind a subset of the final Ca(2+) ions in solution, and then associate weakly with the membrane and bind additional Ca(2+) ions to yield a stronger membrane interaction in the fully assembled tertiary complex. The full complement of Ca(2+) ions is needed for tight binding to the membrane. Thus, even though the three C2 domains are 64% identical, differences in Ca(2+) affinity, stoichiometry, and cooperativity are observed, demonstrating that these closely related C2 domains are specialized for their individual functions and contexts.