TM Segments Research Articles

Dissecting movement of the transmembrane segments of non-gastric proton pump mutants with voltage-clamp fluorometry.

The Na+,K+-ATPase (NKA) extrudes 3 Na+ and imports 2 K+ across the plasmalemma of every animal cell. The non-gastric H+,K+- ATPase (ngHKA) exports one H+ and imports one K+ in the apical membrane of several epithelia. Both P-type pumps have ∼65% identity and nearly identical catalytic cycles driven by binding of different ions to two major conformations (E1, high affinity for Na+ or H+ and E2 higher affinity for K+). Recently, we reported the structures of the wild type (WT) ngHKA and its electrogenic mutant K794A and the NKA-like K794S/A797P/W940/R949C (SPWC) mutant, in the K+-occluded E2 state. We also reported SPWC's AMPCP-bound E1 Cryo-EM structure, which is nearly identical to that of the E1(3Na+) NKA. We obtained the WT (Na+ and AlF-ADP) and K794A (Na+ and AMPPCP) structures. While the WT structure was identical to E2P state, the AMPPCP-bound K794A structure with (presumably) 2 Na+ bound has a mixed conformation. The P and N domains as well as cytoplasmic portion of transmembrane segment TM4 shows E1-like conformation, but the A domain, TM1-TM3 and the luminal portion of TM4 took an E2-like conformation. To evaluate displacements of the moving TM segments in all three constructs, we introduced a single Cys residue in the loops between TM1-TM2, TM3-4, or TM5-6, labeled them with tetramethylrhodamine maleimide (TMRM) and evaluated currents and fluorescence changes under voltage clamp fluorometry in Xenopus oocytes. Consistent with the stable E2 state in WT, a TMRM introduced in TM1-TM2 showed minimal voltage dependent changes in fluorescence, while the voltage dependence of the TM1-TM2 fluorescent signals observed with K794A and SPWC mutants were progressively closer to signals observed in NKA. Studies with TMRM introduced at TM3-4 and TM5-6 positions are underway. Funded by NSF MCB-2003251.

SLC8 family of sodium/calcium exchangers in GtoPdb v.2021.3

The sodium/calcium exchangers (NCX) use the extracellular sodium concentration to facilitate the extrusion of calcium out of the cell. Alongside the plasma membrane Ca2+-ATPase (PMCA) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), as well as the sodium/potassium/calcium exchangers (NKCX, SLC24 family), NCX allow recovery of intracellular calcium back to basal levels after cellular stimulation. When intracellular sodium ion levels rise, for example, following depolarisation, these transporters can operate in the reverse direction to allow calcium influx and sodium efflux, as an electrogenic mechanism. Structural modelling suggests the presence of 9 TM segments, with a large intracellular loop between the fifth and sixth TM segments [1].

SLC23 family of ascorbic acid transporters in GtoPdb v.2021.3

Predicted to be 12 TM segment proteins, members of this family transport the reduced form of ascorbic acid (while the oxidized form may be handled by members of the SLC2 family (GLUT1/SLC2A1, GLUT3/SLC2A3 and GLUT4/SLC2A4). phloretin is considered a non-selective inhibitor of these transporters, with an affinity in the micromolar range [1].

SLC47 family of multidrug and toxin extrusion transporters in GtoPdb v.2021.3

These proton:organic cation exchangers are predicted to have 13 TM segments [12] and are suggested to be responsible for excretion of many drugs in the liver and kidneys [1].

Residues in Transmembrane Segments of the P2X4 Receptor Contribute to Channel Function and Ethanol Sensitivity.

Mouse models of alcohol use disorder (AUD) revealed purinergic P2X4 receptors (P2X4Rs) as a promising target for AUD drug development. We have previously demonstrated that residues at the transmembrane (TM)–ectodomain interface and within the TM1 segment contribute to the formation of an ethanol action pocket in P2X4Rs. In the present study, we tested the hypothesis that there are more residues in TM1 and TM2 segments that are important for the ethanol sensitivity of P2X4Rs. Using site-directed mutagenesis and two electrode voltage-clamp electrophysiology in Xenopus oocytes, we found that arginine at position 33 (R33) in the TM1 segment plays a role in the ethanol sensitivity of P2X4Rs. Molecular models in both closed and open states provided evidence for interactions between R33 and aspartic acid at position 354 (D354) of the neighboring TM2 segment. The loss of ethanol sensitivity in mixtures of wild-type (WT) and reciprocal single mutants, R33D:WT and D354R:WT, versus the WT-like response in R33D-D354R:WT double mutant provided further support for this interaction. Additional findings indicated that valine at TM1 position 49 plays a role in P2X4R function by providing flexibility/stability during channel opening. Collectively, these findings identified new activity sites and suggest the importance of TM1-TM2 interaction for the function and ethanol sensitivity of P2X4Rs.

Defining the Substrate Spectrum of the TIM22 Complex Identifies Pyruvate Carrier Subunits as Unconventional Cargos.

SummaryIn mitochondria, the carrier translocase (TIM22 complex) facilitates membrane insertion of multi-spanning proteins with internal targeting signals into the inner membrane [1, 2, 3]. Tom70, a subunit of TOM complex, represents the major receptor for these precursors [2, 4, 5, 6]. After transport across the outer membrane, the hydrophobic carriers engage with the small TIM protein complex composed of Tim9 and Tim10 for transport across the intermembrane space (IMS) toward the TIM22 complex [7, 8, 9, 10, 11, 12]. Tim22 represents the pore-forming core unit of the complex [13, 14]. Only a small subset of TIM22 cargo molecules, containing four or six transmembrane spans, have been experimentally defined. Here, we used a tim22 temperature-conditional mutant to define the TIM22 substrate spectrum. Along with carrier-like cargo proteins, we identified subunits of the mitochondrial pyruvate carrier (MPC) as unconventional TIM22 cargos. MPC proteins represent substrates with atypical topology for this transport pathway. In agreement with this, a patient affected in TIM22 function displays reduced MPC levels. Our findings broaden the repertoire of carrier pathway substrates and challenge current concepts of TIM22-mediated transport processes.

Molecular basis of force-from-lipids gating in the mechanosensitive channel MscS.

Prokaryotic mechanosensitive (MS) channels open by sensing the physical state of the membrane. As such, lipid-protein interactions represent the defining molecular process underlying mechanotransduction. Here, we describe cryo-electron microscopy (cryo-EM) structures of the E. coli small-conductance mechanosensitive channel (MscS) in nanodiscs (ND). They reveal a novel membrane-anchoring fold that plays a significant role in channel activation and establish a new location for the lipid bilayer, shifted ~14 Å from previous consensus placements. Two types of lipid densities are explicitly observed. A phospholipid that 'hooks' the top of each TM2-TM3 hairpin and likely plays a role in force sensing, and a bundle of acyl chains occluding the permeation path above the L105 cuff. These observations reshape our understanding of force-from-lipids gating in MscS and highlight the key role of allosteric interactions between TM segments and phospholipids bound to key dynamic components of the channel.

SLC8 family of sodium/calcium exchangers (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The sodium/calcium exchangers (NCX) use the extracellular sodium concentration to facilitate the extrusion of calcium out of the cell. Alongside the plasma membrane Ca2+-ATPase (PMCA) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), as well as the sodium/potassium/calcium exchangers (NKCX, SLC24 family), NCX allow recovery of intracellular calcium back to basal levels after cellular stimulation. When intracellular sodium ion levels rise, for example, following depolarisation, these transporters can operate in the reverse direction to allow calcium influx and sodium efflux, as an electrogenic mechanism. Structural modelling suggests the presence of 9 TM segments, with a large intracellular loop between the fifth and sixth TM segments.

SLC6 neurotransmitter transporter family (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Members of the solute carrier family 6 (SLC6) of sodium- and (sometimes chloride-) dependent neurotransmitter transporters [29, 22, 70] are primarily plasma membrane located and may be divided into four subfamilies that transport monoamines, GABA, glycine and neutral amino acids, plus the related bacterial NSS transporters [99]. The members of this superfamily share a structural motif of 10 TM segments that has been observed in crystal structures of the NSS bacterial homolog LeuTAa, a Na+-dependent amino acid transporter from Aquiflex aeolicus [126] and in several other transporter families structurally related to LeuT [45].

SLC47 family of multidrug and toxin extrusion transporters (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

These proton:organic cation exchangers are predicted to have 13 TM segments [10] and are suggested to be responsible for excretion of many drugs in the liver and kidneys.

SLC23 family of ascorbic acid transporters (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Predicted to be 12 TM segment proteins, members of this family transport the reduced form of ascorbic acid (while the oxidized form may be handled by members of the SLC2 family (GLUT1/SLC2A1, GLUT3/SLC2A3 and GLUT4/SLC2A4). phloretin is considered a non-selective inhibitor of these transporters, with an affinity in the micromolar range.

Dropping Out and Other Fates of Transmembrane Segments Inserted by the SecA ATPase

Type II single-span membrane proteins, such as CadC or RodZ, lacking a signal sequence and having a far-downstream hydrophobic segment, require the SecA secretion motor for insertion into the inner membrane of Escherichia coli. Using two chimeric single-span proteins containing a designed hydrophobic segment H, we have determined the requirements for SecA-mediated secretion, the molecular distinction between TM domains and signal peptides, and the propensity for hydrophobic H-segments to remain embedded within the bilayer after targeting. By means of engineered H-segments and a strategically placed SPase I cleavage site, we determined how targeting and stability of the chimeric proteins are affected by the length and hydrophobicity of the H-segment. Very hydrophobic segments (e.g., 16 Leu) are stably incorporated into the inner membrane, resulting in a C-terminal anchored membrane protein, while a 24L construct was not targeted to the membrane by SecA and remained in the cytoplasm. However, a construct carrying preMalE at the N-terminus led to SecA targeting to SecYEG via the native signal sequence and stable insertion of the downstream 24L H-segment. We show that the RseP intramembrane protease degrades weakly stable H-segments and is a useful tool for investigating the borderline between stable and unstable TM segments. Using RseP− cells, we find that moderately hydrophobic sequences (e.g., 5Leu + 11Ala) are targeted to SecYEG by SecA and inserted, but subsequently drop out of the membrane into the cytoplasm. Therefore, the free energy of transfer from translocon to bilayer is different from the transfer free energy from membrane to water.

Iodide Binding in Sodium-Coupled Cotransporters

Several apical iodide translocation pathways have been proposed for iodide efflux out of thyroid follicular cells, including a pathway mediated by the sodium-coupled monocarboxylate transporter 1 (SMCT1), which remains controversial. Herein, we evaluate structural and functional similarities between SMCT1 and the well-studied sodium-iodide symporter (NIS) that mediates the first step of iodide entry into the thyroid. Free-energy calculations using a force field with electronic polarizability verify the presence of a conserved iodide-binding pocket between the TM2, TM3, and TM7 segments in hNIS, where iodide is coordinated by Phe67, Gln72, Cys91, and Gln94. We demonstrate the mutation of residue Gly93 of hNIS to a larger amino acid expels the side chain of a critical tryptophan residue (Trp255) into the interior of the binding pocket, partially occluding the iodide binding site and reducing iodide affinity, which is consistent with previous reports associating mutation of this residue with iodide uptake deficiency and hypothyroidism. Furthermore, we find that the position of Trp255 in this hNIS mutant mirrors that of Trp253 in wild-type hSMCT1, where a threonine (Thr91) occupies the position homologous to that occupied by glycine in wild-type hNIS (Gly93). Correspondingly, mutation of Thr91 to glycine in hSMCT1 makes the pocket structure more like that of wild-type hNIS, increasing its iodide affinity. These results suggest that wild-type hSMCT1 in the inward-facing conformation may bind iodide only very weakly, which may have implications for its ability to transport iodide.

Signal Sensing and Transduction by Histidine Kinases as Unveiled through Studies on a Temperature Sensor.

Histidine kinases (HK) are the sensory proteins of two-component systems, responsible for a large fraction of bacterial responses to stimuli and environmental changes. Prototypical HKs are membrane-bound proteins that phosphorylate cognate response regulator proteins in the cytoplasm upon signal detection in the membrane or periplasm. HKs stand as potential drug targets but also constitute fascinating systems for studying proteins at work, specifically regarding the chemistry and mechanics of signal detection, transduction through the membrane, and regulation of catalytic outputs. In this Account, we focus on Bacillus subtilis DesK, a membrane-bound HK part of a two-component system that maintains appropriate membrane fluidity at low growth temperatures. Unlike most HKs, DesK has no extracytoplasmic signal-sensing domains; instead, sensing is carried out by 10 transmembrane helices (coming from two protomers) arranged in an unknown structure. The fifth transmembrane helix from each protomer connects, without any of the intermediate domains found in other HKs, into the dimerization and histidine phosphotransfer (DHp) domain located in the cytoplasm, which is followed by the ATP-binding domains (ABD). Throughout the years, genetic, biochemical, structural, and computational studies on wild-type, mutant, and truncated versions of DesK allowed us to dissect several aspects of DesK's functioning, pushing forward a more general understanding of its own structure/function relationships as well as those of other HKs. We have shown that the sensing mechanism is rooted in temperature-dependent membrane properties, most likely a combination of thickness, fluidity, and water permeability, and we have proposed possible mechanisms by which DesK senses these properties and transduces the signals. X-ray structures and computational models have revealed structural features of TM and cytoplasmic regions in DesK's kinase- and phosphatase-competent states. Biochemical and genetic experiments and molecular simulations further showed that reversible formation of a two-helix coiled coil in the fifth TM segment and the N-terminus of the cytoplasmic domain is essential for the sensing and signal transduction mechanisms. Together with other structural and functional works, the emerging picture suggests that diverse HKs possess distinct sensing and transduction mechanisms but share as rather general features (i) a symmetric phosphatase state and an asymmetric kinase state and (ii) similar functional outputs on the conserved DHp and ABD domains, achieved through different mechanisms that depend on the nature of the initial signal. We here advance (iii) an important role for TM prolines in transducing the initial signals to the cytoplasmic coiled coils, based on simulations of DesK's TM helices and our previous work on a related HK, PhoQ. Lastly, evidence for DesK, PhoQ, BvgS, and DctB HKs shows that (iv) overall catalytic output is tuned by a delicate balance between hydration potentials, coiled coil stability, and exposure of hydrophobic surface patches at their cytoplasmic coiled coils and at the N-terminal and C-terminal sides of their TM helices. This balance is so delicate that small perturbations, either physiological signals or induced by mutations, lead to large remodeling of the underlying conformational landscape achieving clear-cut changes in catalytic output, mirroring the required response speed of these systems for proper biological function.

SmFRET Reveals Structural Basis for Conformational Misfolding of a Cystic Fibrosis Mutation in CFTR

Cystic fibrosis (CF) is the most common lethal genetic disease among the Western population caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). The development of effective therapeutic agents against CF requires a molecular understanding of how mutation-related structural alterations lead to impaired functioning of CFTR. The main cause for CF are processing mutations frequently found within CFTR-transmembrane (TM) segments which affect co-translational folding and insertion. However, the underlying molecular mechanisms for misfolding remain obscure, mainly because of the unavailability of high-resolution structures and a lack of methods suitable for studying membrane protein structure-function relationships.Devising a divide-and-conquer approach in combination with single-molecule FRET (smFRET), we present here a strategy to gain novel insights into the structural basis for conformational misfolding in CFTR. Using this approach, we study the CF-phenotypic polar mutation V232D located within the fourth TM helix (TM4) of CFTR. In vivo folding and insertion of TM4 is functionally coupled with TM3 where both TM segments are co-translationally inserted into the membrane as a TM helix-loop-helix hairpin motif (TM3/4). Utilizing this minimal folding unit, we employ smFRET as a molecular ruler to readout structural alterations imposed on the helical packing of the TM3/4 hairpin upon mutation. In contrast to earlier findings that suggested a TM lock between TM3 and TM4 by a non-native H-bond, our results reveal that V232D TM3/4 associates with membranes in an open conformation. This implies that the charged residue imposes an energy penalty on membrane insertion of the hairpin. We propose a model for CF pathogenesis where partitioning of V232D TM3/4 into the interfacial region leads to misfolding of CFTR during co-translational membrane insertion and inhibits maturation by trapping the protein as a partially folded intermediate.

Polarity and Charge as Determinants for Translocase Requirement for Membrane Protein Insertion

Membrane protein biogenesis in bacteria is a well-studied but poorly understood area in the field of membrane proteins. In bacteria, the Sec translocon and the YidC insertase promote the membrane insertion process. Both proteins are essential, and universally conserved, and together insert about 95% of the proteins in E.coli. Membrane insertion of SecYEG-dependent proteins likely occurs at the lateral gate of SecY, the channel-forming subunit of the bacterial Sec translocon. On the other hand, YidC catalyzes insertion by possessing a hydrophilic groove, which can accommodate the hydrophilic region of the substrate that inserts across the membrane. Recently, Soman et al (2014) have reported that increasing the charge of the translocated region of procoat can route substrates from the YidC pathway into the Sec pathway. Here, we have further tested this polarity/charge hypothesis by showing that the major coat protein of bacteriophage M13 can become increasingly YidC/Sec dependent by making the periplasmic loop highly polar in the absence of charged residues. We also show that adding hydrophobic amino acids to highly polar or charged loop can decrease the Sec-dependence of the otherwise strictly Sec-dependent substrates. Additionally we demonstrate that increasing the driving force of insertion by adding four leucyl residues to the transmembrane segment leads to translocation of a highly charged region that was not inserted by native TM segment. Finally, we show that the length of procoat loop is a determinant for Sec dependent insertion. The combined results support the polarity/charge hypothesis and is consistent with the notion that membrane insertion occurs at the interface of SecYEG and YidC.

Transmembrane topology of the arsenite permease Acr3 from Saccharomyces cerevisiae

Acr3 is a plasma membrane transporter, a member of the bile/arsenite/riboflavin transporter (BART) superfamily, which confers high-level resistance to arsenicals in the yeast Saccharomyces cerevisiae. We have previously shown that the yeast Acr3 acts as a low affinity As(III)/H+ and Sb(III)/H+ antiporter. We have also identified several amino acid residues that are localized in putative transmembrane helices (TM) and appeared to be critical for the Acr3 activity. In the present study, the topology of Acr3 was investigated by insertion of glycosylation and factor Xa protease cleavage sites at predicted hydrophilic regions. The analysis of the glycosylation pattern and factor Xa cleavage products of resulting Acr3 fusion constructs provide evidence supporting a topological model of Acr3 with 10 TM segments and cytoplasmically oriented N- and C-terminal domains. Next, we investigated the role of the hydrophilic loop connecting TM8 and TM9, the large size of which is unique to members of the yeast Acr3 family of metalloid transporters. We found that a 28 amino acid deletion in this region does not affect Acr3 folding, trafficking substrate binding, or transport activity. Finally, we constructed a homology-based structural model of Acr3 using the crystal structure of the Yersinia frederiksenii homologue of the human bile acid sodium symporter ASBT.

The in silico identification and characterization of a bread wheat/Triticum militinae introgression line.

SummaryThe capacity of the bread wheat (Triticum aestivum) genome to tolerate introgression from related genomes can be exploited for wheat improvement. A resistance to powdery mildew expressed by a derivative of the cross‐bread wheat cv. Tähti × T. militinae (Tm) is known to be due to the incorporation of a Tm segment into the long arm of chromosome 4A. Here, a newly developed in silico method termed rearrangement identification and characterization (RICh) has been applied to characterize the introgression. A virtual gene order, assembled using the GenomeZipper approach, was obtained for the native copy of chromosome 4A; it incorporated 570 4A DArTseq markers to produce a zipper comprising 2132 loci. A comparison between the native and introgressed forms of the 4AL chromosome arm showed that the introgressed region is located at the distal part of the arm. The Tm segment, derived from chromosome 7G, harbours 131 homoeologs of the 357 genes present on the corresponding region of Chinese Spring 4AL. The estimated number of Tm genes transferred along with the disease resistance gene was 169. Characterizing the introgression's position, gene content and internal gene order should not only facilitate gene isolation, but may also be informative with respect to chromatin structure and behaviour studies.

Multiscale simulations on conformational dynamics and membrane interactions of the non-structural 2 (NS2) transmembrane domain

Hepatitis C virus (HCV) is one of the most crucial global health issues, in which the HCV non-structural protein 2 (NS2), particularly its three transmembrane segments, plays a crucial role in HCV assembly. In this context, multiscale MD simulations have been applied to investigate the preferred orientation of transmembrane domain of NS2 protein (TNS2) in a POPC bilayer, structural stability and characteristic of intramembrane protein-lipid and protein-protein interaction. Our study indicates that NS2 protein adopts three trans-membrane segments with highly stable α-helix structure in a POPC bilayer and a short helical luminal segment. While the first and second TM segment involved in continuous helical domain, the third TM segment is however cleaved into two sub-segments with different tilt angles via a kink at L87G88. Salt bridges K81-E45, R32-PO4 and R43-PO4 are determined as the key factor to stabilize the structure of TM2 and TM3 which consist of charged residues located in the hydrophobic region of the membrane.

Structural Model of the Bilitranslocase Transmembrane Domain Supported by NMR and FRET Data.

We present a 3D model of the four transmembrane (TM) helical regions of bilitranslocase (BTL), a structurally uncharacterized protein that transports organic anions across the cell membrane. The model was computed by considering helix-helix interactions as primary constraints, using Monte Carlo simulations. The interactions between the TM2 and TM3 segments have been confirmed by Förster resonance energy transfer (FRET) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, increasing our confidence in the model. Several insights into the BTL transport mechanism were obtained by analyzing the model. For example, the observed cis-trans Leu-Pro peptide bond isomerization in the TM3 fragment may indicate a key conformational change during anion transport by BTL. Our structural model of BTL may facilitate further studies, including drug discovery.