Biology subjects

Svergun, D. I.

Publications and source records attributed to Svergun, D. I..

17 recordsLinked to original sources

bioRxiv2022

Structural determinants of homophilic adhesion by receptor tyrosine phosphatases

The type IIB receptor protein tyrosine phosphatases (R2B RPTPs) are cell surface transmembrane proteins that engage in cell adhesion via their extracellular domains (ECDs) and cell signaling via their cytoplasmic phosphatase domains. The ECDs of R2B RPTPs form stable, homophilic, trans interactions between adjacent cell membranes. Previous work has demonstrated how one family member, PTPRM, forms homodimers; however, the determinants of homophilic specificity remain unknown. We have solved the X-ray crystal structure of the membrane-distal, N-terminal domains of PTPRK that form a head-to-tail dimer consistent with intermembrane adhesion. Comparison with the PTPRM structure demonstrates inter-domain conformational differences that may define homophilic specificity. Using small-angle X-ray scattering we determined the solution structures of the full-length ECDs of PTPRM and PTPRK, identifying that both are rigid, extended molecules that differ in their overall long-range conformation. Furthermore, we identify one residue, W351, within the interaction interface that differs between PTPRM and PTPRK and show that mutation to glycine, the equivalent residue in PTPRM, abolishes PTPRK dimer formation in vitro. This comparison of two members of the receptor tyrosine phosphatase family suggest that homophilic specificity is driven by a combination of shape complementarity and specific but limited sequence differences. SIGNIFICANCE STATEMENTCell-cell contacts are dynamically regulated, in part, by the actions of tyrosine kinases and phosphatases. The R2B family of receptor tyrosine phosphatases combine an adhesive extracellular domain with intracellular catalytic domains that bind and dephosphorylate key cell adhesion and polarity proteins. Previous work demonstrated that the extracellular domains form head-to-tail homodimers but, as the interface was composed of residues conserved across the family, homophilic specificity determinants remained unclear. We have used a range of structural techniques including X-ray crystallography, small angle X-ray scattering and AlphaFold modelling to demonstrate that, despite their similarity, two members of the R2B family possess significant differences in their overall shape. Our results support that a combination of subtle shape and sequence variations may determine homophilic binding.

biochemistry↗

bioRxiv2022

Herpes simplex virus 1 protein pUL21 stimulates cellular ceramide transport by activating CERT

Herpes simplex virus (HSV)-1 dramatically alters the architecture and protein composition of cellular membranes during infection, but its effects upon membrane lipid composition remain unclear. HSV-1 pUL21 is a virus-encoded protein phosphatase adaptor that promotes dephosphorylation of multiple cellular and virus proteins, including the cellular ceramide transport protein CERT. CERT mediates non- vesicular transport of ceramide from the ER to the trans-Golgi network, whereupon ceramide is converted to sphingomyelin and other sphingolipids that play important roles in cell proliferation, cell signalling and membrane trafficking. Using click chemistry to profile the kinetics of sphingolipid metabolism in cultured cells, we show that pUL21-mediated dephosphorylation activates CERT and increases the rate of ceramide to sphingomyelin conversion. Purified pUL21 and full-length CERT interact with sub-micromolar affinity and we map the domains responsible for the interaction. Solving the solution structure of the pUL21 C-terminal domain in complex with the CERT PH and START domains using small-angle X-ray scattering allows us to identify a single amino acid mutation on the surface of pUL21 that disrupts CERT binding in vitro and in cultured cells. Sphingolipid profiling demonstrates that ceramide to sphingomyelin conversion is severely diminished in the context of HSV- 1 infection, a defect that is compounded when infecting with a virus encoding the mutated form of pUL21 that lacks the ability to activate CERT. However, virus replication and spread are not significantly altered when pUL21-mediated CERT dephosphorylation is abolished, highlighting that dephosphorylation of other cellular and/or viral targets underpins the important role of pUL21 in HSV-1 biology. SignificanceHerpes simplex virus (HSV)-1 causes a life-long dormant infection of neurons, sporadically reactivating to manifest as cold-sores or genital herpes. While the impact of HSV-1 upon the protein content of infected cells has been well studied, we know relatively little about its impact upon cellular lipids. Using bioorthogonal labelling in cultured cells we show that HSV-1 protein pUL21 activates the key cellular lipid transport protein CERT to accelerate the conversion of ceramide to sphingomyelin. HSV-1 infection dramatically alters the kinetics of ceramide metabolism, leading to ceramide accumulation. Mutation of HSV-1 pUL21 to prevent CERT activation further enhances ceramide accumulation but this does not alter the replication or spread of HSV-1, highlighting that other cellular and/or viral proteins represent the critical targets of pUL21-mediated dephosphorylation in cultured cells.

microbiology↗

bioRxiv2022

Cryo-EM structure of an atypical proton-coupled peptide transporter: Di- and tripeptide permease C

Proton-coupled Oligopeptide Transporters (POTs) of the Major Facilitator Superfamily (MFS) mediate the uptake of short di- and tripeptides in all phyla of life. POTs are thought to constitute the most promiscuous class of MFS transporters, with the potential to transport more than 8400 unique substrates. Over the past two decades, transport assays and biophysical studies have shown that various orthologues and paralogues display differences in substrate selectivity. The E. coli genome codes for four different POTs, known as Di- and Tripeptide permeases A-D (DtpA-D). DtpC was shown previously to favor positively charged peptides as substrates. In this study, we describe, how we determined the structure of the 53 kDa DtpC by cryogenic electron microscopy (cryo-EM), and provide structural insights into the ligand specificity of this atypical POT. We collected and analyzed data on the transporter fused to split superfolder GFP (split sfGFP), in complex with a 52 kDa macrobody and with a 13 kDa nanobody. The latter sample was more stable, rigid and a significant fraction dimeric, allowing us to reconstruct a 3D volume of DtpC at a resolution of 2.7 [A]. This work provides a molecular explanation for the selectivity of DtpC, and highlights the value of small and rigid fiducial markers such as nanobodies for structure determination of low molecular weight integral membrane proteins lacking soluble domains.

biophysics↗

bioRxiv2021

Chaperoning of the histone octamer by the acidic domain of DNA repair factor APLF

Nucleosome assembly requires the coordinated deposition of histone complexes H3-H4 and H2A-H2B to form a histone octamer on DNA. In the current paradigm, specific histone chaperones guide the deposition of first H3-H4 and then H2A-H2B(1-5). Here, we show that the acidic domain of DNA repair factor APLF (APLFAD) can assemble the histone octamer in a single step, and deposit it on DNA to form nucleosomes. The crystal structure of the APLFAD-histone octamer complex shows that APLFAD tethers the histones in their nucleosomal conformation. Mutations of key aromatic anchor residues in APLFAD affect chaperone activity in vitro and in cells. Together, we propose that chaperoning of the histone octamer is a mechanism for histone chaperone function at sites where chromatin is temporarily disrupted. one sentence summaryHistone chaperone APLF assembles histones H2A-H2B/H3-H4 into histone octamers to deposit them onto DNA and form nucleosomes.

biochemistry↗

bioRxiv2021

N-terminal phosphorylation regulates the activity of Glycogen Synthase Kinase 3 from Plasmodium falciparum

As the decline of malaria cases stalled over the last five years, novel targets in Plasmodium falciparum are necessary for the development of new drugs. Glycogen Synthase Kinase (PfGSK3) has been identified as a potential target, since its selective inhibitors were shown to disrupt the parasites life cycle. Here, we show that PfGSK3 exhibits autophosphorylation, leading to an extensive phosphorylation both in vitro and in the parasite. In the uncanonical N-terminal region of the parasite enzyme, we identified several autophosphorylation sites that regulate the activity of PfGSK3. By combining molecular modeling with experimental small-angle X-ray scattering data, we show that increased PfGSK3 activity is promoted by conformational changes in the PfGSK3 N-terminus, triggered by N-terminal phosphorylation. Our work provides novel insights into the structure and regulation of the malarial PfGSK3.

biochemistry↗

bioRxiv2021

pUL21 is a viral phosphatase adaptor that promotes herpes simplex virus replication and spread

The herpes simplex virus (HSV)-1 protein pUL21 is essential for efficient virus replication and dissemination. While pUL21 has been shown to promote multiple steps of virus assembly and spread, the molecular basis of its function remained unclear. Here we identify that pUL21 is a virus-encoded adaptor of protein phosphatase 1 (PP1). pUL21 directs the dephosphorylation of cellular and virus proteins, including components of the viral nuclear egress complex, and we define a conserved non-canonical linear motif in pUL21 that is essential for PP1 recruitment. In vitro evolution experiments reveal that pUL21 directly antagonises the activity of the virus-encoded kinase pUS3, with growth and spread of pUL21 PP1-binding mutant viruses being restored when pUS3 activity is disrupted. This study shows that virus-directed phosphatase activity is essential for efficient herpesvirus assembly and spread, highlighting the fine balance between kinase and phosphatase activity required for optimal virus replication.

microbiology↗

bioRxiv2021

A non-structural pure enzyme protein forms an LCST type of stimuli-responsive and reversible hydrogel with novel structure and catalytic activity

Hydrogels have a wide range of applications such as in biomedicine, cosmetics and soft electronics. Compared to polymer hydrogels based on covalent bonding, protein hydrogels offer distinct advantages owing to their biocompatibility and better access to molecular engineering. However, pure and natural protein hydrogels have been seldom reported except for structural proteins like collagen and silk fibrin. Here, we report the unusual ability and mechanism of a unique natural enzyme, lipoate-protein ligase A (LplA) of E. coli to self-assemble into a stimuli-responsive and reversible hydrogel of the low critical solution temperature (LCST) type. This is the first globular and catalytic protein found to form a hydrogel in response to temperature, pH and the presence of ions. Protein structure based analysis reveals the key residues responsible for the gel formation and mutational studies confirms the essential roles of hydrogen bonding between the C-terminal domains and electrostatic interactions in the N-terminal domains. Characterization of phase transitions of wild type LplA and its mutants using small angle X-ray scattering (SAXS) yields details of the gelation process from initial dimer formation over a pre-gel-state to full network development. Further electron microscopic analyses and modeling of SAXS data suggest an unusual interlinked ladder-like structure of the macroscopic crosslinking network with dimers as ladder steps. The unique features of this first reported protein hydrogel may open up hitherto inaccessible applications, especially those taking advantage of the inherent catalytic activity of LplA.

biochemistry↗

bioRxiv2020

Structure of the endocytic adaptor complex reveals the basis for efficient membrane anchoring during clathrin-mediated endocytosis

During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process, middle coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryoEM structure of a 16-mer complex of membrane binding domains from Sla2 and Ent1 that anchors to the plasma membrane. Detailed mutagenesis in vitro and in vivo of the tetramer interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate the biological relevance of these interactions. Finally, time-resolved experiments in solution suggest that adaptors have evolved to achieve a fast subsecond timescale assembly in the presence of PIP2. Together, these findings provide a molecular understanding of an essential piece for the molecular puzzle of clathrin-coated sites.

biophysics↗

bioRxiv2020

The allosteric modulation of Complement C5 by knob domain peptides

To overcome limited germline combinatorial diversity, bovines have evolved a subset of antibodies with ultra-long CDRH3 regions that harbour cysteine-rich knob domains. To produce affinity-maturated peptides, we previously isolated autonomous 3-6 kDa knob domains from bovine antibodies. Here, we show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Allosteric mechanisms predominated, with one peptide selectively inhibiting C5 cleavage by the alternative pathway C5 convertase, revealing a targetable mechanistic difference between the classical and alternative pathway C5 convertases. Taking a hybrid biophysical approach, we present C5-knob domain co-crystal structures and, by solution methods, observed allosteric effects propagating >50 [A] from the binding sites. This study expands the therapeutic scope of C5, presents new inhibitors and introduces knob domains as new, low molecular weight antibody fragments, with therapeutic potential.

immunology↗

bioRxiv2020

Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 {micro}g/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed a novel conformation of the spike where two RBDs are in the up ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.

biophysics↗

bioRxiv2020

Structure-based identification and functional characterization of an essential lipocalin in the malaria parasite Plasmodium falciparum

Proteins of the lipocalin family are known to bind small hydrophobic ligands and are involved in various physiological processes ranging from lipid transport to oxidative stress responses. The genome of the malaria parasite Plasmodium falciparum contains a single protein PF3D7_0925900 with a lipocalin signature. Using crystallography and small-angle X-ray scattering, we show that the protein has a tetrameric structure of typical lipocalin monomers, hence we name it P. falciparum lipocalin (PfLCN), the first lipocalin structurally and functionally characterized in a single-celled eukaryote. We show that PfLCN is expressed in the intraerythrocytic stages of the parasite and localizes to the parasitophorous and food vacuoles. Conditional knockdown of PfLCN impairs parasite development, which can be rescued by treatment with the radical scavenger Trolox or by temporal inhibition of hemoglobin digestion. This suggests a key function of PfLCN in counteracting oxidative stress induced cell damage during multiplication of parasites within red blood cells.

cell biology↗

bioRxiv2019

Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats

MORN (Membrane Occupation and Recognition Nexus) repeat proteins have a wide taxonomic distribution, being found in both prokaryotes and eukaryotes. Despite this ubiquity, they remain poorly characterised at both a structural and a functional level compared to other common repeats. In functional terms, they are often assumed to be lipid-binding modules that mediate membrane targeting. We addressed this putative activity by focusing on a protein composed solely of MORN repeats - Trypanosoma brucei MORN1. Surprisingly, no evidence for binding to membranes or lipid vesicles by TbMORN1 could be obtained either in vivo or in vitro. Conversely, TbMORN1 did interact with individual phospholipids. High- and low-resolution structures of the MORN1 protein from Trypanosoma brucei and homologous proteins from the parasites Toxoplasma gondii and Plasmodium falciparum were obtained using a combination of macromolecular crystallography, small-angle X-ray scattering, and electron microscopy. This enabled a first structure-based definition of the MORN repeat itself. Furthermore, all three structures dimerised via their C-termini in an antiparallel configuration. The dimers could form extended or V-shaped quaternary structures depending on the presence of specific interface residues. This work provides a new perspective on MORN repeats, showing that they are protein-protein interaction modules capable of mediating both dimerisation and oligomerisation.

biochemistry↗

bioRxiv2019

Structure of herpes simplex virus pUL7:pUL51, a conserved complex required for efficient herpesvirus assembly

Herpesviruses are an ancient family of highly-prevalent human and animal pathogens that acquire their membrane envelopes in the cytoplasm of infected cells. While multiple conserved viral proteins are known to be required for efficient herpesvirus production, many of these proteins lack identifiable structural homologues and the molecular details of herpesvirus assembly remain unclear. We have characterized the complex of assembly proteins pUL7 and pUL51 from herpes simplex virus (HSV)-1, an -herpesvirus, using multi-angle light scattering and small-angle X-ray scattering with chemical crosslinking. HSV-1 pUL7 and pUL51 form a stable 1:2 complex that is capable of higher-order oligomerization in solution. We solved the crystal structure of this complex, revealing a core heterodimer comprising pUL7 bound to residues 41-125 of pUL51. While pUL7 adopts a previously-unseen compact fold, the extended helix-turn-helix conformation of pUL51 resembles the cellular endosomal complex required for transport (ESCRT)-III component CHMP4B, suggesting a direct role for pUL51 in promoting membrane scission during virus assembly. We demonstrate that the interaction between pUL7 and pUL51 homologues is conserved across human -, {beta}- and {gamma}-herpesviruses, as is their association with trans-Golgi membranes in cultured cells. However, pUL7 and pUL51 homologues do not form complexes with their respective partners from different virus families, suggesting that the molecular details of the interaction interface have diverged. Our results demonstrate that the pUL7:pUL51 complex is conserved across the herpesviruses and provide a structural framework for understanding its role in herpesvirus assembly.\n\nSignificance StatementHerpesviruses are extremely common human pathogens that cause diseases ranging from cold sores to cancer. Herpesvirus acquire their membrane envelope in the cytoplasm via a conserved pathway, the molecular details of which remain unclear. We have solved the structure of a complex between herpes simplex virus (HSV)-1 proteins pUL7 and pUL51, two proteins that are required for efficient HSV-1 assembly. We show that formation of this complex is conserved across distantly-related human herpesviruses, as is the association of these homologues with cellular membranes that are used for virion assembly. While pUL7 adopts a previously-unseen fold, pUL51 resembles key cellular membrane-remodeling complex components, suggesting that the pUL7:pUL51 complex may play a direct role in deforming membranes to promote virion assembly.

microbiology↗

bioRxiv2019

An automated data processing and analysis pipeline for transmembrane proteins in detergent solutions

The application of small angle X-ray scattering (SAXS) to the structural characterization of transmembrane proteins (MPs) in detergent solutions has become a routine procedure at the most synchrotron BioSAXS beamlines around the world. SAXS provides overall parameters and low resolution shapes of solubilized MPs, but is also meaningfully employed in hybrid modeling procedures that combine scattering data with information provided by high-resolution techniques (eg. macromolecular crystallography, nuclear magnetic resonance and cryo-electron microscopy). Structural modeling of MPs from SAXS data is non-trivial, and the necessary computational procedures require further formalization and facilitation. We propose an automated pipeline integrated with the laboratory-information management system ISPyB, aimed at preliminary SAXS analysis and the first-step reconstruction of MPs in detergent solutions, in order to streamline high-throughput studies, especially at synchrotron beamlines. The pipeline queries an ISPyB database for available a priori information via dedicated services, estimates model-free SAXS parameters and generates preliminary models utilizing either ab initio, high-resolution-based, or mixed/hybrid methods. The results of the automated analysis can be inspected online using the standard ISPyB interface and the estimated modeling parameters may be utilized for further in-depth modeling beyond the pipeline. Examples of the pipeline results for the modelling of the tetrameric alpha-helical membrane channel Aquaporin0 and mechanosensitive channel T2, solubilized by n-Dodecyl {beta}-D-maltoside are presented. We demonstrate how the increasing amount a priori information improves the model resolution and enables deeper insights into the molecular structure of protein-detergent complexes.\n\nSTATEMENT OF SIGNIFICANCESmall angle X-ray scattering (SAXS) using synchrotron radiation is a powerful technique for the structural characterization of transmembrane proteins (MPs) in detergent solutions Overall structural characterization and modeling of MPs from SAXS data is non-trivial, and the necessary computational procedures require further formalization and facilitation. We propose an automated pipeline integrated with the laboratory-information management system ISPyB, aimed at preliminary SAXS analysis and modelling of MPs in detergent solutions, in order to streamline high-throughput studies, especially at synchrotron beamlines.

molecular biology↗

bioRxiv2018

Utilisation of staphylococcal immune evasion protein Sbi as a novel vaccine adjuvant

Co-ligation of the B cell antigen receptor with complement receptor 2 on B-cells via a C3d-opsonised antigen complex significantly lowers the threshold required for B cell activation. Consequently, fusions of antigens with C3d polymers have shown great potential in vaccine design. However, these linear arrays of C3d multimers do not mimic the natural opsonisation of antigens with C3d. Here we investigate the potential of using the unique complement activating characteristics of Staphylococcal immune-evasion protein Sbi to develop a pro-vaccine approach that spontaneously coats antigens with C3 degradation products in a natural way. We show that Sbi rapidly triggers the alternative complement pathway through recruitment of complement regulators, forming a tripartite complex that acts as a competitive antagonist of factor H, resulting in enhanced complement consumption. These functional results are corroborated by the structure of this complement activating Sbi-III-IV:C3d:FHR-1 complex. Finally, we demonstrate that Sbi, fused with Mycobacterium tuberculosis antigen Ag85b, causes efficient opsonisation with C3 fragments, thereby enhancing the immune response significantly beyond that of Ag85b alone, providing proof of concept for our pro-vaccine approach.

biochemistry↗

bioRxiv2018

Functional interaction of low-homology FRPs from different cyanobacteria with Synechocystis OCP

Photosynthesis requires a balance between efficient light harvesting and protection against photodamage. The cyanobacterial photoprotection system uniquely relies on the functioning of the photoactive orange carotenoid protein (OCP) that under intense illumination provides fluorescence quenching of the light-harvesting antenna complexes, phycobilisomes. The recently identified fluorescence recovery protein (FRP) binds to the photoactivated OCP and accelerates its relaxation into the basal form, completing the regulatory circle. The molecular mechanism of FRP functioning is largely controversial. Moreover, since the available knowledge has mainly been gained from studying Synechocystis proteins, the cross-species conservation of the FRP mechanism remains unexplored. Besides phylogenetic analysis, we performed a detailed structural-functional analysis of two selected low-homology FRPs by comparing them with Synechocystis FRP (SynFRP). While adopting similar dimeric conformations in solution and preserving binding preferences of SynFRP toward various OCP variants, the low-homology FRPs demonstrated distinct binding stoichiometries and differentially accentuated features of this functional interaction. By providing clues to understand the FRP mechanism universally, our results also establish foundations for upcoming structural investigations necessary to elucidate the FRP-dependent regulatory mechanism.

biochemistry↗

bioRxiv2016

Structural reorganization of the chromatin remodeling enzyme Chd1 upon engagement with nucleosomes.

The yeast Chd1 protein acts to position nucleosomes across genomes. Here we model the structure of the Chd1 protein in solution and when bound to nucleosomes. In the apo state the DNA binding domain contacts the edge of the nucleosome while in the presence of the non-hydrolyzable ATP analog, ADP-beryllium fluoride, we observe additional interactions between the ATPase domain and the adjacent DNA gyre 1.5 helical turns from the dyad axis of symmetry. Binding in this conformation involves unravelling the outer turn of nucleosomal DNA and requires substantial reorientation of the DNA binding domain with respect to the ATPase domains. The orientation of the DNA-binding domain is mediated by sequences in the N-terminus and mutations to this part of the protein have positive and negative effects on Chd1 activity. These observations indicate that the unfavourable alignment of C-terminal DNA binding region in solution contributes to an auto-inhibited state.

molecular biology↗