Their Transmembrane Domains Research Articles

Deep mutational scanning reveals transmembrane features governing surface expression of the B cell antigen receptor.

B cells surveil the body for foreign matter using their surface-expressed B cell antigen receptor (BCR), a tetrameric complex comprising a membrane-tethered antibody (mIg) that binds antigens and a signaling dimer (CD79AB) that conveys this interaction to the B cell. Recent cryogenic electron microscopy (cryo-EM) structures of IgM and IgG isotype BCRs provide the first complete views of their architecture, revealing that the largest interaction surfaces between the mIg and CD79AB are in their transmembrane domains (TMDs). These structures support decades of biochemical work interrogating the requirements for assembly of a functional BCR and provide the basis for explaining the effects of mutations. Here we report a focused saturating mutagenesis to comprehensively characterize the nature of the interactions in the mIg TMD that are required for BCR surface expression. We examined the effects of 600 single-amino-acid changes simultaneously in a pooled competition assay and quantified their effects by next-generation sequencing. Our deep mutational scanning results reflect a feature-rich TMD sequence, with some positions completely intolerant to mutation and others requiring specific biochemical properties such as charge, polarity or hydrophobicity, emphasizing the high value of saturating mutagenesis over, for example, alanine scanning. The data agree closely with published mutagenesis and the cryo-EM structures, while also highlighting several positions and surfaces that have not previously been characterized or have effects that are difficult to rationalize purely based on structure. This unbiased and complete mutagenesis dataset serves as a reference and framework for informed hypothesis testing, design of therapeutics to regulate BCR surface expression and to annotate patient mutations.

Inconspicuous Yet Indispensable: The Coronavirus Spike Transmembrane Domain.

Membrane-spanning portions of proteins' polypeptide chains are commonly known as their transmembrane domains (TMDs). The structural organisation and dynamic behaviour of TMDs from proteins of various families, be that receptors, ion channels, enzymes etc., have been under scrutiny on the part of the scientific community for the last few decades. The reason for such attention is that, apart from their obvious role as an "anchor" in ensuring the correct orientation of the protein's extra-membrane domains (in most cases functionally important), TMDs often actively and directly contribute to the operation of "the protein machine". They are capable of transmitting signals across the membrane, interacting with adjacent TMDs and membrane-proximal domains, as well as with various ligands, etc. Structural data on TMD arrangement are still fragmentary at best due to their complex molecular organisation as, most commonly, dynamic oligomers, as well as due to the challenges related to experimental studies thereof. Inter alia, this is especially true for viral fusion proteins, which have been the focus of numerous studies for quite some time, but have provoked unprecedented interest in view of the SARS-CoV-2 pandemic. However, despite numerous structure-centred studies of the spike (S) protein effectuating target cell entry in coronaviruses, structural data on the TMD as part of the entire spike protein are still incomplete, whereas this segment is known to be crucial to the spike's fusogenic activity. Therefore, in attempting to bring together currently available data on the structure and dynamics of spike proteins' TMDs, the present review aims to tackle a highly pertinent task and contribute to a better understanding of the molecular mechanisms underlying virus-mediated fusion, also offering a rationale for the design of novel efficacious methods for the treatment of infectious diseases caused by SARS-CoV-2 and related viruses.

EMC rectifies the topology of multipass membrane proteins

Most eukaryotic multipass membrane proteins are inserted into the membrane of the endoplasmic reticulum. Their transmembrane domains (TMDs) are thought to be inserted co-translationally as they emerge from a membrane-bound ribosome. Here we find that TMDs near the carboxyl terminus of mammalian multipass proteins are inserted post-translationally by the endoplasmic reticulum membrane protein complex (EMC). Site-specific crosslinking shows that the EMC’s cytosol-facing hydrophilic vestibule is adjacent to a pre-translocated C-terminal tail. EMC-mediated insertion is mostly agnostic to TMD hydrophobicity, favored for short uncharged C-tails and stimulated by a preceding unassembled TMD bundle. Thus, multipass membrane proteins can be released by the ribosome–translocon complex in an incompletely inserted state, requiring a separate EMC-mediated post-translational insertion step to rectify their topology, complete biogenesis and evade quality control. This sequential co-translational and post-translational mechanism may apply to ~250 diverse multipass proteins, including subunits of the pentameric ion channel family that are crucial for neurotransmission.

Interaction of Substrates with γ-Secretase at the Level of Individual Transmembrane Helices-A Methodological Approach.

Intramembrane proteases, such as γ secretase, typically recruit multiple substrates from an excess of single-span membrane proteins. It is currently unclear to which extent substrate recognition depends on specific interactions of their transmembrane domains (TMDs) with TMDs of a protease. Here, we investigated a large number of potential pairwise interactions between TMDs of γ secretase and a diverse set of its substrates using two different configurations of BLaTM, a genetic reporter system. Our results reveal significant interactions between TMD2 of presenilin, the enzymatic subunit of γ secretase, and the TMD of the amyloid precursor protein, as well as of several other substrates. Presenilin TMD2 is a prime candidate for substrate recruitment, as has been shown from previous studies. In addition, the amyloid precursor protein TMD enters interactions with presenilin TMD 4 as well as with the TMD of nicastrin. Interestingly, the Gly-rich interfaces between the amyloid precursor protein TMD and presenilin TMDs 2 and 4 are highly similar to its homodimerization interface. In terms of methodology, the economics of the newly developed library-based method could prove to be a useful feature in related future work for identifying heterotypic TMD-TMD interactions within other biological contexts.

Structure of Mycobacterium tuberculosis Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases.

Mycobacterium tuberculosis adenylyl cyclase (AC) Rv1625c/Cya is an evolutionary ancestor of the mammalian membrane ACs and a model system for studies of their structure and function. Although the vital role of ACs in cellular signalling is well established, the function of their transmembrane (TM) regions remains unknown. Here, we describe the cryo-EM structure of Cya bound to a stabilizing nanobody at 3.6 Å resolution. The TM helices 1-5 form a structurally conserved domain that facilitates the assembly of the helical and catalytic domains. The TM region contains discrete pockets accessible from the extracellular and cytosolic side of the membrane. Neutralization of the negatively charged extracellular pocket Ex1 destabilizes the cytosolic helical domain and reduces the catalytic activity of the enzyme. The TM domain acts as a functional component of Cya, guiding the assembly of the catalytic domain and providing the means for direct regulation of catalytic activity in response to extracellular ligands.

Structural and Functional Insights into the Transmembrane Domain Association of Eph Receptors.

Eph receptors are the largest family of receptor tyrosine kinases and by interactions with ephrin ligands mediate a myriad of processes from embryonic development to adult tissue homeostasis. The interaction of Eph receptors, especially at their transmembrane (TM) domains is key to understanding their mechanism of signal transduction across cellular membranes. We review the structural and functional aspects of EphA1/A2 association and the techniques used to investigate their TM domains: NMR, molecular modelling/dynamics simulations and fluorescence. We also introduce transmembrane peptides, which can be used to alter Eph receptor signaling and we provide a perspective for future studies.

Chaperoning transmembrane helices in the lipid bilayer.

Elimination of membrane proteins often requires recognition of their transmembrane domains (TMDs) in the lipid bilayer. In this issue, Arines et al. (2020. J. Cell Biol.https://doi.org/10.1083/jcb.202001116) show that in Saccharomyces cerevisiae, the vacuole-associated Rsp5 ubiquitin ligase uses a TMD in substrate adaptor Ssh4 to recognize membrane helices in Ypq1, which targets this lysine transporter for lysosomal degradation during lysine starvation.

Structural and functional diversity calls for a new classification of ABC transporters.

Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs.

Experimental determination and data-driven prediction of homotypic transmembrane domain interfaces

Interactions between their transmembrane domains (TMDs) frequently support the assembly of single-pass membrane proteins to non-covalent complexes. Yet, the TMD-TMD interactome remains largely uncharted. With a view to predicting homotypic TMD-TMD interfaces from primary structure, we performed a systematic analysis of their physical and evolutionary properties. To this end, we generated a dataset of 50 self-interacting TMDs. This dataset contains interfaces of nine TMDs from bitopic human proteins (Ire1, Armcx6, Tie1, ATP1B1, PTPRO, PTPRU, PTPRG, DDR1, and Siglec7) that were experimentally identified here and combined with literature data. We show that interfacial residues of these homotypic TMD-TMD interfaces tend to be more conserved, coevolved and polar than non-interfacial residues. Further, we suggest for the first time that interface positions are deficient in β-branched residues, and likely to be located deep in the hydrophobic core of the membrane. Overrepresentation of the GxxxG motif at interfaces is strong, but that of (small)xxx(small) motifs is weak. The multiplicity of these features and the individual character of TMD-TMD interfaces, as uncovered here, prompted us to train a machine learning algorithm. The resulting prediction method, THOIPA (www.thoipa.org), excels in the prediction of key interface residues from evolutionary sequence data.

Conformationally Flexible Sites within the Transmembrane Helices of Amyloid Precursor Protein and Notch1 Receptor.

Intramembrane proteases typically cleave multiple substrates within their transmembrane domains (TMDs). Because substrate TMDs lack a consensus sequence around their scissile sites, it remains unclear how the enzyme discriminates substrates from nonsubstrates at the level of their TMDs. Here, we compare the previously well investigated TMDs of γ-secretase substrates C99 and Notch1 in terms of helix flexibility. Our results reveal that the low-stability site neigboring a functionally relevant diglycine hinge of C99 has an equivalent in the Notch1 TMD. This suggests that the tetra-alanine motif of Notch1 also functions as a hinge which may facilitate its cleavage.

In Silico Insights into HIV-1 Vpu-Tetherin Interactions and Its Mutational Counterparts.

Tetherin, an interferon-induced host protein encoded by the bone marrow stromal antigen 2 (BST2/CD317/HM1.24) gene, is involved in obstructing the release of many retroviruses and other enveloped viruses by cross-linking the budding virus particles to the cell surface. This activity is antagonized in the case of human immunodeficiency virus (HIV)-1 wherein its accessory protein Viral Protein U (Vpu) interacts with tetherin, causing its downregulation from the cell surface. Vpu and tetherin connect through their transmembrane (TM) domains, culminating into events leading to tetherin degradation by recruitment of β-TrCP2. However, mutations in the TM domains of both proteins are reported to act as a resistance mechanism to Vpu countermeasure impacting tetherin’s sensitivity towards Vpu but retaining its antiviral activity. Our study illustrates the binding aspects of blood-derived, brain-derived, and consensus HIV-1 Vpu with tetherin through protein–protein docking. The analysis of the bound complexes confirms the blood-derived Vpu–tetherin complex to have the best binding affinity as compared to other two. The mutations in tetherin and Vpu are devised computationally and are subjected to protein–protein interactions. The complexes are tested for their binding affinities, residue connections, hydrophobic forces, and, finally, the effect of mutation on their interactions. The single point mutations in tetherin at positions L23Y, L24T, and P40T, and triple mutations at {L22S, F44Y, L37I} and {L23T, L37T, T45I}, while single point mutations in Vpu at positions A19H and W23Y and triplet of mutations at {V10K, A11L, A19T}, {V14T, I18T, I26S}, and {A11T, V14L, A15T} have revealed no polar contacts with minimal hydrophobic interactions between Vpu and tetherin, resulting in reduced binding affinity. Additionally, we have explored the aggregation potential of tetherin and its association with the brain-derived Vpu protein. This work is a possible step toward an understanding of Vpu–tetherin interactions.

On the contributing role of the transmembrane domain for subunit-specific sensitivity of integrin activation

Integrins are α/β heterodimeric transmembrane adhesion receptors. Evidence exists that their transmembrane domain (TMD) separates upon activation. Subunit-specific differences in activation sensitivity of integrins were reported. However, whether sequence variations in the TMD lead to differential TMD association has remained elusive. Here, we show by molecular dynamics simulations and association free energy calculations on TMDs of integrin αIIbβ3, αvβ3, and α5β1 that αIIbβ3 TMD is most stably associated; this difference is related to interaction differences across the TMDs. The order of TMD association stability is paralleled by the basal activity of these integrins, which suggests that TMD differences can have a decisive effect on integrin conformational free energies. We also identified a specific order of clasp disintegration upon TMD dissociation, which suggests that the closed state of integrins may comprise several microstates. Our results provide unprecedented insights into a possibly contributing role of TMD towards subunit-specific sensitivity of integrin activation.

Insights into eukaryotic evolution from transmembrane domain lengths

Biological membranes, comprised of proteins anchored by their trans-membrane domains (TMDs) creating a semi-permeable phase with lipid constituents, serve as ‘checkposts’ for not only intracellular trafficking in eukaryotic cells but also for material transactions of all living cells with external environments. Hydropathy (or hydrophobicity) plots of ‘bitopic’ proteins (i.e. having single alpha-helical TMDs) are routinely utilized in biochemistry texts for predicting their TMDs. The number of amino acids (i.e. TMD length) embedded as alpha-helices may serve as indicators of thickness of biological membranes in which they reside under assumptions that are universally applied for fixing window sizes for identifying TMDs using hydropathy plots. In this work we explore variations in thickness of different eukaryotic biological membranes (reflected by TMD lengths of their resident proteins) over evolutionary time scales. Rigorous in silico analyses of over 23,000 non-redundant membrane proteins residing in different subcellular locations from over 200 genomes of fungi, plants, non-mammalian vertebrates and mammals, reveal that differences in plasma membrane and organellar TMD lengths have decreased over time (scales) of eukaryotic cellular evolution. While earlier work has indicated decreasing differences in TMD lengths with increasing ‘perceived’ organismal complexity, this work is the first report on TMD length variations as a function of evolutionary time of eukaryotic cellular systems. We report that differences in TMD lengths of bitopic proteins residing in plasma membranes and other intra-cellular locations have decreased with evolutionary time, suggesting better/more avenues of intracellular trafficking in the emergence of eukaryotic organisms.

A single amino acid in MRGPRX2 necessary for binding and activation by pruritogens

A single amino acid in MRGPRX2 necessary for binding and activation by pruritogens

Transmembrane features governing Fc receptor CD16A assembly with CD16A signaling adaptor molecules

Many activating immunoreceptors associate with signaling adaptor molecules like FcεR1γ or CD247. FcεR1γ and CD247 share high sequence homology and form disulphide-linked homodimers that contain a pair of acidic aspartic acid residues in their transmembrane (TM) domains that mediate assembly, via interaction with an arginine residue at a similar register to these aspartic acids, with the activating immunoreceptors. However, this model cannot hold true for receptors like CD16A, whose TM domains do not contain basic residues. We have carried out an extensive site-directed mutagenesis analysis of the CD16A receptor complex and now report that the association of receptor with the signaling adaptor depends on a network of polar and aromatic residues along the length of the TM domain. Molecular modeling indicates that CD16A TM residues F202, D205, and T206 form the core of the membrane-embedded trimeric interface by establishing highly favorable contacts to the signaling modules through rearrangement of a hydrogen bond network previously identified in the CD247 TM dimer solution NMR structure. Strikingly, the amino acid D205 also regulates the turnover and surface expression of CD16A in the absence of FcεR1γ or CD247. Modeling studies indicate that similar features underlie the association of other activating immune receptors, including CD64 and FcεR1α, with signaling adaptor molecules, and we confirm experimentally that equivalent F, D, and T residues in the TM domain of FcεR1α markedly influence the biology of this receptor and its association with FcεR1γ.

Getting membrane proteins on and off the shuttle bus between the endoplasmic reticulum and the Golgi complex.

Secretory proteins exit the endoplasmic reticulum (ER) in coat protein complex II (COPII)-coated vesicles and then progress through the Golgi complex before delivery to their final destination. Soluble cargo can be recruited to ER exit sites by signal-mediated processes (cargo capture) or by bulk flow. For membrane proteins, a third mechanism, based on the interaction of their transmembrane domain (TMD) with lipid microdomains, must also be considered. In this Commentary, I review evidence in favor of the idea that partitioning of TMDs into bilayer domains that are endowed with distinct physico-chemical properties plays a pivotal role in the transport of membrane proteins within the early secretory pathway. The combination of such self-organizational phenomena with canonical intermolecular interactions is most likely to control the release of membrane proteins from the ER into the secretory pathway.

Transmembrane Domain Lengths Serve as Signatures of Organismal Complexity and Viral Transport Mechanisms.

It is known that membrane proteins are important in various secretory pathways, with a possible role of their transmembrane domains (TMDs) as sorting determinant factors. One key aspect of TMDs associated with various “checkposts” (i.e. organelles) of intracellular trafficking is their length. To explore possible linkages in organisms with varying “complexity” and differences in TMD lengths of membrane proteins associated with different organelles (such as Endoplasmic Reticulum, Golgi, Endosomes, Nucleus, Plasma Membrane), we analyzed ~70000 membrane protein sequences in over 300 genomes of fungi, plants, non-mammalian vertebrates and mammals. We report that as we move from simpler to complex organisms, variation in organellar TMD lengths decreases, especially compared to their respective plasma membranes, with increasing organismal complexity. This suggests an evolutionary pressure in modulating length of TMDs of membrane proteins with increasing complexity of communication between sub-cellular compartments. We also report functional applications of our findings by discovering remarkable distinctions in TMD lengths of membrane proteins associated with different intracellular transport pathways. Finally, we show that TMD lengths extracted from viral proteins can serve as somewhat weak indicators of viral replication sites in plant cells but very strong indicators of different entry pathways employed by animal viruses.

Predicting the Role of a Single Amino Acids in the Dimerization of Transmembrane Helices

Single-span membrane proteins take part in biological processes. In many cases their function has been shown to require dimerization via their transmembrane domains (TMDs). Research into the dimerization of TMDs has revealed sequence dependence, driven by a combination of amino acids. The helix interactions of multi-pass membrane proteins are well understood, as analysis of crystal structures shows that helix interfaces tend to contain small, polar and highly conserved residues. Unfortunately, for single-pass proteins structural data is scarce.The aim of this study is to characterize interfaces of TMDs that show strong self-interaction. In one study of this kind, we examine amino acid composition and conservation in order to identify patterns characteristic of interface residues. Our aim is to develop a method to predict the TMD interfaces from sequences alone.We used the ToxR assay in Escherichia coli to measure TMD homodimerization. In this assay, dimerization driven by TMDs results in the transcriptional activation of ctx promoter, inducing the expression of a reporter enzyme. The amount of reporter enzyme activity is a measure of the strength of the dimerization.Here, we present the scanning mutagenesis followed by ToxR analysis of 10 TMDs with strong self-interaction. Mutations were made to all amino acids within the TM helix. Some TMD interfaces were found to contain sequence patterns previously associated with parallel helix interactions, such as the GxxxG motif. However, in other cases motif searching would not have identified the interface, and the key residues in the interactions were diverse. The conservation of each amino acid in each TMD was calculated based on amino acid alignments against homologs. Preliminary analysis shows that amino acid conservation is a good indicator for the interface of some, but not all interacting TMDs.

Sequence Analysis of a Subset of Plasma Membrane Raft Proteome Containing CXXC Metal Binding Motifs

In an attempt to identify the metal sensing proteins localized to mammalian plasma membrane, the authors screened a list of 300 raft associated proteins that are involved in cellular signaling mechanisms by searching the presence of metal thionin (CXXC) motifs. 50 proteins were found to possess CXXC motifs that could act as potential metal sensing proteins. The authors determined membrane topologies of the above CXXC motif containing proteins using TM-pred and analyzed the positions of their transmembrane (TM) domains using Bio-edit software. Based on the topology of CXXC domains, the authors classified all the raft-associated metal sensing proteins into six categories. They are (i) Exoplasmic tails with CXXC motif, (ii) Exoplasmic loops with CXXC motif, (iii) Cytosolic tails with CXXC motif, (iv) Cytosolic loop with CXXC motif, (v) TM domains with CXXC motifs, (vi) Proteins with multiple topologies of CXXC motif. The authors' study will lead to understanding of the raft-mediated mechanism of heavy metal sensing and signaling in mammalian cells.

Vacuolar SNARE Protein Transmembrane Domains Serve as Nonspecific Membrane Anchors with Unequal Roles in Lipid Mixing

Membrane fusion is induced by SNARE complexes that are anchored in both fusion partners. SNAREs zipper up from the N to C terminus bringing the two membranes into close apposition. Their transmembrane domains (TMDs) might be mere anchoring devices, deforming bilayers by mechanical force. Structural studies suggested that TMDs might also perturb lipid structure by undergoing conformational transitions or by zipping up into the bilayer. Here, we tested this latter hypothesis, which predicts that the activity of SNAREs should depend on the primary sequence of their TMDs. We replaced the TMDs of all vacuolar SNAREs (Nyv1, Vam3, and Vti1) by a lipid anchor, by a TMD from a protein unrelated to the membrane fusion machinery, or by artificial leucine-valine sequences. Individual exchange of the native SNARE TMDs against an unrelated transmembrane anchor or an artificial leucine-valine sequence yielded normal fusion activities. Fusion activity was also preserved upon pairwise exchange of the TMDs against unrelated peptides, which eliminates the possibility for specific TMD-TMD interactions. Thus, a specific primary sequence or zippering beyond the SNARE domains is not a prerequisite for fusion. Lipid-anchored Vti1 was fully active, and lipid-anchored Nyv1 permitted the reaction to proceed up to hemifusion, and lipid-anchored Vam3 interfered already before hemifusion. The unequal contribution of proteinaceous TMDs on Vam3 and Nyv1 suggests that Q- and R-SNAREs might make different contributions to the hemifusion intermediate and the opening of the fusion pore. Furthermore, our data support the view that SNARE TMDs serve as nonspecific membrane anchors in vacuole fusion.