Principles and Methods in Computational Membrane Protein Design

After decades of progress in computational protein design, the design of proteins folding and functioning in lipid membranes appears today as the next frontier. Some notable successes in the de novo design of simplified model membrane protein systems have helped articulate fundamental principles of protein folding, architecture and interaction in the hydrophobic lipid environment. These principles are reviewed here, together with the computational methods and approaches that were used to identify them. We provide an overview of the methodological innovations in the generation of new protein structures and functions and in the development of membrane-specific energy functions. We highlight the opportunities offered by new machine learning approaches applied to protein design, and by new experimental characterization techniques applied to membrane proteins. Although membrane protein design is in its infancy, it appears more reachable than previously thought.     

Single-Molecule Force Spectroscopy of Membrane Protein Folding

Single-molecule force spectroscopy is a unique method that can probe the structural changes of single proteins at a high spatiotemporal resolution while mechanically manipulating them over a wide force range. Here, we review the current understanding of membrane protein folding learned by using the force spectroscopy approach. Membrane protein folding in lipid bilayers is one of the most complex biological processes in which diverse lipid molecules and chaperone proteins are intricately involved. The approach of single protein forced unfolding in lipid bilayers has produced important findings and insights into membrane protein folding. This review provides an overview of the forced unfolding approach, including recent achievements and technical advances. Progress in the methods can reveal more interesting cases of membrane protein folding and clarify general mechanisms and principles.     

The Extraction Mechanism of Monoubiquitinated PEX5 from the Peroxisomal Membrane

The AAA ATPases PEX1?PEX6 extract PEX5, the peroxisomal protein shuttling receptor, from the peroxisomal membrane so that a new protein transport cycle can start. Extraction requires ubiquitination of PEX5 at residue 11 and involves a threading mechanism, but how exactly this occurs is unclear. We used a cell-free in vitro system and a variety of engineered PEX5 and ubiquitin molecules to challenge the extraction machinery. We show that PEX5 modified with a single ubiquitin is a substrate for extraction and extend previous findings proposing that neither the N- nor the C-terminus of PEX5 are required for extraction. Chimeric PEX5 molecules possessing a branched polypeptide structure at their C-terminal domains can still be extracted from the peroxisomal membrane thus suggesting that the extraction machinery can thread more than one polypeptide chain simultaneously. Importantly, we found that the PEX5-linked monoubiquitin is unfolded at a pre-extraction stage and, accordingly, an intra-molecularly cross-linked ubiquitin blocked extraction when conjugated to residue 11 of PEX5. Collectively, our data suggest that the PEX5-linked monoubiquitin is the extraction initiator and that the complete ubiquitin-PEX5 conjugate is threaded by PEX1?PEX6.     

Towards Decoding the Sequence-Based Grammar Governing the Functions of Intrinsically Disordered Protein Regions

A substantial portion of the proteome consists of intrinsically disordered regions (IDRs) that do not fold into well-defined 3D structures yet perform numerous biological functions and are associated with a broad range of diseases. It has been a long-standing enigma how different IDRs successfully execute their specific functions. Further putting a spotlight on IDRs are recent discoveries of functionally relevant biomolecular assemblies, which in some cases form through liquid-liquid phase separation. At the molecular level, the formation of biomolecular assemblies is largely driven by weak, multivalent, but selective IDR-IDR interactions. Emerging experimental and computational studies suggest that the primary amino acid sequences of IDRs encode a variety of their interaction behaviors. In this review, we focus on findings and insights that connect sequence-derived features of IDRs to their conformations, propensities to form biomolecular assemblies, selectivity of interaction partners, functions in the context of physiology and disease, and regulation of function. We also discuss directions of future research to facilitate establishing a comprehensive sequence-function paradigm that will eventually allow prediction of selective interactions and specificity of function mediated by IDRs.     

Fine Sampling of Sequence Space for Membrane Protein Structural Biology

We describe an enhancement of traditional genomics-based approaches to improve the success of structure determination of membrane proteins. Following a broad screen of sequence space to identify initial expression-positive targets, we employ a second step to select orthologs with closely related sequences to these hits. We demonstrate that a greater percentage of these latter targets express well and are stable in detergent, increasing the likelihood of identifying candidates that will ultimately yield structural information.     

Probing Interdomain Linkers and Protein Supertertiary Structure In Vitro and in Live Cells with Fluorescent Protein Resonance Energy Transfer

Many proteins are composed of independently-folded domains connected by flexible linkers. The primary sequence and length of such linkers can set the effective concentration for the tethered domains, which impacts rates of association and enzyme activity. The length of such linkers can be sensitive to environmental conditions, which raises questions as to how studies in dilute buffer relate to the highly-crowded cellular environment. To examine the role of linkers in domain separation, we measured Fluorescent Protein-Fluorescence Resonance Energy Transfer (FP-FRET) for a series of tandem FPs that varied in the length of their interdomain linkers. We used discrete molecular dynamics to map the underlying conformational distribution, which revealed intramolecular contact states that we confirmed with single molecule FRET. Simulations found that attached FPs increased linker length and slowed conformational dynamics relative to the bare linkers. This makes the CLYs poor sensors of inherent linker properties. However, we also showed that FP-FRET in CLYs was sensitive to solvent quality and macromolecular crowding making them potent environmental sensors. Finally, we targeted the same proteins to the plasma membrane of living mammalian cells to measure FP-FRET in cellulo. The measured FP-FRET when tethered to the plasma membrane was the same as that in dilute buffer. While caveats remain regarding photophysics, this suggests that the supertertiary conformational ensemble of these CLY proteins may not be affected by this specific cellular environment.     

Towards Molecular Understanding of the pH Dependence Characterizing NhaA of Which Structural Fold is Shared by Other Transporters

Na⁺/H⁺ antiporters comprise a super-family (CPA) of membrane proteins that are found in all kingdoms of life and are essential in cellular homeostasis of pH, Na⁺ and volume. Their activity is strictly dependent on pH, a property that underpins their role in pH homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystal structure provided insight into the architecture of this molecular machine. However, the mechanism of the strict pH dependence of NhaA is missing. Here, as a follow up of a recent evolutionary analysis that identified a ‘CPA motif’, we rationally designed three E. coli NhaA mutants: D133S, I134T, and the double mutant D133S-I134T. Exploring growth phenotype, transport activity and Li⁺-binding of the mutants, we revealed that Asp133 does not participate directly in proton binding, nor does it directly dictate the pH-dependent transport of NhaA. Strikingly, the variant I134T lost some of the pH control, and the D133S-Il134T double mutant retained Li⁺ binding in a pH independent fashion. Concurrent to loss of pH control, these mutants bound Li⁺ more strongly than the WT. Both positions are in close vicinity to the ion-binding site of the antiporter, attributing the results to electrostatic interaction between these residues and Asp164 of the ion-binding site. This is consistent with pH sensing resulting from direct coupling between cation binding and deprotonation in Asp164, which applies also to other CPA antiporters that are involved in human diseases.     

Impact on S. aureus and E. coli Membranes of Treatment with Chlorhexidine and Alcohol Solutions: Insights from Molecular Simulations and Nuclear Magnetic Resonance

Membranes form the first line of defence of bacteria against potentially harmful molecules in the surrounding environment. Understanding the protective properties of these membranes represents an important step towards development of targeted anti-bacterial agents such as sanitizers. Use of propanol, isopropanol and chlorhexidine can significantly decrease the threat imposed by bacteria in the face of growing anti-bacterial resistance via mechanisms that include membrane disruption. Here we have employed molecular dynamics simulations and nuclear magnetic resonance to explore the impact of chlorhexidine and alcohol on the S. aureus cell membrane, as well as the E. coli inner and outer membranes. We identify how sanitizer components partition into these bacterial membranes, and show that chlorhexidine is instrumental in this process.     

Membrane Binding and Homodimerization of Atg16 Via Two Distinct Protein Regions is Essential for Autophagy in Yeast

Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12–Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12–Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113–131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64–99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.     

Structural Insights into the Mechanism of Human T-cell Leukemia Virus Type 1 Gag Targeting to the Plasma Membrane for Assembly

Retroviral Gag targeting to the plasma membrane (PM) for assembly is mediated by the N-terminal matrix (MA) domain. For many retroviruses, Gag–PM interaction is dependent on phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). However, it has been shown that for human T-cell leukemia virus type 1 (HTLV-1), Gag binding to membranes is less dependent on PI(4,5)P₂ than HIV-1, suggesting that other factors may modulate Gag assembly. To elucidate the mechanism by which HTLV-1 Gag binds to the PM, we employed NMR techniques to determine the structure of unmyristoylated MA (myr(–)MA) and to characterize its interactions with lipids and liposomes. The MA structure consists of four α-helices and unstructured N- and C-termini. We show that myr(–)MA binds to PI(4,5)P₂ via the polar head and that binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups on the inositol ring, indicating that the MA–IP binding is governed by charge–charge interactions. The IP binding site was mapped to a well-defined basic patch formed by lysine and arginine residues. Using an NMR-based liposome binding assay, we show that PI(4,5)P₂ and phosphatidylserine enhance myr(–)MA binding in a synergistic fashion. Confocal microscopy data revealed formation of puncta on the PM of Gag expressing cells. However, G2A-Gag mutant, lacking myristoylation, is diffuse and cytoplasmic. These results suggest that although myr(–)MA binds to membranes, myristoylation appears to be key for formation of HTLV-1 Gag puncta on the PM. Altogether, these findings advance our understanding of a key mechanism in retroviral assembly.     

Towards Visual Proteomics at High Resolution

Traditionally, structural biologists approach the complexity of cellular proteomes in a reductionist manner. Proteomes are fractionated, their molecular components purified and studied one-by-one using the experimental methods for structure determination at their disposal. Visual proteomics aims at obtaining a holistic picture of cellular proteomes by studying them in situ, ideally in unperturbed cellular environments. The method that enables doing this at highest resolution is cryo-electron tomography. It allows to visualize cellular landscapes with molecular resolution generating maps or atlases revealing the interaction networks which underlie cellular functions in health and in disease states. Current implementations of cryo ET do not yet realize the full potential of the method in terms of resolution and interpretability. To this end, further improvements in technology and methodology are needed. This review describes the state of the art as well as measures which we expect will help overcoming current limitations.     

Microsecond Dynamics During the Binding-induced Folding of an Intrinsically Disordered Protein

Tau is an intrinsically disordered protein implicated in many neurodegenerative diseases. The repeat domain fragment of tau, tau-K18, is known to undergo a disorder to order transition in the presence of lipid micelles and vesicles, in which helices form in each of the repeat domains. Here, the mechanism of helical structure formation, induced by a phospholipid mimetic, sodium dodecyl sulfate (SDS) at sub-micellar concentrations, has been studied using multiple biophysical probes. A study of the conformational dynamics of the disordered state, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS) has indicated the presence of an intermediate state, I, in equilibrium with the unfolded state, U. The cooperative binding of the ligand (L), SDS, to I has been shown to induce the formation of a compact, helical intermediate (IL₅) within the dead time (∼37 µs) of a continuous flow mixer. Quantitative analysis of the PET-FCS data and the ensemble microsecond kinetic data, suggests that the mechanism of induction of helical structure can be described by a U ↔ I ↔ IL₅ ↔ FL₅ mechanism, in which the final helical state, FL₅, forms from IL₅ with a time constant of 50–200 µs. Finally, it has been shown that the helical conformation is an aggregation-competent state that can directly form amyloid fibrils.     

Molecular Characterisation of Titin N2A and Its Binding of CARP Reveals a Titin/Actin Cross-linking Mechanism

Striated muscle responds to mechanical overload by rapidly up-regulating the expression of the cardiac ankyrin repeat protein, CARP, which then targets the sarcomere by binding to titin N2A in the I-band region. To date, the role of this interaction in the stress response of muscle remains poorly understood. Here, we characterise the molecular structure of the CARP-receptor site in titin (UN2A) and its binding of CARP. We find that titin UN2A contains a central three-helix bundle fold (ca 45 residues in length) that is joined to N- and C-terminal flanking immunoglobulin domains by long, flexible linkers with partial helical content. CARP binds titin by engaging an α-hairpin in the three-helix fold of UN2A, the C-terminal linker sequence, and the BC loop in Ig81, which jointly form a broad binding interface. Mutagenesis showed that the CARP/N2A association withstands sequence variations in titin N2A and we use this information to evaluate 85 human single nucleotide variants. In addition, actin co-sedimentation, co-transfection in C2C12 cells, proteomics on heart lysates, and the mechanical response of CARP-soaked myofibrils imply that CARP induces the cross-linking of titin and actin myofilaments, thereby increasing myofibril stiffness. We conclude that CARP acts as a regulator of force output in the sarcomere that preserves muscle mechanical performance upon overload stress.     

Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

Molecular Determinants of Human T-cell Leukemia Virus Type 1 Gag Targeting to the Plasma Membrane for Assembly

Assembly of human T-cell leukemia virus type 1 (HTLV-1) particles is initiated by the trafficking of virally encoded Gag polyproteins to the inner leaflet of the plasma membrane (PM). Gag–PM interactions are mediated by the matrix (MA) domain, which contains a myristoyl group (myr) and a basic patch formed by lysine and arginine residues. For many retroviruses, Gag–PM interactions are mediated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]; however, previous studies suggested that HTLV-1 Gag–PM interactions and therefore virus assembly are less dependent on PI(4,5)P₂. We have recently shown that PI(4,5)P₂ binds directly to HTLV-1 unmyristoylated MA [myr(–)MA] and that myr(–)MA binding to membranes is significantly enhanced by inclusion of phosphatidylserine (PS) and PI(4,5)P₂. Herein, we employed structural, biophysical, biochemical, mutagenesis, and cell-based assays to identify residues involved in MA–membrane interactions. Our data revealed that the lysine-rich motif (Lys47, Lys48, and Lys51) constitutes the primary PI(4,5)P₂–binding site. Furthermore, we show that arginine residues 3, 7, 14 and 17 located in the unstructured N-terminus are essential for MA binding to membranes containing PS and/or PI(4,5)P₂. Substitution of lysine and arginine residues severely attenuated virus-like particle production, but only the lysine residues could be clearly correlated with reduced PM binding. These results support a mechanism by which HTLV-1 Gag targeting to the PM is mediated by a trio engagement of the myr group, Arg-rich and Lys-rich motifs. These findings advance our understanding of a key step in retroviral particle assembly.     

The PCDDB (Protein Circular Dichroism Data Bank): A Bioinformatics Resource for Protein Characterisations and Methods Development

The Protein Circular Dichroism Data Bank (PCDDB) [https://pcddb.cryst.bbk.ac.uk] is an established resource for the biological, biophysical, chemical, bioinformatics, and molecular biology communities. It is a freely-accessible repository of validated protein circular dichroism (CD) spectra and associated sample and metadata, with entries having links to other bioinformatics resources including, amongst others, structure (PDB), AlphaFold, and sequence (UniProt) databases, as well as to published papers which produced the data and cite the database entries. It includes primary (unprocessed) and final (processed) spectral data, which are available in both text and pictorial formats, as well as detailed sample and validation information produced for each of the entries. Recently the metadata content associated with each of the entries, as well as the number and structural breadth of the protein components included, have been expanded. The PCDDB includes data on both wild-type and mutant proteins, and because CD studies primarily examine proteins in solution, it also contains examples of the effects of different environments on their structures, plus thermal unfolding/folding series. Methods for both sequence and spectral comparisons are included.The data included in the PCDDB complement results from crystal, cryo-electron microscopy, NMR spectroscopy, bioinformatics characterisations and classifications, and other structural information available for the proteins via links to other databases. The entries in the PCDDB have been used for the development of new analytical methodologies, for interpreting spectral and other biophysical data, and for providing insight into structures and functions of individual soluble and membrane proteins and protein complexes.     

Liquid–Liquid Phase Separation at the Plasma Membrane–Cytosol Interface: Common Players in Adhesion,Motility, and Synaptic Function

Networks of scaffold proteins and enzymes assemble at the interface between the cytosol and specific sites of the plasma membrane, where these networks guide distinct cellular functions. Some of these plasma membrane–associated platforms (PMAPs) include shared core components that are able to establish specific protein–protein interactions, to produce distinct supramolecular assemblies regulating dynamic processes as diverse as cell adhesion and motility, or the formation and function of neuronal synapses. How cells organize such dynamic networks is still an open question. In this review we introduce molecular networks assembling at the edge of migrating cells, and at pre– and postsynaptic sites, which share molecular players that can drive the assembly of biomolecular condensates. Very recent experimental evidence has highlighted the emerging role of some of these multidomain/scaffold proteins belonging to the GIT, liprin-α and ELKS/ERC families as drivers of liquid–liquid phase separation (LLPS). The data point to an important role of LLPS: (i) in the formation of PMAPs at the edge of migrating cells, where LLPS appears to be involved in promoting protrusion and the turnover of integrin–mediated adhesions, to allow forward cell translocation; (ii) in the assembly of the presynaptic active zone and of the postsynaptic density deputed to the release and reception of neurotransmitter signals, respectively. The recent results indicate that LLPS at cytosol–membrane interfaces is suitable not only for the regulation of active cellular processes, but also for the continuous spatial rearrangements of the molecular interactions involved in these dynamic processes.