Transmembrane Helix Interactions Research Articles

Mutational analysis in Corynebacterium stationis MFS transporters for improving nucleotide bioproduction

Product secretion from an engineered cell can be advantageous for microbial cell factories. Extensive work on nucleotide manufacturing, one of the most successful microbial fermentation processes, has enabled Corynebacterium stationis to transport nucleotides outside the cell by random mutagenesis; however, the underlying mechanism has not been elucidated, hindering its applications in transporter engineering. Herein, we report the nucleotide-exporting major facilitator superfamily (MFS) transporter from the C. stationis genome and its hyperactive mutation at the G64 residue. Structural estimation and molecular dynamics simulations suggested that the activity of this transporter improved via two mechanisms: (1) enhancing interactions between transmembrane helices through the conserved “RxxQG” motif along with substrate binding and (2) trapping substrate-interacting residue for easier release from the cavity. Our results provide novel insights into how MFS transporters change their conformation from inward- to outward-facing states upon substrate binding to facilitate efflux and can contribute to the development of rational design approaches for efflux improvements in microbial cell factories.Keypoints• An MFS transporter from C. stationis genome and its mutation at residue G64 were assessed• It enhanced the transporter activity by strengthening transmembrane helix interactions and trapped substrate-interacting residues• Our results contribute to rational design approach development for efflux improvementGraphical

Bacterial One- and Two-Hybrid Assays to Monitor Transmembrane Helix Interactions.

In transenvelope multiprotein machines such as bacterial secretion systems, protein-protein interactions not only occur between soluble domains but might also be mediated by helix-helix contacts in the inner membrane. Several assays have been therefore developed to test homotypic and heterotypic interactions between transmembrane α-helices in their native membrane environment. Here, we provide detailed protocols for two genetic assays, TOXCAT and GALLEX, which are based on the reconstitution of dimeric regulators allowing the control of expression of reporter genes.

Hydrophobic mismatch and sequence specificity compete when transmembrane helix-helix interactions are measured with the TOXCAT assay.

Genetic assays capable of measuring the propensity of transmembrane helices to oligomerize within the cytoplasmic membrane of the bacterium E. coli are frequently used when sequence-specificity in transmembrane helix-helix interactions is investigated. In the present study, dimerization of the well-investigated wild-type and G83I-mutated transmembrane helix of the human glycophorin A protein was studied. Gradual prolongation of the transmembrane helix at the C-terminus with Leu residues lead to pronounced changes in the dimerization propensity when measured with the TOXCAT assay. Thus, besides sequence specificity, hydrophobic mismatch between the hydrophobic core of a studied transmembrane helix and the E. coli membrane can impact the oligomerization propensity of a transmembrane helix. This suggests that the results of genetic assays aiming at determining interactions of heterologous transmembrane helices within the E. coli membrane do not necessarily solely reflect sequence specificity in transmembrane helix-helix interactions, but might be additionally modulated by topological and structural effects caused by hydrophobic mismatch.

Solid-State NMR Investigations of Transmembrane Helix Interactions

Transmembrane proteins are vital to numerous biological processes, including signal transduction and ion conduction. In many cases, the behavior of potentially ionizable residues in the hydrophobic interior of the membrane is crucial to understanding protein function. We have employed a low-dynamic model α-helical transmembrane peptide, GWALP23 (acetyl-GGALWLALALALALALALWLAGA-amide) as a framework to investigate the possibility of (+/-) ion-pair interactions between arginine and glutamic acid side chains located on different and independent (separately diffusing) transmembrane helices, by 2H NMR spectroscopy. One peptide with an Arg residue, GWALP23-R14 (acetyl-GGALWLALALALARALALWLAGA-amide), and another with a Glu residue, GWALP23-E14 (acetyl-GGALWLALALALAEALALWLAGA-amide), were incorporated into DOPC bilayers together (1:67 peptide:lipid ratio). Past experiments have shown that GWALP23-R14 alone is well-aligned in DOPC bilayers, gives narrow resonances for 2H-labeled alanines and remains charged, even under alkaline pH conditions (JACS 2010, 132(16):5803). By contrast, 2H-Ala-labeled GWALP23-E14 exhibits much broader 2H NMR spectra. We now find that the spectra of labeled GWALP23-R14 become noticeably broadened and weakened, without changing the quadrupolar splittings, when even 16 mol% GWALP23-E14 is included with GWALP23-R14 in the DOPC bilayer samples. A control peptide, GWALP23, has little effect on GWALP23-R14 at 16 mol%, but spectral broadening and weakening occur with at 50 mol%. Conversely, the addition of GWALP-R14 (16 or 50 mol%), has no effect on the already broad spectra of labeled GWALP-E14 in DOPC bilayers. Preliminary results, therefore, suggest that while GWALP-E14 causes concentration-dependent effects on the spectra of GWALP-R14, the complex or possibly multi-state behavior of GWALP-E14 is not ‘rescued’ by the addition of GWALP-R14 in DOPC bilayers.

Fusion Reporter Approaches to Monitoring Transmembrane Helix Interactions in Bacterial Membranes.

In transenvelope multiprotein machines such as bacterial secretion systems, protein-protein interactions not only occur between soluble domains but might also be mediated by helix-helix contacts in the inner membrane. Here we describe genetic assays commonly used to test interactions between transmembrane α-helices in their native membrane environment. These assays are based on the reconstitution of dimeric regulators allowing the control of expression of reporter genes. We provide detailed protocols for the TOXCAT and GALLEX assays used to monitor homotypic and heterotypic transmembrane helix-helix interactions.

Death Receptor 5 Networks Require Membrane Cholesterol for Proper Structure and Function

Death receptor 5 (DR5) is an apoptosis-inducing member of the tumor necrosis factor receptor superfamily, whose activity has been linked to membrane cholesterol content. Upon ligand binding, DR5 forms large clusters within the plasma membrane that have often been assumed to be manifestations of receptor co-localization in cholesterol-rich membrane domains. However, we have recently shown that DR5 clusters are more than just randomly aggregated receptors. Instead, these are highly structured networks held together by receptor dimers. These dimers are stabilized by specific transmembrane helix–helix interactions, including a disulfide bond in the long isoform of the receptor. The complex relationships among DR5 network formation, transmembrane helix dimerization, membrane cholesterol, and receptor activity has not been established. It is unknown whether the membrane itself plays an active role in driving DR5 transmembrane helix interactions or in the formation of the networks. We show that cholesterol depletion in cells does not inhibit the formation of DR5 networks. However, the networks that form in cholesterol-depleted cells fail to induce caspase cleavage. These results suggest a potential structural difference between active and inactive networks. As evidence, we show that cholesterol is necessary for the covalent dimerization of DR5 transmembrane domains. Molecular simulations and experiments in synthetic vesicles on the DR5 transmembrane dimer suggest that dimerization is facilitated by increased helicity in a thicker bilayer.

Synthesis and characterization of β-peptide helices as transmembrane domains in lipid model membranes.

Aggregation, orientation and dynamics of transmembrane helices are of relevance for protein function and transmembrane signaling. To explore the interactions of transmembrane helices and the interdependence of peptide structure and lipid composition of the membranes, β-peptides were explored as model transmembrane domains. Various hydrophobic β-peptide sequences were synthesized by solid phase peptide synthesis. Conformational analyses of β-peptide helices were performed in organic solvents (methanol and 2,2,2-trifluoroethanol) and in large unilamellar liposomes (dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and dioleoylphosphatidylcholine) indicating 12- and 14-helix conformations, depending on β3 -amino acid sequences. The intrinsic tryptophan fluorescence of β3 -homotryptophan units inserted in the center or near the end of the sequence was used to verify the membrane insertion of the β-peptides. A characteristic blue shift with peripheral β3 -homotryptophan compared with β-peptides with central tryptophan served as indication for a transmembrane orientation of the β-peptides within the lipid bilayer. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Impact of Differential Detergent Interactions on Transmembrane Helix Dimerization Affinities.

Interactions between transmembrane (TM) helices play a critical role in the fundamental processes required for cells to communicate and exchange materials with their surroundings. Our understanding of the factors that promote TM helix interactions has greatly benefited from our ability to study these interactions in the solution phase through the use of membrane-mimetic micelles. However, less is known about the potential influence of juxtamembrane regions flanking the interacting TM helices that may modulate dimerization affinities, even when the interacting surface itself is not altered. To investigate this question, we used solution NMR to quantitate the dimerization affinity of the major coat protein from the M13 bacteriophage in sodium dodecyl sulfate (SDS), a well-characterized model of a single-spanning self-associating TM protein. Here, we showed that a shorter construct lacking the N-terminal amphipathic helix has a higher dimerization affinity relative to that of the full-length protein, with no change in the helical structure between the monomeric and dimeric states in both cases. Although this translated into a 0.6 kcal/mol difference in free energy when the SDS solvent was approximated as a continuous phase, there were deviations from this model at high protein to detergent ratios. Instead, the equilibria were better fit to a model that treats the empty micelle as an active participant in the reaction, giving rise to standard free energies of association that were the same for both full-length and TM-segment constructs. According to this model, the higher apparent affinity of the shorter peptide could be completely explained by the enhanced detergent binding by the monomer relative to that bound by the dimer. Therefore, differential detergent binding between the monomeric and dimeric states provides a mechanism by which TM helix interactions can be modulated by noninteracting juxtamembrane regions.

Screening for transmembrane association in divisome proteins using TOXGREEN, a high-throughput variant of the TOXCAT assay

TOXCAT is a widely used genetic assay to study interactions of transmembrane helices within the inner membrane of the bacterium Escherichia coli. TOXCAT is based on a fusion construct that links a transmembrane domain of interest with a cytoplasmic DNA-binding domain from the Vibrio cholerae ToxR protein. Interaction driven by the transmembrane domain results in dimerization of the ToxR domain, which, in turn, activates the expression of the reporter gene chloramphenicol acetyl transferase (CAT). Quantification of CAT is used as a measure of the ability of the transmembrane domain to self-associate. Because the quantification of CAT is relatively laborious, we developed a high-throughput variant of the assay, TOXGREEN, based on the expression of super-folded GFP and detection of fluorescence directly in unprocessed cell cultures. Careful side-by-side comparison of TOXCAT and TOXGREEN demonstrates that the methods have comparable response, dynamic range, sensitivity and intrinsic variability both in LB and minimal media. The greatly enhanced workflow makes TOXGREEN much more scalable and ideal for screening, since hundreds of constructs can be rapidly assessed in 96 well plates. Even for small scale investigations, TOXGREEN significantly reduces time, labor and cost associated with the procedure. We demonstrate applicability with a large screening for self-association among the transmembrane domains of bitopic proteins of the divisome (FtsL, FtsB, FtsQ, FtsI, FtsN, ZipA and EzrA) belonging to 11 bacterial species. The analysis confirms a previously reported tendency for FtsB to self-associate, and suggests that the transmembrane domains of ZipA, EzrA and FtsN may also possibly oligomerize.

Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex.

Mitochondrial Ca(2+) uptake, a process crucial for bioenergetics and Ca(2+) signaling, is catalyzed by the mitochondrial calcium uniporter. The uniporter is a multi-subunit Ca(2+)-activated Ca(2+) channel, with the Ca(2+) pore formed by the MCU protein and Ca(2+)-dependent activation mediated by MICU subunits. Recently, a mitochondrial inner membrane protein EMRE was identified as a uniporter subunit absolutely required for Ca(2+) permeation. However, the molecular mechanism and regulatory purpose of EMRE remain largely unexplored. Here, we determine the transmembrane orientation of EMRE, and show that its known MCU-activating function is mediated by the interaction of transmembrane helices from both proteins. We also reveal a second function of EMRE: to maintain tight MICU regulation of the MCU pore, a role that requires EMRE to bind MICU1 using its conserved C-terminal polyaspartate tail. This dual functionality of EMRE ensures that all transport-competent uniporters are tightly regulated, responding appropriately to a dynamic intracellular Ca(2+) landscape.

Determination of the Interaction of γ-Secretase with its βCTF Substrate

A key step in the pathogenesis of Alzheimer's disease (AD) is γ-secretase's proteolysis of βCTF (C99) substrate, resulting in the formation of different sized Aβ peptides, incuding Aβ42, which is assumed to be the principale component of the characteristical cerebral plaques of the disease.It was proposed that the oligomeric state of C99 might play a role for its proteolytic processing by the γ-secretase enzyme. γ-Secretase is a multi-protein complex consisting of presenilin (PS), nicastrin (Nct), anterior pharynx defective-1 (Aph-1) and presenilin enhancer-2 (Pen-2). Of these, presenilin is the catalytic component and thus of special interest.Despite the importance of the presenilin subunit for the generation of Alzheimer's disease, there are still a number of important questions. The exact location of the C99 substrate binding site and the question if, or if not dimerization of the substrate plays a role for C99 substrate recognition and therefor intramembrane-cleavage is still under big debate.To answer the question which role dimerization plays, we designed four single-cysteine mutants of C99. These proteins were crosslinked to form covalent dimers, which were tested as substrates in an in-vitro γ-secretase assay. We show here that the covalently crosslinked C99 homodimer cannot be cleaved by γ-secretase, suggesting that dimerization may not be important for cleavage. To help identify the γ-secretase binding site, we used an in-vivo reporter assay to test for interaction between the transmembrane helix of C99 substrate and the isolated transmembrane helices of presenilin. The role of transmembrane helix interaction in the recognition of the C99 substrate is discussed.

One-pot system for synthesis, assembly, and display of functional single-span membrane proteins on oil–water interfaces

Single-span membrane proteins (ssMPs) represent approximately one-half of all membrane proteins and play important roles in cellular communications. However, like all membrane proteins, ssMPs are prone to misfolding and aggregation because of the hydrophobicity of transmembrane helices, making them difficult to study using common aqueous solution-based approaches. Detergents and membrane mimetics can solubilize membrane proteins but do not always result in proper folding and functionality. Here, we use cell-free protein synthesis in the presence of oil drops to create a one-pot system for the synthesis, assembly, and display of functional ssMPs. Our studies suggest that oil drops prevent aggregation of some in vitro-synthesized ssMPs by allowing these ssMPs to localize on oil surfaces. We speculate that oil drops may provide a hydrophobic interior for cotranslational insertion of the transmembrane helices and a fluidic surface for proper assembly and display of the ectodomains. These functionalized oil drop surfaces could mimic cell surfaces and allow ssMPs to interact with cell surface receptors under an environment closest to cell-cell communication. Using this approach, we showed that apoptosis-inducing human transmembrane proteins, FasL and TRAIL, synthesized and displayed on oil drops induce apoptosis of cultured tumor cells. In addition, we take advantage of hydrophobic interactions of transmembrane helices to manipulate the assembly of ssMPs and create artificial clusters on oil drop surfaces. Thus, by coupling protein synthesis with self-assembly at the water-oil interface, we create a platform that can use recombinant ssMPs to communicate with cells.

Free energy of helical transmembrane peptide dimerization in OPLS-AA/Berger force field simulations: inaccuracy and implications for partner-specific Lennard-Jones parameters between peptides and lipids

Interactions between transmembrane (TM) peptides are important in biophysical chemistry, but there are few studies assessing atomistic simulation parameters concerning the energetics of interactions of TM helical peptides. Our potential of mean force analysis using OPLS-AA protein/Berger lipid force fields (FFs) shows that the dimerisation energy of (AALALAA)3 helical peptides in the dioleoylphosphatidylcholine bilayer is −4.4 kJ/mol, which was much smaller than the reported experimental value (−12.7 kJ/mol), thus calling for improvement of parameters of the combined FFs. As each of the FFs has been independently developed, we then tested the effects of downscaling the Lennard-Jones (LJ) energy terms between the OPLS-AA atoms and Berger lipid atoms, preserving the parameters within each FF. A 0.9-fold rescaling of the LJ energies was found to render the dimerisation energy close to the experimental value. Solvation of backbone atoms as well as side chain atoms in lipids is crucial for the TM helix interaction. In similar analyses, GROMOS 53A6 FF exhibited as weak dimerisation propensity (~−5.2 kJ/mol) as OPLS-AA/Berger, but CHARMM36 showed relatively accurate propensity (~−9.9 kJ/mol). Challenges and strategies in rendering the TM interaction energy realistic within the framework of current FFs are discussed.

Role of GxxxG Motifs in Transmembrane Domain Interactions.

Transmembrane (TM) helices of integral membrane proteins can facilitate strong and specific noncovalent protein-protein interactions. Mutagenesis and structural analyses have revealed numerous examples in which the interaction between TM helices of single-pass membrane proteins is dependent on a GxxxG or (small)xxx(small) motif. It is therefore tempting to use the presence of these simple motifs as an indicator of TM helix interactions. In this Current Topic review, we point out that these motifs are quite common, with more than 50% of single-pass TM domains containing a (small)xxx(small) motif. However, the actual interaction strength of motif-containing helices depends strongly on sequence context and membrane properties. In addition, recent studies have revealed several GxxxG-containing TM domains that interact via alternative interfaces involving hydrophobic, polar, aromatic, or even ionizable residues that do not form recognizable motifs. In multipass membrane proteins, GxxxG motifs can be important for protein folding, and not just oligomerization. Our current knowledge thus suggests that the presence of a GxxxG motif alone is a weak predictor of protein dimerization in the membrane.

Fluorophores, environments, and quantification techniques in the analysis of transmembrane helix interaction using FRET.

Förster resonance energy transfer (FRET) has been widely used as a spectroscopic tool in vitro to study the interactions between transmembrane (TM) helices in detergent and lipid environments. This technique has been instrumental to many studies that have greatly contributed to quantitative understanding of the physical principles that govern helix-helix interactions in the membrane. These studies have also improved our understanding of the biological role of oligomerization in membrane proteins. In this review, we focus on the combinations of fluorophores used, the membrane mimetic environments, and measurement techniques that have been applied to study model systems as well as biological oligomeric complexes in vitro. We highlight the different formalisms used to calculate FRET efficiency and the challenges associated with accurate quantification. The goal is to provide the reader with a comparative summary of the relevant literature for planning and designing FRET experiments aimed at measuring TM helix-helix associations.

Dimerization of the EphA1 receptor tyrosine kinase transmembrane domain: Insights into the mechanism of receptor activation.

EphA1 is a receptor tyrosine kinase (RTK) that plays a key role in developmental processes, including guidance of the migration of axons and cells in the nervous system. EphA1, in common with other RTKs, contains an N-terminal extracellular domain, a single transmembrane (TM) α-helix, and a C-terminal intracellular kinase domain. The TM helix forms a dimer, as seen in recent NMR studies. We have modeled the EphA1 TM dimer using a multiscale approach combining coarse-grain (CG) and atomistic molecular dynamics (MD) simulations. The one-dimensional potential of mean force (PMF) for this system, based on interhelix separation, has been calculated using CG MD simulations. This provides a view of the free energy landscape for helix–helix interactions of the TM dimer in a lipid bilayer. The resulting PMF profiles suggest two states, consistent with a rotation-coupled activation mechanism. The more stable state corresponds to a right-handed helix dimer interacting via an N-terminal glycine zipper motif, consistent with a recent NMR structure (2K1K). A second metastable state corresponds to a structure in which the glycine zipper motif is not involved. Analysis of unrestrained CG MD simulations based on representative models from the PMF calculations or on the NMR structure reveals possible pathways of interconversion between these two states, involving helix rotations about their long axes. This suggests that the interaction of TM helices in EphA1 dimers may be intrinsically dynamic. This provides a potential mechanism for signaling whereby extracellular events drive a shift in the repopulation of the underlying TM helix dimer energy landscape.

S-7 : A new cytokine receptor activation paradigm: Activation of JAK2 by the growth hormone receptor

The conformational changes required to transmit the GH binding signal from the extracellular domain of the GH receptor to its intracellular domain resulting in activation of JAK2 has been enigmatic. We have recently defined the first complete mechanistic model for JAK2 activation based on an archetypal cytokine receptor, the growth hormone receptor. To formulate this model we have used FRET to monitor positioning of the JAK2 binding motif, Jun-zipper receptor constructs to control receptor transmembrane (TM) helix position, atomistic modeling of TM helix interactions and docking of JAK2 kinase and inhibitory pseudokinase crystal structures with an opposing pair in trans [1] . Surprisingly, we found that activation of the receptor dimer induces a separation of its JAK2 binding motifs, driven by a ligand-induced transition from a parallel TM helix pair to a left handed crossover arrangement. This mechanism leads to removal of the pseudokinase domain from the kinase domain of the partner JAK2 and pairing of the two kinase domains, facilitating trans -activation. This model may generalize to other class I cytokine receptors.

Sidekick for Membrane Simulations: Automated Ensemble Molecular Dynamics Simulations of Transmembrane Helices.

The interactions of transmembrane (TM) α-helices with the phospholipid membrane and with one another are central to understanding the structure and stability of integral membrane proteins. These interactions may be analyzed via coarse grained molecular dynamics (CGMD) simulations. To obtain statistically meaningful analysis of TM helix interactions, large (N ca. 100) ensembles of CGMD simulations are needed. To facilitate the running and analysis of such ensembles of simulations, we have developed Sidekick, an automated pipeline software for performing high throughput CGMD simulations of α-helical peptides in lipid bilayer membranes. Through an end-to-end approach, which takes as input a helix sequence and outputs analytical metrics derived from CGMD simulations, we are able to predict the orientation and likelihood of insertion into a lipid bilayer of a given helix of a family of helix sequences. We illustrate this software via analyses of insertion into a membrane of short hydrophobic TM helices containing a single cationic arginine residue positioned at different positions along the length of the helix. From analyses of these ensembles of simulations, we estimate apparent energy barriers to insertion which are comparable to experimentally determined values. In a second application, we use CGMD simulations to examine the self-assembly of dimers of TM helices from the ErbB1 receptor tyrosine kinase and analyze the numbers of simulation repeats necessary to obtain convergence of simple descriptors of the mode of packing of the two helices within a dimer. Our approach offers a proof-of-principle platform for the further employment of automation in large ensemble CGMD simulations of membrane proteins.

Trimeric Transmembrane Domain Interactions in Paramyxovirus Fusion Proteins: ROLES IN PROTEIN FOLDING, STABILITY, AND FUNCTION

Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion.

Self-interaction of transmembrane helices representing pre-clusters from the human single-span membrane proteins

Most integral membrane proteins form dimeric or oligomeric complexes. Oligomerization is frequently supported by the non-covalent interaction of transmembrane helices. It is currently not clear how many high-affinity transmembrane domains (TMD) exist in a proteome and how specific their interactions are with respect to preferred contacting faces and their underlying residue motifs. We first identify a threshold of 55% sequence similarity, which demarcates the border between meaningful alignments of TMDs and chance alignments. Clustering the human single-span membrane proteome using this threshold groups ~40% of the TMDs. The homotypic interaction of the TMDs representing the 33 largest clusters was systematically investigated under standardized conditions. The results reveal a broad distribution of relative affinities. High relative affinity frequently coincides with (i) the existence of a preferred helix-helix interface and (ii) sequence specificity as indicated by reduced affinity after mutating conserved residues. Supplementary data are available at Bioinformatics online.