Transmembrane Domain Dimerization Research Articles

Structural basis for the transmembrane signaling and antidepressant-induced activation of the receptor tyrosine kinase TrkB.

Neurotrophin receptors of the Trk family are involved in the regulation of brain development and neuroplasticity, and therefore can serve as targets for anti-cancer and stroke-recovery drugs, antidepressants, and many others. The structures of Trk protein domains in various states upon activation need to be elucidated to allow rational drug design. However, little is known about the conformations of the transmembrane and juxtamembrane domains of Trk receptors. In the present study, we employ NMR spectroscopy to solve the structure of the TrkB dimeric transmembrane domain in the lipid environment. We verify the structure using mutagenesis and confirm that the conformation corresponds to the active state of the receptor. Subsequent study of TrkB interaction with the antidepressant drug fluoxetine, and the antipsychotic drug chlorpromazine, provides a clear self-consistent model, describing the mechanism by which fluoxetine activates the receptor by binding to its transmembrane domain.

Spontaneous Dimerization and Distinct Packing Modes of Transmembrane Domains in Receptor Tyrosine Kinases.

The insulin receptor (IR) and the insulin-like growth factor-1 receptor (IGF1R) are homodimeric transmembrane glycoproteins that transduce signals across the membrane on binding of extracellular peptide ligands. The structures of IR/IGF1R fragments in apo and liganded states have revealed that the extracellular subunits of these receptors adopt Λ-shaped configurations to which are connected the intracellular tyrosine kinase (TK) domains. The binding of peptide ligands induces structural transitions in the extracellular subunits leading to potential dimerization of transmembrane domains (TMDs) and autophosphorylation in TKs. However, the activation mechanisms of IR/IGF1R, especially the role of TMDs in coordinating signal-inducing structural transitions, remain poorly understood, in part due to the lack of structures of full-length receptors in apo or liganded states. While atomistic simulations of IR/IGF1R TMDs showed that these domains can dimerize in single component membranes, spontaneous unbiased dimerization in a plasma membrane having a physiologically representative lipid composition has not been observed. We address this limitation by employing coarse-grained (CG) molecular dynamics simulations to probe the dimerization propensity of IR/IGF1R TMDs. We observed that TMDs in both receptors spontaneously dimerized independent of their initial orientations in their dissociated states, signifying their natural propensity for dimerization. In the dimeric state, IR TMDs predominantly adopted X-shaped configurations with asymmetric helical packing and significant tilt relative to the membrane normal, while IGF1R TMDs adopted symmetric V-shaped or parallel configurations with either no tilt or a small tilt relative to the membrane normal. Our results suggest that IR/IGF1R TMDs spontaneously dimerize and adopt distinct dimerized configurations.

Cryo-EM structure of human class C orphan GPCR GPR179 involved in visual processing

GPR179, an orphan class C GPCR, is expressed at the dendritic tips of ON-bipolar cells in the retina. It plays a pivotal role in the initial synaptic transmission of visual signals from photoreceptors, and its deficiency is known to be the cause of complete congenital stationary night blindness. Here, we present the cryo-electron microscopy structure of human GPR179. Notably, the transmembrane domain (TMD) of GPR179 forms a homodimer through the TM1/7 interface with a single inter-protomer disulfide bond, adopting a noncanonical dimerization mode. Furthermore, the TMD dimer exhibits architecture well-suited for the highly curved membrane of the dendritic tip and distinct from the flat membrane arrangement observed in other class C GPCR dimers. Our structure reveals unique structural features of GPR179 TMD, setting it apart from other class C GPCRs. These findings provide a foundation for understanding signal transduction through GPR179 in visual processing and offers insights into the underlying causes of ocular diseases.

Spontaneous Dimerization and Distinct Packing Modes of Transmembrane Domains in Receptor Tyrosine Kinases.

The insulin receptor (IR) and the insulin-like growth factor-1 receptor (IGF1R) are homodimeric transmembrane glycoproteins that transduce signals across the membrane on binding of extracellular peptide ligands. The structures of IR/IGF1R fragments in apo and liganded states have revealed that the extracellular subunits of these receptors adopt Λ-shaped configurations to which are connected the intracellular tyrosine kinase (TK) domains. The binding of peptide ligands induces structural transitions in the extracellular subunits leading to potential dimerization of transmembrane domains (TMDs) and autophosphorylation in TKs. However, the activation mechanisms of IR/IGF1R, especially the role of TMDs in coordinating signal-inducing structural transitions, remain poorly understood, in part due to the lack of structures of full-length receptors in apo or liganded states. While atomistic simulations of IR/IGF1R TMDs showed that these domains can dimerize in single component membranes, spontaneous unbiased dimerization in a plasma membrane having physiologically representative lipid composition has not been observed. We address this limitation by employing coarse-grained (CG) molecular dynamics simulations to probe the dimerization propensity of IR/IGF1R TMDs. We observed that TMDs in both receptors spontaneously dimerized independent of their initial orientations in their dissociated states, signifying their natural propensity for dimerization. In the dimeric state, IR TMDs predominantly adopted X-shaped configurations with asymmetric helical packing and significant tilt relative to the membrane normal, while IGF1R TMDs adopted symmetric V-shaped or parallel configurations with either no tilt or a small tilt relative to the membrane normal. Our results suggest that IR/IGF1R TMDs spontaneously dimerize and adopt distinct dimerized configurations.

Ligand bias underlies differential signaling of multiple FGFs via FGFR1.

The differential signaling of multiple FGF ligands through a single fibroblast growth factor (FGF) receptor (FGFR) plays an important role in embryonic development. Here, we use quantitative biophysical tools to uncover the mechanism behind differences in FGFR1c signaling in response to FGF4, FGF8, and FGF9, a process which is relevant for limb bud outgrowth. We find that FGF8 preferentially induces FRS2 phosphorylation and extracellular matrix loss, while FGF4 and FGF9 preferentially induce FGFR1c phosphorylation and cell growth arrest. Thus, we demonstrate that FGF8 is a biased FGFR1c ligand, as compared to FGF4 and FGF9. Förster resonance energy transfer experiments reveal a correlation between biased signaling and the conformation of the FGFR1c transmembrane domain dimer. Our findings expand the mechanistic understanding of FGF signaling during development and bring the poorly understood concept of receptor tyrosine kinase ligand bias into the spotlight.

Standard binding free-energy calculation of glycophorin a dimer in non-isotropic media sheds light on the transmembrane alpha-helices association mechanism.

The transmembrane (TM) protein domain recognition and association processes happening in lipid bilayers are pivotal for understanding membrane protein biogenesis. The most straightforward system to study the transmembrane protein binding mechanism is the glycophorin A (GpA) dimeric TM domain. The GpA is a glycoprotein ubiquitous to the human erythrocyte membrane, which forms a non-covalent dimer through the reversible association of its membrane-spanning domain sequences, creating a crossing angle between two alpha-helices. In our study, the membrane protein complex is primarily located in the phosphatidylcholine (POPC) lipid bilayer, surrounded by water. Applying a rigorous theoretical framework of the standard binding free-energy calculation that combines molecular dynamics and potential-of-mean-force calculations, the so-called “geometrical route”, we investigated this complex in non-isotropic media. Our analysis shows that at large separation distances, the helices are pushed together by the lipids, which triggers the interhelical interactions to occur. This observation is in agreement with the “two-stage” model of integral membrane protein folding. Additionally, we observed that the monomers, while reversibly associated, go through a meta-stable state, where the GpA alpha-helices form a different shape compared to the initial one. From the theoretical point of view, we suggest that for the membrane proteins in the non-isotropic environment, the standard binding free-energy estimation via the geometrical route should be analyzed from another perspective compared to those of protein-protein complexes in water media. We revised the separation PMF evaluation, assuming that the lipid bilayer forms a pseudo-cylindrical zone for the reversible separation of the dimer. The methodology employed herein successfully addresses the challenging transmembrane protein-protein binding problem while offering promising perspectives for the binding affinity characterization of different protein complexes in lipid bilayers.

Efficient sampling of TrkA transmembrane domain dimerization captures functionally distinct structural ensembles.

Upon ligand binding, the transmembrane domains (TMD) of the receptor tyrosine kinase (RTK) dimerize, induce conformational change in the juxtamembrane domain (JMD), and trigger downstream phosphorylation that eventually determines fate of the cell. To overcome the experimental difficulty in probing the TMD dimerization events in membrane bilayers, we employed all-atom molecular dynamics with a novel membrane mimetic solvent Simple Carbon Solvent Ethane (SCSE) to efficiently sample the TMD dimerization processes for tropomyosin receptor kinase A (TrkA). Spontaneous and stable TMD dimers were observed in less than 50 ns of simulation time from an initially separated grid configuration. We then performed hierarchical clustering using a customized distance metric based on TMD contact residues, and revealed major structural ensembles of TMD dimers. We demonstrated the validity of the resolved TMD dimer structures by performing MM-GBSA free energy calculation and molecular dynamics with full-tail lipid bilayers. As the residues in contact in the most populated TMD dimer ensemble from our SCSE simulations overlap with those found to be critical for kinase activation, we hypothesized that the captured TMD dimer corresponds to the active state, while the previously characterized NMR structure is the inactive state. To understand the role of different TMD dimer conformations in downstream signaling, we assembled TMD+JMD constructs using the captured in simulation dimer structure and the NMR structure. We modelled the system with Highly Mobile Membrane Mimetic (HMMM) for accelerated peptide-lipid interactions and found that the captured TMD dimer leads to JMD conformations distinct from those tethered to the NMR dimer structure, which may enable the kinase domain for cross-phosphorylation. Our method represents a new approach to study RTK dimerization and sample physiologically relevant dimer structures.

Modulation of the Human Thrombopoietin Receptor Conformation Uncouples JAK2 V617F-Driven from Cytokine-Induced Activation

Myeloproliferative Neoplasms are clonal diseases where mutations in hematopoietic stem cells (HSCs) result in cytokine-independent activation of the Thrombopoietin Receptor (TpoR) and aberrant proliferation and differentiation of HSCs and myeloid progenitors1. The most prevalent mutation, JAK2 V617F, is the basis of >95% of Polycythemia Vera (PV) and 60% of Essential Thrombocythemia (ET) and Myelofibrosis cases2. Current treatment strategies have focused on direct inhibition of JAK2 with little or no success and with poor specificity for the V617F mutant. Unlike JAK2, the TpoR is present at the cell surface and is more easily targetable. Using a system of controlled dimerization, we previously reported that different dimeric orientations of murine TpoR result in distinct signaling pathways and variable activation intensity3. Recently, others have demonstrated that extracellular ligands (diabodies) are able to modulate human TpoR conformation to influence downstream signaling4. Here, we investigated the possibility to modulate human TpoR conformation to specifically target JAK2 V617F driven activation while preserving natural cytokine-induced signaling. Using our system of controlled dimerization of TpoR transmembrane (TM) domain, we characterized the active and inactive conformation of human TpoR in presence of JAK2 WT or JAK2 V617F in hematopoietic cells. While murine TpoR is active in almost all (6 out of 7) dimeric conformations, only 3 conformations allowed activation of human TpoR. The discrepancy between murine and human TpoR is due to sequence differences in the TM domain that modulates dimerization. Remarkably, we discovered that different dimeric conformations mediate activation of JAK2 WT and JAK2 V617F in human but not in murine TpoR. Using a cysteine cross-linking assay on live cells, we validated that TpoR dimers exhibit a different conformation in presence of JAK2 V617F than this induced by the thrombopoietin (Tpo). Given the different dimeric conformations of TpoR induced by Tpo and JAK2 V617F, we hypothesize that modulation of TpoR conformation could allow specific inhibition of JAK2 V617F-driven activation while preserving Tpo-induced activation. We probed this by introducing a glycine zipper GxxxG motif to favor dimerization in a conformation that renders JAK2 V617F inactive but allows Tpo induced activation. Conversely, we mutated residues of the TM domain to specifically disrupt the conformation required for JAK2 V617F-mediated activation. Both strategies resulted in complete inactivation of JAK2-V617F driven activation of TpoR but preserved its full activation by Tpo and cell surface localization. Together, these results provide a first demonstration that modulation of TpoR dimeric conformation is a suitable strategy for specific inhibition of the JAK2 V617F-driven activation and opens the road for novel therapeutic avenues. 1. Vainchenker W, Kralovics R. Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms. Blood. 2017;129(6):667-679. 2. Constantinescu SN, Vainchenker W, Levy G, Papadopoulos N. Functional Consequences of Mutations in Myeloproliferative Neoplasms. Hemasphere. 2021;5(6):e578. 3. Staerk J, Defour JP, Pecquet C, et al. Orientation-specific signalling by thrombopoietin receptor dimers. Embo j. 2011;30(21):4398-4413. 4. Cui L, Moraga I, Lerbs T, et al. Tuning MPL signaling to influence hematopoietic stem cell differentiation and inhibit essential thrombocythemia progenitors. Proceedings of the National Academy of Sciences. 2021;118(2):e2017849118. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Lipid-Mediated Association of the Slg1 Transmembrane Domains in Yeast Plasma Membranes.

Clustering of transmembrane proteins underlies a multitude of fundamental biological processes at the plasma membrane (PM) such as receptor activation, lateral domain formation, and mechanotransduction. The self-association of the respective transmembrane domains (TMDs) has also been suggested to be responsible for the micron-scaled patterns seen for integral membrane proteins in the budding yeast PM. However, the underlying interplay between the local lipid composition and the TMD identity is still not mechanistically understood. In this work, we combined coarse-grained molecular dynamics simulations of simplified bilayer systems with high-resolution live-cell microscopy to analyze the distribution of a representative helical yeast TMD from the PM sensor Slg1 within different lipid environments. In our simulations, we specifically evaluated the effects of acyl chain saturation and anionic lipid head groups on the association of two TMDs. We found that weak lipid-protein interactions significantly affect the configuration of TMD dimers and the free energy of association. Increased amounts of unsaturated phospholipids (PLs) strongly reduced the helix-helix interaction, while the presence of anionic phosphatidylserine (PS) hardly affected the dimer formation. We could experimentally confirm this surprising lack of effect of PS using the network factor, a mesoscopic measure of PM pattern formation in yeast cells. Simulations also showed that the formation of TMD dimers in turn increased the order parameter of the surrounding lipids and induced long-range perturbations in lipid organization. In summary, our results shed new light on the mechanisms of lipid-mediated dimerization of TMDs in complex lipid mixtures.

How transmembrane helices of type 1 receptors transmit information: prediction of dimers with the Martini 3 coarse grained model

Determination of the structure and dynamics of transmembrane (TM) domains of single-transmembrane receptors is key to understanding their mechanism of signal transduction across the plasma membrane. Although many studies have been performed on isolated soluble extra- and intracellular receptor domains in aqueous solutions, limited knowledge exists on the lipid embedded TM region. In this study, we predict the assembly of alternate configurations of receptor TM domain dimers using the Martini 3 force field for coarse-grain (CG) molecular dynamic simulations.

Multiple dimerizing motifs at different locations modulate the dimerization of the syndecan transmembrane domains

Syndecans (SDCs) are a family of four members of integral membrane proteins, which play important roles in cell-cell interactions. Dimerization/oligomerization generated by transmembrane domains (TMDs) appears to crucially regulate several functional behaviors of all syndecan members. The different levels of protein-protein interactions mediated by Syndecan TMDs may lead to a rather complicated function of Syndecans. The molecular mechanism of the different dimerization tendencies in each type of SDCs remains unclear. Here, the self-assembly process of syndecan TMD homodimers and heterodimers was studied in molecular details by molecular dynamics simulations. Our computational results showed that the SDC2 forms the most stable homodimer, which is consistent with previous experimental results. Detailed analysis suggests that instead of the conserved dimerizing motif G8XXXG12 in all four SDCs involved in homo- and hetero-dimerization of SDCs. The different locations of GXXXA motif affect the stability of SDC dimers. In addition, we found that A3XXXA7 can stabilize the dimerization, making the dimer of SDC2 the most stable among these SDC dimers. Our results shed light on the complex effect of multiple dimerizing motifs on the dimerization of transmembrane domains.

Revising the mechanism of p75NTR activation: intrinsically monomeric state of death domains invokes the "helper" hypothesis

The neurotrophin receptor p75NTR plays crucial roles in neuron development and regulates important neuronal processes like degeneration, apoptosis and cell survival. At the same time the detailed mechanism of signal transduction is unclear. One of the main hypotheses known as the snail-tong mechanism assumes that in the inactive state, the death domains interact with each other and in response to ligand binding there is a conformational change leading to their exposure. Here, we show that neither rat nor human p75NTR death domains homodimerize in solution. Moreover, there is no interaction between the death domains in a more native context: the dimerization of transmembrane domains in liposomes and the presence of activating mutation in extracellular juxtamembrane region do not lead to intracellular domain interaction. These findings suggest that the activation mechanism of p75NTR should be revised. Thus, we propose a novel model of p75NTR functioning based on interaction with “helper” protein.

An evolutionarily conserved motif is required for Plasmodesmata-located protein 5 to regulate cell-to-cell movement

Numerous cell surface receptors and receptor-like proteins (RLPs) undergo activation or deactivation via a transmembrane domain (TMD). A subset of plant RLPs distinctively localizes to the plasma membrane-lined pores called plasmodesmata. Those RLPs include the Arabidopsis thaliana Plasmodesmata-located protein (PDLP) 5, which is well known for its vital function regulating plasmodesmal gating and molecular movement between cells. In this study, we report that the TMD, although not a determining factor for the plasmodesmal targeting, serves essential roles for the PDLP5 function. In addition to its role for membrane anchoring, the TMD mediates PDLP5 self-interaction and carries an evolutionarily conserved motif that is essential for PDLP5 to regulate cell-to-cell movement. Computational modeling-based analyses suggest that PDLP TMDs have high propensities to dimerize. We discuss how a specific mode(s) of TMD dimerization might serve as a common mechanism for PDLP5 and other PDLP members to regulate cell-to-cell movement.

Dimerization of the Transmembrane Domain of Mucin 16

Mucin 16 (MUC16) is a transmembrane protein produced by epithelial cells, and its main role is to contribute to the formation of a protective mucous barrier to inhibit debris, pathogens and viruses from entering the cells. However, when overexpressed, MUC16 can lead to cancers of the breast and ovary. Recent studies have shown that in cancer cells, MUC16 is trafficked to the nucleus, but little is known regarding the mechanism of this trafficking. One possible explanation may involve dimerization of MUC16, as shown in the case of mucin 1 (MUC1). Overexpression of the transmembrane MUC1 is also associated with various forms of cancer. Recent studies have shown that MUC1 is trafficked to the nucleus, where it may act as a transcription factor regulating the expression of genes associated with cell growth. The nuclear trafficking of MUC1 is dependent on the formation of MUC1 dimers, formed through inter‐disulfide bonds by two cysteine residues near the end of the transmembrane domain (TMD) of MUC1.The TMD of MUC16 (FWAVILIGLAGLLGVIT CLI C) has two cysteine residues that reside in the end of the TMD, which led us to the hypothesis that MUC16 may also form cysteine‐mediated dimers. The ToxR assay, a technique to measure transmembrane domain dimerization, was used to investigate the roles of the cysteine residues in MUC16 TMD dimerization. Our preliminary results show that the cysteine residues in the TMD play a role in dimerization, but not as significant as in the case of MUC1 TMD. These results suggest that noncovalent interactions may be important for MUC16 TMD dimerization. Understanding the formation of MUC16 dimers, similar to MUC1, is important because dimerization may serve as a precursor to the nuclear trafficking of MUC16.

Structural basis of the transmembrane domain dimerization and rotation in the activation mechanism of the TRKA receptor by nerve growth factor

Tropomyosin-receptor kinases (TRKs) are essential for the development of the nervous system. The molecular mechanism of TRKA activation by its ligand nerve growth factor (NGF) is still unsolved. Recent results indicate that at endogenous levels most of TRKA is in a monomer-dimer equilibrium and that the binding of NGF induces an increase of the dimeric and oligomeric forms of this receptor. An unsolved issue is the role of the TRKA transmembrane domain (TMD) in the dimerization of TRKA and the structural details of the TMD in the active dimer receptor. Here, we found that the TRKA-TMD can form dimers, identified the structural determinants of the dimer interface in the active receptor, and validated this interface through site-directed mutagenesis together with functional and cell differentiation studies. Using in vivo cross-linking, we found that the extracellular juxtamembrane region is reordered after ligand binding. Replacement of some residues in the juxtamembrane region with cysteine resulted in ligand-independent active dimers and revealed the preferred dimer interface. Moreover, insertion of leucine residues into the TMD helix induced a ligand-independent TRKA activation, suggesting that a rotation of the TMD dimers underlies NGF-induced TRKA activation. Altogether, our findings indicate that the transmembrane and juxtamembrane regions of TRKA play key roles in its dimerization and activation by NGF.

Investigating the Role of the Transmembrane Helix of EphA2 in Signal Transduction across the Plasma Membrane

Receptor tyrosine kinases (RTKs), the second largest class of membrane receptors, transduce biochemical signals across the cell membrane via lateral dimerization or oligomerization. RTKs control cell growth, differentiation and motility, and are involved in many diseases such as cancer. The single transmembrane domains of RTKs have been hypothesized to play important roles in signal transduction. First, sequence-specific interactions between the transmembrane domains are believed to contribute to the overall stability of RTK dimers and oligomers. Second, it has been proposed that the transmembrane domain dimer controls the spatial separation and orientation of the catalytic domains in the RTK oligomers. In this study, we focus on the transmembrane domain of EphA2, an RTK involved in many critical physiological processes, including tissue homeostasis, angiogenesis and tumorigenesis. Previous studies in our lab have shown that EphA2 can associate into two types of dimers, as well as clusters, depending on the type of ligand (dimeric EphrinA1-Fc, Ephrin A1 and the engineered peptide YSA), with distinct interaction interfaces in the extracellular region. By mutating key interaction motifs in the transmembrane domain and utilizing a quantitative FRET methodology, we seek to determine if the interaction between the transmembrane domains is affected by the type of bound ligands. These experiments will give new insights into the role of the transmembrane domain in EphA2 signaling.

Transmembrane domain dimerization induces cholesterol rafts in curved lipid bilayers.

Are the dimerization of transmembrane (TM) domains and the reorganization of the lipid bilayer two independent events? Does one event induce or interfere with the other? In this work, we have performed well-tempered metadynamics simulations to calculate the free energy cost to bend a model ternary lipid bilayer in the presence of a TM peptide in its dimer form. We have compared this result with the free energy cost needed to bend a bilayer-only system. Additionally, we have calculated the free energy cost to form a model TM peptide dimer quantitatively describing how lipids reorganize themselves in response to the increase of the membrane curvature and to the lipid-peptide interactions. Our results indicate that the formation of the peptide dimer inside the bilayer increases the cost of the membrane bending due to the spontaneous clustering of cholesterol molecules.

The dimerization of PSGL-1 is driven by the transmembrane domain via a leucine zipper motif.

P-selectin glycoprotein ligand-1 (PSGL-1) is a homodimeric mucin ligand that is important to mediate the earliest adhesive event during an inflammatory response by rapidly forming and dissociating the selectin-ligand adhesive bonds. Recent research indicates that the noncovalent associations between the PSGL-1 transmembrane domains (TMDs) can substitute for the C320-dependent covalent bond to mediate the dimerization of PSGL-1. In this article, we combined TOXCAT assays and molecular dynamics (MD) simulations to probe the mechanism of PSGL-1 dimerization. The results of TOXCAT assays and Martini coarse-grained molecular dynamics (CG MD) simulations demonstrated that PSGL-1 TMDs strongly dimerized in a natural membrane and a leucine zipper motif was responsible for the noncovalent dimerization of PSGL-1 TMD since mutations of the residues that occupied a or d positions in an (abcdefg)n leucine heptad repeat motif significantly reduced the dimer activity. Furthermore, we studied the effects of the disulfide bond on the PSGL-1 dimer using MD simulations. The disulfide bond was critical to form the leucine zipper structure, by which the disulfide bond further improved the stability of the PSGL-1 dimer. These findings provide insights to understand the transmembrane association of PSGL-1 that is an important structural basis for PSGL-1 preferentially binding to P-selectin to achieve its biochemical and biophysical functions.

Structural basis of the signal transduction via transmembrane domain of the human growth hormone receptor

BackgroundPrior studies of the human growth hormone receptor (GHR) revealed a distinct role of spatial rearrangements of its dimeric transmembrane domain in signal transduction across membrane. Detailed structural information obtained in the present study allowed elucidating the bases of such rearrangement and provided novel insights into receptor functioning. MethodsWe investigated the dimerization of recombinant TMD fragment GHR254–294 by means of high-resolution NMR in DPC micelles and molecular dynamics in explicit POPC membrane. ResultsWe resolved two distinct dimeric structures of GHR TMD coexisting in membrane-mimicking micellar environment and providing left- and right-handed helix-helix association via different dimerization motifs. Based on the available mutagenesis data, the conformations correspond to the dormant and active receptor states and are distinguished by cis-trans isomerization of Phe-Pro266 bond in the transmembrane helix entry. Molecular dynamic relaxations of the structures in lipid bilayer revealed the role of the proline residue in functionally significant rearrangements of the adjacent juxtamembrane region supporting alternation between protein-protein and protein-lipid interactions of this region that can be triggered by ligand binding. Also, the importance of juxtamembrane SS bonding for signal persistency, and somewhat unusual aspects of transmembrane region interaction with water molecules were demonstrated. ConclusionsTwo alternative dimeric structures of GHR TMD attributed to dormant and active receptor states interchange via allosteric rearrangements of transmembrane helices and extracellular juxtamembrane regions that support coordination between protein-protein and protein-lipid interactions. General significanceThis study provides a holistic vision of GHR signal transduction across the membrane emphasizing the role of protein-lipid interactions.

Excessive aggregation of membrane proteins in the Martini model.

The coarse-grained Martini model is employed extensively to study membrane protein oligomerization. While this approach is exceptionally promising given its computational efficiency, it is alarming that a significant fraction of these studies demonstrate unrealistic protein clusters, whose formation is essentially an irreversible process. This suggests that the protein–protein interactions are exaggerated in the Martini model. If this held true, then it would limit the applicability of Martini to study multi-protein complexes, as the rapidly clustering proteins would not be able to properly sample the correct dimerization conformations. In this work we first demonstrate the excessive protein aggregation by comparing the dimerization free energies of helical transmembrane peptides obtained with the Martini model to those determined from FRET experiments. Second, we show that the predictions provided by the Martini model for the structures of transmembrane domain dimers are in poor agreement with the corresponding structures resolved using NMR. Next, we demonstrate that the first issue can be overcome by slightly scaling down the Martini protein–protein interactions in a manner, which does not interfere with the other Martini interaction parameters. By preventing excessive, irreversible, and non-selective aggregation of membrane proteins, this approach renders the consideration of lateral dynamics and protein–lipid interactions in crowded membranes by the Martini model more realistic. However, this adjusted model does not lead to an improvement in the predicted dimer structures. This implicates that the poor agreement between the Martini model and NMR structures cannot be cured by simply uniformly reducing the interactions between all protein beads. Instead, a careful amino-acid specific adjustment of the protein–protein interactions is likely required.