Structure, Dynamics and Implied Gating Mechanism of a Human Cyclic Nucleotide-Gated Channel

Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels, essential for visual and olfactory sensory transduction. Although the channels include voltage-sensor domains (VSDs), their conductance is thought to be independent of the membrane potential, and their gating regulated by cytosolic cyclic nucleotide–binding domains. Mutations in these channels result in severe, degenerative retinal diseases, which remain untreatable. The lack of structural information on CNG channels has prevented mechanistic understanding of disease-causing mutations, precluded structure-based drug design, and hampered in silico investigation of the gating mechanism. To address this, we built a 3D model of the cone tetrameric CNG channel, based on homology to two distinct templates with known structures: the transmembrane (TM) domain of a bacterial channel, and the cyclic nucleotide-binding domain of the mouse HCN2 channel. Since the TM-domain template had low sequence-similarity to the TM domains of the CNG channels, and to reconcile conflicts between the two templates, we developed a novel, hybrid approach, combining homology modeling with evolutionary coupling constraints. Next, we used elastic network analysis of the model structure to investigate global motions of the channel and to elucidate its gating mechanism. We found the following: (i) In the main mode of motion, the TM and cytosolic domains counter-rotated around the membrane normal. We related this motion to gating, a proposition that is supported by previous experimental data, and by comparison to the known gating mechanism of the bacterial KirBac channel. (ii) The VSDs could facilitate gating (supplementing the pore gate), explaining their presence in such ‘voltage-insensitive’ channels. (iii) Our elastic network model analysis of the CNGA3 channel supports a modular model of allosteric gating, according to which protein domains are quasi-independent: they can move independently, but are coupled to each other allosterically

Acquisition of MRSA Through Sexual Transmission and Treatment of Carrier Status: A Case Report

Introduction. Community acquired Methicillin resistant Staphylococcus aureus (CA-MRSA), a major cause of cutaneous and systemic infection, is becoming increasingly prevalent. Cook et al found heterosexual transmission of CA MRSA in three households. These results are important because sexual intercourse appears to be a relatively new and significant vehicle for the transmission of CA MRSA among family members and to the rest of the community. Case Report. 22 year-old immunocompetent male presented asymptomatic but requested a test for MRSA since his girlfriend, whom he recently had sexual relations with, had gluteal lesions, which were previously diagnosed as MRSA. A thorough physical exam revealed no abnormalities but a nasal swab was taken and sent to the lab as a precaution. Within 5 days, the lab report came back positive for MRSA and Group B Streptococcus species. Presumably, the patient acquired MRSA through vaginal contact (i.e. oral sex), evidenced by the Group B colonization of nasal mucosa. The patient was given a prescription for Mupirocin 2% Ointment. Five days later, he was free of MRSA. Discussion. Awareness of the possibility of sexual transmission of MRSA is essential to containment of the organism. This case showed that MRSA can be transmitted through oral sex. Although there has not been major research work on the subject, results have shown that when MRSA is sexually transmitted, the isolates are unrecognizable strains and potentially invasive. In fact, this novel form of transmission may explain the ability of new strains to become established in the community

The Transmembrane Helix Tilt May Be Determined by the Balance between Precession Entropy and Lipid Perturbation

Hydrophobic helical peptides interact with lipid bilayers in various modes, determined by the match between the length of the helix’s hydrophobic core and the thickness of the hydrocarbon region of the bilayer. For example, long helices may tilt with respect to the membrane normal to bury their hydrophobic cores in the membrane, and the lipid bilayer may stretch to match the helix length. Recent molecular dynamics simulations and potential of mean force calculations have shown that some TM helices whose lengths are equal to, or even shorter than, the bilayer thickness may also tilt. The tilt is driven by a gain in the helix precession entropy, which compensates for the free energy penalty resulting from membrane deformation. Using this free energy balance, we derived theoretically an equation of state, describing the dependence of the tilt on the helix length and membrane thickness. To this end, we conducted coarse-grained Monte Carlo simulations of the interaction of helices of various lengths with lipid bilayers of various thicknesses, reproducing and expanding the previous molecular dynamics simulations. Insight from the simulations facilitated the derivation of the theoretical model. The tilt angles calculated using the theoretical model agree well with our simulations and with previous calculations and measurements

Membrane integration of a mitochondrial signal-anchored protein does not require additional proteinaceous factors

The MOM (mitochondrial outer membrane) contains SA (signal-anchored) proteins that bear at their N-terminus a single hydrophobic segment that serves as both a mitochondrial targeting signal and an anchor at the membrane. These proteins, like the vast majority of mitochondrial proteins, are encoded in the nucleus and have to be imported into the organelle. Currently, the mechanisms by which they are targeted to and inserted into the OM (outer membrane) are unclear. To shed light on these issues, we employed a recombinant version of the SA protein OM45 and a synthetic peptide corresponding to its signal-anchor segment. Both forms are associated with isolated mitochondria independently of cytosolic factors. Interaction with mitochondria was diminished when a mutated form of the signal-anchor was employed. We demonstrate that the signal-anchor peptide acquires an α-helical structure in a lipid environment and adopted a TM (transmembrane) topology within artificial lipid bilayers. Moreover, the peptide’s affinity to artificial membranes with OM-like lipid composition was much higher than that of membranes with ER (endoplasmic reticulum)-like lipid composition. Collectively, our results suggest that SA proteins are specifically inserted into the MOM by a process that is not dependent on additional proteins, but is rather facilitated by the distinct lipid composition of this membrane.

A combined pulse EPR and Monte Carlo simulation study provides molecular insight on peptide−membrane interactions

We present a new approach to obtain details on the distribution and average structure and locations of membrane−associated peptides. The approach combines (i) pulse double electron−electron resonance (DEER) to determine intramolecular distances between residues in spin labeled peptides, (ii) electron spin echo envelope modulation (ESEEM) experiments to measure water exposure and the direct interaction of spin labeled peptides with deuterium nuclei on the phospholipid molecules, and (iii) Monte Carlo (MC) simulations to derive the peptide−membrane populations, energetics, and average conformation of the native peptide and mutants mimicking the spin labeling. To demonstrate the approach, we investigated the membrane−bound and solution state of the well−known antimicrobial peptide melittin, used as a model system. A good agreement was obtained between the experimental results and the MC simulations regarding the distribution of distances between the labeled amino acids, the side chain mobility, and the peptide's orientation. A good agreement in the extent of membrane penetration of amino acids in the peptide core was obtained as well, but the EPR data reported a somewhat deeper membrane penetration of the termini compared to the simulations. Overall, melittin adsorbed on the membrane surface, in a monomeric state, as an amphipatic helix with its hydrophobic residues in the hydrocarbon region of the membrane and its charged and polar residues in the lipid headgroup region

Modeling of the full-length cone channel in its resting state, on the basis of two separate templates.

<p>(A) Side view of the TM region of the bacterial channel MlotiK1 in a closed state, PDB entry 3BEH <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003976#pcbi.1003976-Clayton1" target="_blank">[10]</a>. This structure was the template for modeling the TM region of the cone channel. (B) Side view of the mouse HCN2 CNBDs in a resting state, PDB entry 1Q3E <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003976#pcbi.1003976-Zagotta1" target="_blank">[15]</a>. This structure served as a template for the cytosolic domain of the cone channel. (A, B) The regions of conflict between the templates are in red. (C) Side view of the resultant model structure of the human cone channel, shown in cartoon representation. CNGA3 subunits are colored cyan (light and dark); CNGB3 subunits are colored orange (light and dark).</p

Motion I of CNGA3 appears to describe channel gating.

<p>For clarity, only helices S5 and S6 (or corresponding KirBac helices) of two juxtaposed subunits are shown in each panel. (A, B) The similarity between the predicted conformations in panel B and the crystal structures in panel A is apparent, verifying the relation between these conformations and channel gating. (A) Side view of the KirBac3.1 crystal structures in open (pale green, PDB entry 3ZRS <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003976#pcbi.1003976-Bavro1" target="_blank">[43]</a>) and closed (pale red, PDB entry 2WLJ <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003976#pcbi.1003976-Clarke1" target="_blank">[44]</a>) states. The α-carbons of the two gate-residues, namely G120 and Y132, are shown as a space-filling model. (B–D) The edge conformations of KirBac3.1 (B), CNGA3 (C) and CNGA3 lacking the VSDs (D), as predicted by the elastic network models in the slowest mode of motion. The two edge conformations are shown in red and green. (C) The CNGA3 conformations resemble the conformations predicted for the KirBac channel (panel B), but the pore region is rigid. (D) The CNGA3 without VSD conformations are identical to the KirBac conformations (panel B).</p

Evolutionary coupling (EC) calculations support the model.

<p>Contact maps of top-ranked predicted ECs (red) overlaid on monomer (light grey) and intermonomer (dark grey) contacts from (A) the model structure of the TM domain of CNGA3; (B) the model structure of the cytosolic domain of CNGA3. The insets show the contact maps of alternative CNGA3 model structures; the orange arrows point to evolutionary couplings between amino acid pairs that are not in contact in the alternative models, but are in contact in our final model. Clearly, the overlay of the ECs with the contact map is far better for the chosen model structure than for the alternatives.</p