Transmembrane allosteric energetics characterization for strong coupling between proton and potassium ion binding in the KcsA channel.

The slow spontaneous inactivation of potassium channels exhibits classic signatures of transmembrane allostery. A variety of data support a model in which the loss of K+ ions from the selectivity filter is a major factor in promoting inactivation, which defeats transmission, and is allosterically coupled to protonation of key channel activation residues, more than 30 Å from the K+ ion binding site. We show that proton binding at the intracellular pH sensor perturbs the potassium affinity at the extracellular selectivity filter by more than three orders of magnitude for the full-length wild-type KcsA, a pH-gated bacterial channel, in membrane bilayers. Studies of F103 in the hinge of the inner helix suggest an important role for its bulky sidechain in the allosteric mechanism; we show that the energetic strength of coupling of the gates is strongly altered when this residue is mutated to alanine. These results provide quantitative site-specific measurements of allostery in a bilayer environment, and highlight the power of describing ion channel gating through the lens of allosteric coupling.

Structure and Function of the Transmembrane Domain of NsaS, an Antibiotic Sensing Histidine Kinase in Staphylococcus aureus.

NsaS is one of four intramembrane histidine kinases (HKs) in Staphylococcus aureus that mediate the pathogen's response to membrane active antimicrobials and human innate immunity. We describe the first integrative structural study of NsaS using a combination of solution state NMR spectroscopy, chemical-cross-linking, molecular modeling and dynamics. Three key structural features emerge: First, NsaS has a short N-terminal amphiphilic helix that anchors its transmembrane (TM) bundle into the inner leaflet of the membrane such that it might sense neighboring proteins or membrane deformations. Second, the transmembrane domain of NsaS is a 4-helix bundle with significant dynamics and structural deformations at the membrane interface. Third, the intracellular linker connecting the TM domain to the cytoplasmic catalytic domains of NsaS is a marginally stable helical dimer, with one state likely to be a coiled-coil. Data from chemical shifts, heteronuclear NOE, H/D exchange measurements and molecular modeling suggest that this linker might adopt different conformations during antibiotic induced signaling.

Transmembrane allosteric coupling of the gates in a potassium channel.

It has been hypothesized that transmembrane allostery is the basis for inactivation of the potassium channel KcsA: opening the intracellular gate is spontaneously followed by ion expulsion at the extracellular selectivity filter. This suggests a corollary: following ion expulsion at neutral pH, a spontaneous global conformation change of the transmembrane helices, similar to the motion involved in opening, is expected. Consequently, both the low potassium state and the low pH state of the system could provide useful models for the inactivated state. Unique NMR studies of full-length KcsA in hydrated bilayers provide strong evidence for such a mutual coupling across the bilayer: namely, upon removing ambient potassium ions, changes are seen in the NMR shifts of carboxylates E118 and E120 in the pH gate in the hinges of the inner transmembrane helix (98-103), and in the selectivity filter, all of which resemble changes seen upon acid-induced opening and inhibition and suggest that ion release can trigger channel helix opening.

Protonation state of E71 in KcsA and its role for channel collapse and inactivation.

The prototypical prokaryotic potassium channel KcsA alters its pore depending on the ambient potassium; at high potassium, it exists in a conductive form, and at low potassium, it collapses into a nonconductive structure with reduced ion occupancy. We present solid-state NMR studies of KcsA in which we test the hypothesis that an important channel-inactivation process, known as C-type inactivation, proceeds via a state similar to this collapsed state. We test this using an inactivation-resistant mutant E71A, and show that E71A is unable to collapse its pore at both low potassium and low pH, suggesting that the collapsed state is structurally similar to the inactivated state. We also show that E71A has a disordered selectivity filter. Using site-specific K(+) titrations, we detect a local change at E71 that is coupled to channel collapse at low K(+). To gain more insight into this change, we site specifically measure the chemical shift tensors of the side-chain carboxyls of E71 and its hydrogen bond partner D80, and use the tensors to assign protonation states to E71 and D80 at high K(+) and neutral pH. Our measurements show that E71 is protonated at pH 7.5 and must have an unusually perturbed pK(a) (> 7.5) suggesting that the change at E71 is a structural rearrangement rather than a protonation event. The results offer new mechanistic insights into why the widely used mutant KcsA-E71A does not inactivate and establish the ambient K(+) level as a means to populate the inactivated state of KcsA in a controlled way.

Regulatory phosphorylation induces extracellular conformational changes in a CLC anion channel.

CLH-3b is a CLC-1/2/Ka/Kb channel homolog activated by meiotic cell cycle progression and cell swelling. Channel inhibition occurs by GCK-3 kinase-mediated phosphorylation of serine residues on the cytoplasmic C-terminus linker connecting CBS1 and CBS2. Two conserved aromatic amino acid residues located on the intracellular loop connecting membrane helices H and I and α1 of CBS2 are required for transducing phosphorylation changes into changes in channel activity. Helices H and I form part of the interface between the two subunits that comprise functional CLC channels. Using a cysteine-less CLH-3b mutant, we demonstrate that the sulfhydryl reagent reactivity of substituted cysteines at the subunit interface changes dramatically during GCK-3-mediated channel inhibition and that these changes are prevented by mutation of the H-I loop/CBS2 α1 signal transduction domain. We also show that GCK-3 modifies Zn(2+) inhibition, which is thought to be mediated by the common gating process. These and other results suggest that phosphorylation of the cytoplasmic C-terminus inhibits CLH-3b by inducing subunit interface conformation changes that activate the common gate. Our findings have important implications for understanding CLC regulation by diverse signaling mechanisms and for understanding the structure/function relationships that mediate intraprotein communication in this important family of Cl(-) transport proteins.

Preparation of uniformly isotope labeled KcsA for solid state NMR: expression, purification, reconstitution into liposomes and functional assay.

We report the expression, purification, liposome reconstitution and functional validation of uniformly (13)C and (15)N isotope labeled KcsA, a bacterial potassium channel that has high homology with mammalian channels, for solid-state NMR studies. The expression and purification is optimized for an average yield of ∼35-40mg/L of M9 media in a time-efficient way. The protein purity is confirmed by gel electrophoresis and the protein concentration is quantified by UV-vis absorption spectroscopy. Protocols to efficiently reconstitute KcsA into liposomes are also presented. The presence of liposomes is confirmed by cryo-electron microscopy images and the effect of magic angle spinning on liposome packing is shown. High-resolution solid-state NMR spectra of uniformly isotope labeled KcsA in these liposomes reveal that our protocol yields to a very homogenous KcsA sample with high signal to noise and several well-resolved residues in NMR spectra. Electrophysiology of our samples before and after solid-state NMR show that channel function and selectivity remain intact after the solid-state NMR.

Solvation and hydrogen bonding in alanine- and glycine-containing dipeptides probed using solution- and solid-state NMR spectroscopy.

The NMR chemical shift is a sensitive reporter of peptide secondary structure and its solvation environment, and it is potentially rich with information about both backbone dihedral angles and hydrogen bonding. We report results from solution- and solid-state (13)C and (15)N NMR studies of four zwitterionic model dipeptides, L-alanyl-L-alanine, L-alanyl-glycine, glycyl-L-alanine, and glycyl-glycine, in which we attempt to isolate structural and environmental contributions to the chemical shift. We have mapped hydrogen-bonding patterns in the crystalline states of these dipeptides using the published crystal structures and correlated them with (13)C and (15)N magic angle spinning chemical shift data. To aid in the interpretation of the solvated chemical shifts, we performed ab initio quantum chemical calculations to determine the low-energy conformers and their chemical shifts. Assuming low energy barriers to interconversion between thermally accessible conformers, we compare the Boltzmann-averaged chemical shifts with the experimentally determined solvated-state shifts. The results allow us to correlate the observed differences in chemical shifts between the crystalline and solvated states to changes in conformation and hydrogen bonding that occur upon solvation.

Signal transduction in histidine kinases: insights from new structures.

Histidine kinases (HKs) are major players in bacterial signaling. There has been an explosion of new HK crystal structures in the last 5 years. We globally analyze the structures of HKs to yield insights into the mechanisms by which signals are transmitted to and across protein structures in this family. We interpret known enzymological data in the context of new structural data to show how asymmetry across the dimer interface is a key feature of signal transduction in HKs, and discuss how different HK domains undergo asymmetric to symmetric transitions during signal transduction and catalysis. A thermodynamic framework for signaling that encompasses these various properties is presented, and the consequences of weak thermodynamic coupling are discussed. The synthesis of observations from enzymology, structural biology, protein engineering, and thermodynamics paves the way for a deeper molecular understanding of HK signal transduction.

De novo design of a transmembrane Zn²âº-transporting four-helix bundle.

The design of functional membrane proteins from first principles represents a grand challenge in chemistry and structural biology. Here, we report the design of a membrane-spanning, four-helical bundle that transports first-row transition metal ions Zn(2+) and Co(2+), but not Ca(2+), across membranes. The conduction path was designed to contain two di-metal binding sites that bind with negative cooperativity. X-ray crystallography and solid-state and solution nuclear magnetic resonance indicate that the overall helical bundle is formed from two tightly interacting pairs of helices, which form individual domains that interact weakly along a more dynamic interface. Vesicle flux experiments show that as Zn(2+) ions diffuse down their concentration gradients, protons are antiported. These experiments illustrate the feasibility of designing membrane proteins with predefined structural and dynamic properties.

Conformational dynamics in the selectivity filter of KcsA in response to potassium ion concentration.

Conformational change in the selectivity filter of KcsA as a function of ambient potassium concentration is studied with solid-state NMR. This highly conserved region of the protein is known to chelate potassium ions selectively. We report solid-state NMR chemical shift fingerprints of two distinct conformations of the selectivity filter; significant changes are observed in the chemical shifts of key residues in the filter as the potassium ion concentration is changed from 50 mM to 1 muM. Potassium ion titration studies reveal that the site-specific K(d) for K(+) binding at the key pore residue Val76 is on the order of approximately 7 muM and that a relatively high sample hydration is necessary to observe the low-K(+) conformer. Simultaneous detection of both conformers at low ambient potassium concentration suggests that the high-K(+) and low-K(+) states are in slow exchange on the NMR timescale (k(ex)<500 s(-)(1)). The slow rate and tight binding for evacuating both inner sites simultaneously differ from prior observations in detergent in solution, but agree well with measurements by electrophysiology and appear to result from our use of a hydrated bilayer environment. These observations strongly support a common assumption that the low-K(+) state is not involved in ion transmission, and that during transmission one of the two inner sites is always occupied. On the other hand, these kinetic and thermodynamic characteristics of the evacuation of the inner sites certainly could be compatible with participation in a control mechanism at low ion concentration such as C-type inactivation, a process that is coupled to activation and involves closing of the outer mouth of the channel.