Transmembrane Helical Segments Research Articles

Comparing 2 crystal structures and 12 AlphaFold2-predicted human membrane glucose transporters and their water-soluble glutamine, threonine and tyrosine variants.

Membrane transporters including glucose transporters (GLUTs) are involved in cellular energy supplies, cell metabolism and other vital biological activities. They have also been implicated in cancer proliferation and metastasis, thus they represent an important target in combatting cancer. However, membrane transporters are very difficult to study due to their multispan transmembrane properties. The new computational tool, AlphaFold2, offers highly accurate predictions of three-dimensional protein structures. The glutamine, threonine and tyrosine (QTY) code provides a systematic method of rendering hydrophobic sequences into hydrophilic ones. Here, we present computational studies of native integral membrane GLUTs with 12 transmembrane helical segments determined by X-ray crystallography and CryoEM, comparing the AlphaFold2-predicted native structure to their water-soluble QTY variants predicted by AlphaFold2. In the native structures of the transmembrane helices, there are hydrophobic amino acids leucine (L), isoleucine (I), valine (V) and phenylalanine (F). Applying the QTY code, these hydrophobic amino acids are systematically replaced by hydrophilic amino acids, glutamine (Q), threonine (T) and tyrosine (Y) rendering them water-soluble. We present the superposed structures of native GLUTs and their water-soluble QTY variants. The superposed structures show remarkable similar residue mean square distance values between 0.47 and 3.6Å (most about 1-2Å) despite >44% transmembrane amino acid differences. We also show the differences of hydrophobicity patches between the native membrane transporters and their QTY variants. We explain the rationale why the membrane protein QTY variants become water-soluble. Our study provides insight into the differences between the hydrophobic helices and hydrophilic helices, and offers confirmation of the QTY method for studying multispan transmembrane proteins and other aggregated proteins through their water-soluble variants.

Analysis of M4 Transmembrane Segments in NMDA Receptor Function: A Negative Allosteric Modulatory Site at the GluN1 M4 is Determining the Efficiency of Neurosteroid Modulation.

Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated ion channels that play a crucial role in excitatory synaptic transmission in the central nervous system. Each subunit contributes with three helical transmembrane segments (M1, M3, and M4) and a pore loop (M2) to form the channel pore. Recent studies suggest that the architecture of all eukaryotic iGluRs derives from a common prokaryotic ancestral receptor that lacks M4 and consists only of transmembrane segments M1 and M3. Although significant contribution has emerged in the last years, the role of this additionally evolved transmembrane segment in iGluR assembly and function remains unclear. Here, we have investigated how deletions and mutations of M4 in members of the NMDA receptor (NMDAR) subfamily, the conventional heteromeric GluN1/GluN2 and glycine-gated GluN1/GluN3 NMDARs, affect expression and function in Xenopus oocytes. We show that deletion of M4 in the GluN1, GluN2A, or GluN3A subunit, despite retained receptor assembly and cell surface expression, results in nonfunctional membrane receptors. Coexpression of the corresponding M4 as an isolated peptide in M4-deleted receptors rescued receptor function of GluN1/GluN2A NMDARs without altering the apparent affinity of glutamate or glycine. Electrophysiological analyses of agonist-induced receptor function and its modulation by the neurosteroid pregnenolone sulfate (PS) at mutations of the GluN1-M4/GluN2/3-transmembrane interfaces indicate a crucial role of position M813 in M4 of GluN1 for functional coupling to the core receptor and the negative modulatory effects of PS. Substitution of residues and insertion of interhelical disulfide bridges confirmed interhelical interactions of positions in M4 of GluN1 with residues of transmembrane segments of neighboring subunits. Our results show that although M4s in NMDARs are not important for receptor assembly and surface expression, the residues at the subunit interface are substantially involved in M4 recognition of the core receptor and regulation of PS efficacy. Because mutations in the M4 of GluN1 specifically resulted in loss of PS-induced inhibition of GluN1/GluN2A and GluN1/GluN3A NMDAR currents, our results point to distinct roles of M4s in NMDAR modulation and highlight the importance of the evolutionarily newly evolved M4 for selective in vivo modulation of glutamate- and glycine-activated NMDARs by steroids.

NADPH oxidase involved in immune response via regulating the expression of antioxidant genes in Litopenaeus vannamei

Free radicals such as reactive oxygen species (ROS) play a significant role in the innate immune system. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Noxes) are found to be involved in ROS production and played crucial roles in host biological processes of humans, insects, and plants. However, there are relatively few studies on the function of Noxes in crustaceans. In this study, the full-length cDNA of Nox was cloned from Litopenaeus vannamei by rapid amplification of cDNA ends (RACE) to obtain a 3790 base pairs (bp) cDNA sequence. LvNox cDNA comprised a 5’-untranslated region (UTR) of 454 bp, a 3’-UTR of 333 bp, and a putative open reading frame (ORF) of 3006 bp. Structural analysis showed that LvNox had a transmembrane helical segment (THS) at positions 233-255, a flavin adenine dinucleotide (FAD) binding domain (at positions 447-579), and a nicotinamide adenine dinucleotide (NAD) binding domain (at positions 815-990). Tissue distribution analysis revealed that LvNox mRNA was expressed in heart, intestine, muscle, gill, hepatopancreas, stomach, epidermis, hemocyte, and eyestalk. High expression levels of LvNox were observed in epidermis, hemocyte and eyestalk. The expression levels of LvNox were significantly induced after Vibrio alginolyticus injection for 6, 12, 24 and 48 h, and after Cu exposure for 24 and 48 h. Furthermore, the RNA interference of LvNox significantly reduced the production of ROS and the expression levels of copper/zinc superoxide dismutase (Cu-Zn SOD), catalase (CAT) and nitric oxide synthase (NOS). These results indicated that LvNox could be induced by V. alginolyticus infection and Cu exposure, which might be involved in immune response of shrimp via regulating the expression of antioxidant genes. These findings provide a solid foundation for further study of the function of Noxes in crustaceans.

Interaction fingerprint of transmembrane segments in voltage sensor domains

Voltage sensor domain (VSD) in channel and non-channel membrane proteins shares a common function in the detection of changes in the transmembrane electric potential. The VSD is made of four helical transmembrane segments (S1–S4) that form a structurally conserved scaffold through inter-transmembrane residue-residue interactions. Details about these interactions are yet to be fully understood in the context of the unique structural and physical characteristics of the voltage sensor unit. In this study, molecular dynamics simulations were carried out to investigate transmembrane helix-helix interactions via residue-based nonbonding energies using the activated and resting state conformations of VSD from Hv1, CiVSP, KvAP and NavAb. Inter-transmembrane interaction energies within the VSD were determined. Analysis of electrostatic and van der Waals components revealed the strengths and weaknesses of the interactions between each pair of transmembrane segments. In all cases the S4 helix had the highest electrostatic contribution to favor the key role as the voltage sensitive segment. Electrostatic interactions for the S1–S2 pair as well as the S1–S3 pair were relatively weak. Van der Waal interaction energies between adjacent segments were on average greater than that between diagonally opposite segments. Salt bridge interactions between S4-arginines and the negatively charged residues in other segments appear to contribute more to stabilizing the energy than the van der Waals interactions between nonpolar residues. The overall behavior of residue-residue contacts is similar among the transmembrane domains, reflecting the common inter- transmembrane interaction pattern in the VSD. In addition, analysis of the residue positions suggested that subtle differences in the orientation of the salt-bridges can be attributed to the difference in the inter-transmembrane interaction strengths inside the VSDs.

COVID-2019: The role of the nsp2 and nsp3 in its pathogenesis.

Last December 2019, a new virus, named novel Coronavirus (COVID‐2019) causing many cases of severe pneumonia was reported in Wuhan, China. The virus knowledge is limited and especially about COVID‐2019 pathogenesis. The Open Reading Frame 1ab (ORF1ab) of COVID‐2019 has been analyzed to evidence the presence of mutation caused by selective pressure on the virus. For selective pressure analysis fast‐unconstrained Bayesian approximation (FUBAR) was used. Homology modelling has been performed by SwissModel and HHPred servers. The presence of transmembrane helical segments in Coronavirus ORF1ab non structural protein 2 (nsp2) and nsp3 was tested by TMHMM, MEMSAT, and MEMPACK tools. Three‐dimensional structures have been analyzed and displayed using PyMOL. FUBAR analysis revealed the presence of potential sites under positive selective pressure (P < .05). Position 723 in the COVID‐2019 has a serine instead a glycine residue, while at aminoacidic position 1010 a proline instead an isoleucine. Significant (P < .05) pervasive negative selection in 2416 sites (55%) was found. The positive selective pressure could account for some clinical features of this virus compared with severe acute respiratory syndrome (SARS) and Bat SARS‐like CoV. The stabilizing mutation falling in the endosome‐associated‐protein‐like domain of the nsp2 protein could account for COVID‐2019 high ability of contagious, while the destabilizing mutation in nsp3 proteins could suggest a potential mechanism differentiating COVID‐2019 from SARS. These data could be helpful for further investigation aimed to identify potential therapeutic targets or vaccine strategy, especially in the actual moment when the epidemic is ongoing and the scientific community is trying to enrich knowledge about this new viral pathogen.

Characterizing the Alternative Splicing Dynamics of Cav1.3, a Calcium Ion Channel Involved in Autism Spectrum Disorder

Voltage‐gated calcium channels move calcium ions across cellular membranes in response to action potential stimuli, which mediate processes including cellular signaling, neurotransmitter discharge, muscle contraction, and gene expression. They are multimer proteins composed of alpha‐1, beta, alpha‐2/delta, and gamma subunits. Variety in calcium channels is afforded by different subtypes of alpha‐1, including the A, B, C, D, E, and S isoforms.Cav1.3 is a member of the L‐type calcium channels and is categorized in the high‐voltage activated family. It employs the alpha‐1 subunit encoded by the CACNA1D gene. The alpha‐1 subunit forms four homologous repeat domains, each consisting of six transmembrane helical segments. Cacna1d knock out mice suffer from bradycardia and congenital deafness, testifying to the significance of the alpha‐1 subunit in the sinoatrial node and in auditory hair cells. Naturally occurring missense mutations in alternatively spliced regions of CACNA1D, such as the mutually exclusive exons 8a and 8b encoding part of the sixth transmembrane segment of domain I, have been reported to be gain‐of‐function mutations, which cause aldosterone‐producing adenomas, aldosteronism, seizures, and autism spectrum disorder, highlighting the importance of alternative splicing for Cav1.3 function.Although the diseases derived from CACNA1D mutations have been characterized, the splicing dynamics of the CACNA1D pre‐mRNA that gives rise to the different alpha‐1 isoforms is not well understood. Here, we describe the alternatively spliced isoforms of Cacna1d and their distribution in mouse tissues.Support or Funding InformationNational Institute of General Medical Sciences Grant 1R15GM119099‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Main-chain mutagenesis reveals intrahelical coupling in an ion channel voltage-sensor

Membrane proteins are universal signal decoders. The helical transmembrane segments of these proteins play central roles in sensory transduction, yet the mechanistic contributions of secondary structure remain unresolved. To investigate the role of main-chain hydrogen bonding on transmembrane function, we encoded amide-to-ester substitutions at sites throughout the S4 voltage-sensing segment of Shaker potassium channels, a region that undergoes rapid, voltage-driven movement during channel gating. Functional measurements of ester-harboring channels highlight a transitional region between α-helical and 310 segments where hydrogen bond removal is particularly disruptive to voltage-gating. Simulations of an active voltage sensor reveal that this region features a dynamic hydrogen bonding pattern and that its helical structure is reliant upon amide support. Overall, the data highlight the specialized role of main-chain chemistry in the mechanism of voltage-sensing; other catalytic transmembrane segments may enlist similar strategies in signal transduction mechanisms.

Roughness of Transmembrane Helices Reduces Lipid Membrane Dynamics

SummaryThe dynamics of cellular membranes is primarily determined by lipid species forming a bilayer. Proteins are considered mainly as effector molecules of diverse cellular processes. In addition to large assemblies of proteins, which were found to influence properties of fluid membranes, biological membranes are densely populated by small, highly mobile proteins. However, little is known about the effect of such proteins on the dynamics of membranes. Using synthetic peptides, we demonstrate that transmembrane helices interfere with the mobility of membrane components by trapping lipid acyl chains on their rough surfaces. The effect is more pronounced in the presence of cholesterol, which segregates from the rough surface of helical peptides. This may contribute to the formation or stabilization of membrane heterogeneities. Since roughness is a general property of helical transmembrane segments, our results suggest that, independent of their size or cytoskeleton linkage, integral membrane proteins affect local membrane dynamics and organization.

DbSWEET: An Integrated Resource for SWEET Superfamily to Understand, Analyze and Predict the Function of Sugar Transporters in Prokaryotes and Eukaryotes

SWEET (Sweet Will Eventually be Exported Transporter) proteins have been recently discovered and form one of the three major families of sugar transporters. Homologs of SWEET are found in both prokaryotes and eukaryotes. Bacterial SWEET homologs have three transmembrane segments forming a triple-helical bundle and the functional form is dimers. Eukaryotic SWEETs have seven transmembrane helical segments forming two triple-helical bundles with a linker helix. Members of SWEET homologs have been shown to be involved in several important physiological processes in plants. However, not much is known regarding the biological significance of SWEET homologs in prokaryotes and in mammals. We have collected more than 2000 SWEET homologs from both prokaryotes and eukaryotes. For each homolog, we have modeled three different conformational states representing outward open, inward open and occluded states. We have provided details regarding substrate-interacting residues and residues forming the selectivity filter for each SWEET homolog. Several search and analysis options are available. The users can generate a phylogenetic tree and structure-based sequence alignment for selected set of sequences. With no metazoan SWEETs functionally characterized, the features observed in the selectivity filter residues can be used to predict the potential substrates that are likely to be transported across the metazoan SWEETs. We believe that this database will help the researchers to design mutational experiments and simulation studies that will aid to advance our understanding of the physiological role of SWEET homologs. This database is freely available to the scientific community at http://bioinfo.iitk.ac.in/bioinfo/dbSWEET/Home.

Energy landscape of LeuT from molecular simulations.

The bacterial sodium-coupled leucine transporter (LeuT) has been broadly used as a structural model for understanding the structure-dynamics-function of mammalian neurotransmitter transporters as well as other solute carriers that share the same fold (LeuT fold), as the first member of the family crystallographically resolved in multiple states: outward-facing open, outward-facing occluded, and inward-facing open. Yet, a complete picture of the energy landscape of (sub)states visited along the LeuT transport cycle has been elusive. In an attempt to visualize the conformational spectrum of LeuT, we performed extensive simulations of LeuT dimer dynamics in the presence of substrate (Ala or Leu) and co-transported Na(+) ions, in explicit membrane and water. We used both conventional molecular dynamics (MD) simulations (with Anton supercomputing machine) and a recently introduced method, collective MD, that takes advantage of collective modes of motions predicted by the anisotropic network model. Free energy landscapes constructed based on ∼40 μs trajectories reveal multiple substates occluded to the extracellular (EC) and/or intracellular (IC) media, varying in the levels of exposure of LeuT to EC or IC vestibules. The IC-facing transmembrane (TM) helical segment TM1a shows an opening, albeit to a smaller extent and in a slightly different direction than that observed in the inward-facing open crystal structure. The study provides insights into the spectrum of conformational substates and paths accessible to LeuT and highlights the differences between Ala- and Leu-bound substates.

NhaA: A Unique Structural Fold of Secondary Active Transporters

AbstractThe Na+/H+ antiporters transport sodium (or several other monovalent cations) in exchange for H+ across lipid bilayers in all kingdoms of life. They are critical in the pH homeostasis of cytoplasm and/or organelles. In humans, these proteins are associated with the pathophysiology of various diseases. Yet, the most extensively studied Na+/H+ antiporter is Ec‐NhaA, the main Na+/H+ antiporter of Escherichia coli. The crystal structure of an inactive, down‐regulated Ec‐NhaA, determined at acidic pH, has provided the first structural insights into the antiport mechanism and pH regulation of an Na+/H+ antiporter. It reveals a unique structural fold (called the NhaA fold) in which helical transmembrane segments (TMs) are organized in inverted‐topology repeats, including two antiparallel non‐helical extended chain regions that cross each other, forming a delicate electrostatic balance in the middle of the membrane. The NhaA fold contributes to the cation binding site and facilitates rapid conformational changes of Ec‐NhaA. The NhaA fold has now been recognized to be shared by four Na+/H+ antiporters (bacterial and archaeal) and two Na+ symporters. Thus far, no crystal structure of any of the human Na+/H+ antiporters has been determined. Nevertheless, the Ec‐NhaA crystal structure has enabled the structural modeling of NHE1, NHE9, and NHA2, which are involved in human pathophysiology. Future elucidation of structure‐function relationships of eukaryotic and prokaryotic Na+/H+ antiporters are likely to provide insights into the human pathophysiology. Here, we will focus on the NhaA structural fold which underpins the antiporter functionality of Ec‐NhaA and the secondary active transporters.

Dynamics of the Plasma Membrane Proton Pump.

Proton transfer over distances longer than that of a hydrogen bond often requires water molecules and protein motions. Following transfer of the proton from the donor to the acceptor, the change in the charge distribution may alter the dynamics of protein and water. To begin to understand how protonation dynamics couple to protein and water dynamics, here we explore how changes in the protonation state affect water and protein dynamics in the AHA2 proton pump. We find that the protonation state of the proton donor and acceptor groups largely affects the dynamics of internal waters and of specific hydrogen bonds, and the orientation of transmembrane helical segments that couple remote regions of the protein. The primary proton donor/acceptor group D684, can interact with water molecules from the cytoplasmic bulk and/or other protein groups.

Aromatic–aromatic interactions between residues in KCa3.1 pore helix and S5 transmembrane segment control the channel gating process

The Ca(2+)-activated potassium channel KCa3.1 is emerging as a therapeutic target for a large variety of health disorders. One distinguishing feature of KCa3.1 is that the channel open probability at saturating Ca(2+) concentrations (Pomax) is low, typically 0.1-0.2 for KCa3.1 wild type. This observation argues for the binding of Ca(2+) to the calmodulin (CaM)-KCa3.1 complex, promoting the formation of a preopen closed-state configuration leading to channel opening. We have previously shown that the KCa3.1 active gate is most likely located at the level of the selectivity filter. As Ca(2+)-dependent gating of KCa3.1 originates from the binding of Ca(2+) to CaM in the C terminus, the hypothesis of a gate located at the level of the selectivity filter requires that the conformational change initiated in the C terminus be transmitted to the S5 and S6 transmembrane helices, with a resulting effect on the channel pore helix directly connected to the selectivity filter. A study was thus undertaken to determine to what extent the interactions between the channel pore helix with the S5 and S6 transmembrane segments contribute to KCa3.1 gating. Molecular dynamics simulations first revealed that the largest contact area between the pore helix and the S5 plus S6 transmembrane helices involves residue F248 at the C-terminal end of the pore helix. Unitary current recordings next confirmed that modulating aromatic-aromatic interactions between F248 and W216 of the S5 transmembrane helical segment and/or perturbing the interactions between F248 and residues in S6 surrounding the glycine hinge G274 cause important changes in Pomax. This work thus provides the first evidence for a key contribution of the pore helix in setting Pomax by stabilizing the channel closed configuration through aromatic-aromatic interactions involving F248 of the pore helix. We propose that the interface pore helix/S5 constitutes a promising site for designing KCa3.1 potentiators.

Direct Evidence that Scorpion α-Toxins (Site-3) Modulate Sodium Channel Inactivation by Hindrance of Voltage-Sensor Movements

The position of the voltage-sensing transmembrane segment, S4, in voltage-gated ion channels as a function of voltage remains incompletely elucidated. Site-3 toxins bind primarily to the extracellular loops connecting transmembrane helical segments S1-S2 and S3-S4 in Domain 4 (D4) and S5-S6 in Domain 1 (D1) and slow fast-inactivation of voltage-gated sodium channels. As S4 of the human skeletal muscle voltage-gated sodium channel, hNav1.4, moves in response to depolarization from the resting to the inactivated state, two D4S4 reporters (R2C and R3C, Arg1451Cys and Arg1454Cys, respectively) move from internal to external positions as deduced by reactivity to internally or externally applied sulfhydryl group reagents, methane thiosulfonates (MTS). The changes in reporter reactivity, when cycling rapidly between hyperpolarized and depolarized voltages, enabled determination of the positions of the D4 voltage-sensor and of its rate of movement. Scorpion α-toxin binding impedes D4S4 segment movement during inactivation since the modification rates of R3C in hNav1.4 with methanethiosulfonate (CH3SO2SCH2CH2R, where R = -N(CH3)3 + trimethylammonium, MTSET) and benzophenone-4-carboxamidocysteine methanethiosulfonate (BPMTS) were slowed ~10-fold in toxin-modified channels. Based upon the different size, hydrophobicity and charge of the two reagents it is unlikely that the change in reactivity is due to direct or indirect blockage of access of this site to reagent in the presence of toxin (Tx), but rather is the result of inability of this segment to move outward to the normal extent and at the normal rate in the toxin-modified channel. Measurements of availability of R3C to internally applied reagent show decreased access (slower rates of thiol reaction) providing further evidence for encumbered D4S4 movement in the presence of toxins consistent with the assignment of at least part of the toxin binding site to the region of D4S4 region of the voltage-sensor module.

Investigating the Voltage Sensor Domains of Nav1.4, its Structural and Functional Properties via Histidine Scanning Mutagenesis

Mammalian sodium channels are composed of four homologous domains (DI, DII, DIII and DIV). Each domain is composed of six helical transmembrane segments (S1-S6). Together, the folding of S5-S6 segments of all the domains form the pore domain (PD). The S1-S4 segments of each domain form the voltage sensor domain (VSD). Recently, mutations in the VSD of Nav channels have been linked to pathologies such as hypo- and normokalemic periodic paralysis and recently dilated cardiomyopathyGosselin-Badaroudine P., et al.,(2012): PlosONE, 7(5):e38331.Here we use the histidine scanning mutagenesis technique to investigate the role of the positively charged amino acids along the S4 segments of each domain. Detecting a proton current at hyperpolarized potentials indicates that the mutated residue is located in the gating charge transfer center of the protein. Also, proton transport implicates that the mutated residue moves across the gating charge transfer center during activation. Furthermore, a shift in the Q-V curve indicate that the mutated residue plays an important role in the stabilization of the S4 segment in its activated or resting position.The results lead to the creation of the first structural model of the VSDs of a mammalian sodium channel in its resting state. This structural model features hydrophobic septa of different dimensions. Indeed, the VSD of the fourth domain displays a hydrophobic septum much larger than the septa of the other domains. This difference of the VSD of the forth domain provides a rationale for its late onset in the activation sequence and the fact that no gating pore current have been uncovered in the VSD of DIV of mammalian sodium channels.

A Simple Method for Predicting Transmembrane Proteins Based on Wavelet Transform

The increasing protein sequences from the genome project require theoretical methods to predict transmembrane helical segments (TMHs). So far, several prediction methods have been reported, but there are some deficiencies in prediction accuracy and adaptability in these methods. In this paper, a method based on discrete wavelet transform (DWT) has been developed to predict the number and location of TMHs in membrane proteins. PDB coded as 1KQG is chosen as an example to describe the prediction process by this method. 80 proteins with known 3D structure from Mptopo database are chosen at random as data sets (including 325 TMHs) and 80 sequences are divided into 13 groups according to their function and type. TMHs prediction is carried out for each group of membrane protein sequences and obtain satisfactory result. To verify the feasibility of this method, 80 membrane protein sequences are treated as test sets, 308 TMHs can be predicted and the prediction accuracy is 96.3%. Compared with the main prediction results of seven popular prediction methods, the obtained results indicate that the proposed method in this paper has higher prediction accuracy.

Bioinformatic Analyses of Bacterial Mercury Ion (Hg2+) Transporters

Currently, there are five known types of mercury transporters in bacteria: MerC, MerE, MerF, MerH, and MerT. Their general function is to mediate mercuric ion uptake into the cell in preparation for reduction to Hg°. They are present in several bacterial phyla and comprise five distinct families. We have utilized standard statistical bioinformatic tools and the superfamily principle to show that they are related by common descent. After using programs such as Global Alignment Program and SSearch to establish homology, we aligned and analyzed their amino acid sequences to find a single well conserved motif. Although these proteins exhibit 2, 3, or 4 transmembrane helical segments (TMSs), TMSs 1 and 2 are common to all superfamily members. An ancestral sequence was determined, and reliable phylogenetic trees were constructed. The results support the conclusion of homology, establishing that these proteins belong to a single superfamily. This important discovery allows extrapolation of information about structure, function, and mechanism from one protein to all superfamily members to degrees inversely proportional to their phylogenetic distances.

Hinge-bending motions in the pore domain of a bacterial voltage-gated sodium channel

Computational methods and experimental data are used to provide structural models for NaChBac, the homo-tetrameric voltage-gated sodium channel from the bacterium Bacillus halodurans, with a closed and partially open pore domain. Molecular dynamic (MD) simulations on membrane-bound homo-tetrameric NaChBac structures, each comprising six helical transmembrane segments (labeled S1 through S6), reveal that the shape of the lumen, which is defined by the bundle of four alpha-helical S6 segments, is modulated by hinge bending motions around the S6 glycine residues. Mutation of these glycine residues into proline and alanine affects, respectively, the structure and conformational flexibility of the S6 bundle. In the closed channel conformation, a cluster of stacked phenylalanine residues from the four S6 helices hinders diffusion of water molecules and Na+ ions. Activation of the voltage sensor domains causes destabilization of the aforementioned cluster of phenylalanines, leading to a more open structure. The conformational change involving the phenylalanine cluster promotes a kink in S6, suggesting that channel gating likely results from the combined action of hinge-bending motions of the S6 bundle and concerted reorientation of the aromatic phenylalanine side-chains.

Transmembrane helix: simple or complex

Transmembrane helical segments (TMs) can be classified into two groups of so-called ‘simple’ and ‘complex’ TMs. Whereas the first group represents mere hydrophobic anchors with an overrepresentation of aliphatic hydrophobic residues that are likely attributed to convergent evolution in many cases, the complex ones embody ancestral information and tend to have structural and functional roles beyond just membrane immersion. Hence, the sequence homology concept is not applicable on simple TMs. In practice, these simple TMs can attract statistically significant but evolutionarily unrelated hits during similarity searches (whether through BLAST- or HMM-based approaches). This is especially problematic for membrane proteins that contain both globular segments and TMs. As such, we have developed the transmembrane helix: simple or complex (TMSOC) webserver for the identification of simple and complex TMs. By masking simple TM segments in seed sequences prior to sequence similarity searches, the false-discovery rate decreases without sacrificing sensitivity. Therefore, TMSOC is a novel and necessary sequence analytic tool for both the experimentalists and the computational biology community working on membrane proteins. It is freely accessible at http://tmsoc.bii.a-star.edu.sg or available for download.

Properties of the Membrane Binding Component of Catechol-O-methyltransferase Revealed by Atomistic Molecular Dynamics Simulations

We used atomistic simulations to study the membrane-bound form of catechol-O-methyltransferase (MB-COMT). In particular we investigated the 26-residue transmembrane α-helical segment of MB-COMT together with the 24-residue fragment that links the transmembrane component to the main protein unit that was not included in our model. In numerous independent simulations we observed the formation of a salt bridge between ARG27 and GLU40. The salt bridge closed the flexible loop that formed in the linker and kept it in the vicinity of the membrane-water interface. All simulations supported this conclusion that the linker has a clear affinity for the interface and preferentially arranges its residues to reside next to the membrane, without a tendency to relocate into the water phase. Furthermore, an extensive analysis of databases for sequences of membrane proteins that have a single transmembrane helical segment brought about an interesting view that the flexible loop observed in our work can be a common structural element in these types of proteins. In the same spirit we close the article by discussing the role of salt bridges in the formation of three-dimensional structures of membrane proteins that exhibit a single transmembrane helix.