Biology subjects

Carrique, L.

Publications and source records attributed to Carrique, L..

17 recordsLinked to original sources

bioRxiv2026

A recombinant heterotrimeric mini-procollagen I reveals new functional and structural roles for the α2(I) chain

Collagen I provides mechanical stability to organs and plays a critical role in several physiological processes such as wound healing and bone remodeling. Its ability to form fibrils depends on the proteolytic conversion of soluble procollagen I into mature protomers. However, the mechanistic aspects of this critical step have been difficult to analyze due to the lack of recombinant procollagen I suitable for structural and biochemical analyses. Here, we describe the successful production and purification of a heterotrimeric mini-procollagen I [1(I)22(I)] together with its homotrimeric counterpart [1(I)3] in HEK293-F cells. These tools were used to probe the specific role of the 2(I) chain both in procollagen I assembly and proteolytic maturation. While the presence of 2(I) in the heterotrimer does not strengthen trimer interfaces on its own, as revealed by cryo-electron microscopy, it provides a clear functional advantage to the procollagen substrate that is cleaved more efficiently by bone morphogenetic protein 1 (BMP-1) than the homotrimer. Furthermore, we demonstrate that the first cleavage event in the heterotrimer is at the level of the 2(I) chain, through a mechanism involving its C-telopeptide and a specific PRS motif close to the cleavage site. The preferential cleavage of 2(I) by BMP-1 can be partially corrected in the presence of PCPE-1 (procollagen C-proteinase enhancer 1) but the efficacy of the correction depends on its position on the procollagen trimer. Finally, we solved the high-resolution structure of the complex between the heterotrimer and PCPE-1 and proposed a new mechanism to explain the enhancing activity of PCPE-1.

biochemistry↗

bioRxiv2026

Structure of PCPE-2 in complex with the BMP-1 metalloprotease reveals the molecular basis of its mechanism of inhibition

PCPE-2 (procollagen C-proteinase enhancer-2) is an extracellular glycoprotein playing dual functions in the regulation of BMP-1/tolloid-like proteinases. Like its homologue PCPE-1, PCPE-2 can enhance the proteolytic maturation of fibrillar procollagens, but it also acts as a potent and specific inhibitor of BMP-1 through formation of a high-affinity complex with the protease. Here, we investigated the molecular basis of this inhibitory interaction using complementary biochemical, biophysical and structural approaches. We show that the two CUB domains of PCPE-2 must be covalently linked for efficient BMP-1 binding and inhibition, consistent with cooperative engagement of the protease. Cryo-electron microscopy combined with density-guided structural modelling and hydrogen/deuterium-exchange mass spectrometry supports a bipartite interaction complex in which the CUB1 domain of PCPE-2 engages the catalytic domain of BMP-1, while the CUB2 domains of both proteins interact together. The interaction involves the calcium-binding surfaces of the PCPE-2 CUB domains that also participate in procollagen recognition, providing a structural framework for understanding the mutually exclusive interactions of PCPE-2 with BMP-1 and procollagen. Allosteric inhibition is supported by the binding of PCPE-2 CUB1 opposite to the active site cleft, leading to the shielding of the residues surrounding the S1-prime pocket, as observed by HDX-MS. Together, these findings define the architecture of the inhibitory PCPE-2/BMP-1 complex and provide the molecular basis for understanding the distinct regulatory activities of the two procollagen C-proteinase enhancers.

biochemistry↗

bioRxiv2026

Influenza A virus initiates genome selection through promoter-dependent nuclear export

Influenza A virus replicates its segmented RNA genome in the nucleus in the context of viral ribonucleoprotein (vRNP) complexes. Newly synthesised vRNPs are exported to the cytoplasm for virion assembly, whereas structurally similar complementary RNPs (cRNPs), which function as replication intermediates, are not incorporated into virions. The molecular basis for this selectivity remains unclear. Here, combining promoter mutagenesis, single-molecule fluorescence in situ hybridisation (smFISH), virus-like particle (VLP) assays and cryo-electron microscopy (cryo-EM), we find that cRNPs are retained in the nucleus and that export competence is encoded by promoter architecture, with the length of the single-stranded 3' promoter region as the principal contributing feature and promoter sequence and duplex architecture further modulating export efficiency. cRNPs carrying export-competent promoters are incorporated into virus-like particles, demonstrating a functional link between nuclear export and packaging competence. Cryo-EM analyses reveal that both 3' vRNA and cRNA promoters bind a common polymerase surface site but are associated with distinct polymerase conformational landscapes, with only the vRNA promoter being compatible with an encapsidase conformation of the polymerase. Together, our findings identify a promoter-dependent checkpoint that links RNA promoter architecture and polymerase conformation to RNP export and packaging. These results demonstrate that influenza virus initiates genome selection in the nucleus and reveal nuclear export as an early step in genome selection.

microbiology↗

bioRxiv2025

Structural and functional characterisation of the Crimean-Congo Haemorrhagic Fever Virus RNA Dependent RNA Polymerase

Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is found across Africa, Asia, and the Middle East where it can cause Haemorrhagic outbreaks with high case fatality rates. Central to the viral life cycle is the viral L-protein, a crucial and multifunctional protein which both transcribes and replicates the viral genome. Here, we present the cryoEM structures of an RNA free and a 5 promoter bound complex, describing the core catalytic RNA-dependent RNA polymerase (RdRp). We observe an RdRp that is substantially larger than related L-proteins and contains domain insertions unique to the nairovirus family. The 5 RNA promoter is found in a tight RNA hairpin stabilised by a single base pair, with 5 binding triggering the closure of protein over the RNA. Functional analysis of the endonuclease and RdRp activities reveals an enzyme which is capable of both activities and demonstrate RdRp inhibition by known antiviral nucleosides. These data advance our understanding of the molecular mechanisms behind genome replication and transcription, that will help inform future antiviral development.

microbiology↗

bioRxiv2025

Design principles of a membrane-spanning ubiquitin ligase

Receptor-type E3 ubiquitin ligases are membrane-spanning assemblies that enable extracellular signals to directly control ubiquitylation in the cytoplasm. Despite playing widespread roles in tissue patterning and homeostasis, metabolism, and immunity, their structures and mechanisms remain poorly understood. Using cryo-electron microscopy, integrated with biophysical and functional studies, we visualized an E3 complex composed of two transmembrane proteins, MEGF8 and MOSMO, and the intracellular RING-family protein MGRN1. This MEGF8-MOSMO-MGRN1 (MMM) complex regulates left-right patterning of the body axis and the development of multiple organs, partly by attenuating signaling through the Hedgehog pathway. We find that the MMM complex functions like a fishing pole: a long, flexible helix attached to a membrane platform suspends an activated and precisely oriented RING domain--like a fishhook--to ubiquitylate the cytoplasmic surfaces of target receptors. Our structure explains how mutations in MEGF8 cause multi-organ birth defects in humans and defines a paradigm for receptor regulation by ubiquitylation.

biochemistry↗

bioRxiv2024

Di-Gluebodies as covalently-rigidified, modular protein assemblies enable simultaneous determination of high-resolution, low-size, cryo-EM structures

Cryo-EM has become a routine structural biology method, yet elucidation of small proteins (<100 kDa) and increasing throughput remain challenging. Here, we describe covalently-dimerized engineered nanobodies, Di-Gluebodies, as novel and modular imaging scaffolds to address the challenges. They can simultaneously display two copies of the same protein or two distinct proteins, providing sufficient rigidity required by cryo-EM. We validate this method with multiple soluble and membrane targets, demonstrating that this provides a flexible and scalable platform for expanded protein structure determination.

synthetic biology↗

bioRxiv2023

Structural and functional characterisation of the interaction between the influenza A virus RNA polymerase and the CTD of host RNA Polymerase II

Influenza A viruses (IAV), causing seasonal epidemics and occasional pandemics, rely on interactions with host proteins for their RNA genome transcription and replication. The viral RNA polymerase utilizes host RNA polymerase II (Pol II) and interacts with the serine 5 phosphorylated (pS5) C-terminal domain (CTD) of Pol II to initiate transcription. Our study, using single-particle electron cryomicroscopy (cryo-EM), reveals the structure of the 1918 pandemic IAV polymerase bound to a synthetic pS5 CTD heptad repeat peptide. The structure shows that the CTD peptide binds at the C-terminal domain of the PA viral polymerase subunit (PA-C) and reveals a previously unobserved position of the 627 domain of the PB2 subunit near the CTD. We identify crucial residues of the CTD peptide mediating interactions with positively charged cavities on PA-C, explaining the preference of the viral polymerase for pS5 CTD. Functional analysis of mutants targeting the CTD-binding site within PA-C reveals reduced transcriptional function with normal replication, while other mutants display defects in both transcription and replication, highlighting the multifunctional role of PA-C in viral RNA synthesis. Our study provides insights into the structural and functional aspects of the influenza virus polymerase-host Pol II interaction and identifies a target for antiviral development.

microbiology↗

bioRxiv2023

DSIF factor Spt5 coordinates transcription, maturation and exoribonucleolysis of RNA polymerase II transcripts.

The timely termination of RNA polymerase II (Pol II) transcription is critical for polymerase recycling and preventing interference with the expression of the neighbouring genes. Termination of Pol II transcription involves exoribonucleolytic decay of the nascent RNA by 5-3 exonuclease Xrn2. Xrn2 attacks the 5-PO4-end and executes degradation of the nascent RNA generated by the endonucleolytic cleavage at the poly(A) site which eventually leads to Pol II release from DNA. However, the molecular details of when and how during this process Xrn2 interacts with the Pol II elongation complex to mediate its dissociation from DNA is not understood. Here, we demonstrate that Xrn2 interacts with Pol II and Spt5, a conserved transcription factor that controls Pol II processivity and pausing. Importantly, Xrn2 activity is stimulated by Spt5 in vitro and Spt5-depleted cells show defective transcription termination. Our results support a model where Xrn2 first forms a stable complex with the elongating Pol II to acquire its full activity in degrading nascent RNA. Spt5 also promotes premature termination attenuating the expression of non-coding transcripts. By contrast, Spt5 depletion leads to Pol II retention at promoters of protein-coding genes. Pol IIs that transcribe into a gene body in the absence of Spt5 exhibit severely reduced elongation rates and defective pre-mRNA processing. We propose that Spt5 plays a major role in the production of functional mRNA by directly stimulating the activity of the RNA enzymes and preventing entry of Pol II complexes not configured to support transcription and pre-mRNA processing into elongation.

molecular biology↗

bioRxiv2023

Structure of the N-RNA/P interface reveals mode of L/P attachment to the nucleocapsid of human metapneumovirus

Human metapneumovirus (HMPV) is a major cause of respiratory illness in young children. The polymerase complex of HMPV consists of two obligate components, the L polymerase and its cofactor, the phosphoprotein P. During replication and transcription, the L/P complex traverses the viral RNA genome, which is encapsidated within multimerized N nucleoproteins. An essential interaction between N and a C-terminal region of P is required for tethering of the L/P polymerase to the RNA template. This N-P interaction is also involved in the formation of cytoplasmic viral factories in infected cells, called inclusion bodies. To define how L/P recognizes N-encapsidated RNA (N-RNA) we employed cryogenic electron microscopy (cryo-EM) and molecular dynamics simulations, coupled to polymerase activity assays and imaging of inclusion bodies in transfected cells. We report a 2.9 [A] resolution structure of a triple-complex between multimeric N, bound to both RNA and the C-terminal region of P. Furthermore, we also present cryo-EM structures of assembled N in different oligomeric states, highlighting the plasticity of N. Combined with our functional assays, these structural data delineate in molecular detail how P attaches to N-RNA whilst retaining substantial conformational dynamics. Moreover, the N-RNA-P triple complex structure provides a molecular blueprint for the design of therapeutics to potentially disrupt the attachment of L/P to its template.

microbiology↗

bioRxiv2023

Structural Characterization of the Full-length Hantaan Virus Polymerase

Hantaviridae are a family of segmented negative-sense RNA viruses that contains important human and animal pathogens including Andes orthohantavirus and Hantaan orthohantavirus. Hantaviridae contain a viral RNA-dependent RNA polymerase that replicates and transcribes the viral genome in the cytoplasm of infected cells. Here we establish the expression and purification of the polymerase from the Old World Hantaan orthohantavirus and characterise the structure using single particle cryo electron microscopy. We determine a series of structures at resolutions between 2.7 and 3.3 [A] of RNA free polymerase comprising the core, core and endonuclease, and a full-length polymerase. The full-length polymerase structure depicts the location of the cap binding and C-terminal domains which are arranged in a conformation that is incompatible with transcription and in a novel conformation not observed in previous conformations of cap-snatching viral polymerase. We further describe structures with 5' vRNA promoter in the presence and absence of a nucleotide triphosphate. The nucleotide bound structure mimics a replication pre-initiation complex and the nucleotide stabilises the motif E in a conformation distinct from those previously observed. We observe motif E in four distinct conformations including {beta}-sheet, two helical arrangements, and an ordered loop. The insights gained here form a starting point for future detailed mechanistic studies of both the transcription and replication activities of the hantavirus polymerase and for the development of therapeutic targets.

biochemistry↗

bioRxiv2022

LipIDens: Simulation assisted interpretation of lipid densities in cryo-EM structures of membrane proteins.

Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures. The pipeline integrates the implementation and analysis of multi-scale MD simulations for identification, ranking and refinement of lipid binding poses which superpose onto cryo-EM map densities. Thus, LipIDens enables direct integration of experimental and computational structural approaches to facilitate the interpretation of lipid-like cryo-EM densities and to reveal the molecular identities of protein-lipid interactions within a bilayer environment. The LipIDens code is open-source and embedded within a notebook format to assist automation and usability.

biophysics↗

bioRxiv2021

Structure and Mechanism of Hedgehog Acyl Transferase

The iconic Sonic Hedgehog (SHH) morphogen pathway is a fundamental orchestrator of embryonic development and stem cell maintenance, and is implicated in cancers in various organs. A key step in signalling is transfer of a palmitate group to the N-terminal cysteine residue of SHH, catalysed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT) resident in the endoplasmic reticulum (ER). Here, we present the high-resolution cryo-EM structure of HHAT bound to substrate analogue palmityl-coenzyme A and a SHH mimetic megabody. Surprisingly, we identified a heme group bound to an HHAT cysteine residue and show that this modification is essential for HHAT structure and function. A structure of HHAT bound to potent small molecule inhibitor IMP-1575 revealed conformational changes in the active site which occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the novel mechanism by which HHAT adapts the membrane environment to transfer a long chain fatty acid across the ER membrane from cytosolic acyl-CoA to a luminal protein substrate. This structure of a member of the protein-substrate membrane-bound O-acyltransferase (MBOAT) superfamily provides a blueprint for other protein substrate MBOATs, such as WNT morphogen acyltransferase Porcupine and ghrelin O-acyltransferase GOAT, and a template for future drug discovery.

biophysics↗

bioRxiv2020

The SARS-CoV-2 Spike harbours a lipid binding pocket which modulates stability of the prefusion trimer

Large trimeric Spikes decorate SARS-CoV-2 and bind host cells via receptor binding domains (RBDs). We report a conformation in which the trimer is locked into a compact well-ordered form. This differs from previous structures where the RBD can flip up to recognise the receptor. In the locked form regions associated with fusion transitions are stabilised and the RBD harbours curved lipids. The acyl chains bind a hydrophobic pocket in one RBD whilst the polar headgroups attach to an adjacent RBD of the trimer. By functional analogy with enteroviral pocket factors loss of the lipid would destabilise the locked form facilitating receptor attachment, conversion to the postfusion state and virus infection. The nature of lipids available at the site of infection might affect the antigenicity/pathogenicity of released virus. These results reveal a potentially druggable pocket and suggest that the natural prefusion state occludes neutralising RBD epitopes, achieving conformational shielding from antibodies. HighlightsO_LISARS-CoV-2 Spike can adopt a locked conformation with all receptor binding domains (RBDs) down, likely to represent the prefusion resting state C_LIO_LIThis locked conformation is compact and stable, braced by lipid bound within a potentially druggable pocket C_LIO_LIKey neutralization epitopes are shielded in the locked form C_LIO_LILoss of lipid may trigger a cascade of events that lead to cell entry analogous to the role of lipids in enterovirus cell entry C_LI

microbiology↗

bioRxiv2020

Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient

The COVID-19 pandemic has had unprecedented health and economic impact, but currently there are no approved therapies. We have isolated an antibody, EY6A, from a late-stage COVID-19 patient and show it neutralises SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds tightly (KD of 2 nM) the receptor binding domain (RBD) of the viral Spike glycoprotein and a 2.6[A] crystal structure of an RBD/EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues of this epitope are key to stabilising the pre-fusion Spike. Cryo-EM analyses of the pre-fusion Spike incubated with EY6A Fab reveal a complex of the intact trimer with three Fabs bound and two further multimeric forms comprising destabilized Spike attached to Fab. EY6A binds what is probably a major neutralising epitope, making it a candidate therapeutic for COVID-19.

microbiology↗

bioRxiv2020

A high resolution view of an adolescent flavivirus

Mosquito-transmitted flaviviruses, such as Dengue virus (DENV) or Zika virus (ZIKV), are responsible for significant economic damage and human misery. In infected cells, flaviviruses first assemble into an immature form within the endoplasmatic reticulum (ER), and then undergo further processing by furin protease in the trans-Golgi. Despite substantial efforts, previous cryogenic electron microscopy (cryo-EM) studies of immature flaviviruses were restricted to low to medium resolutions, limiting our understanding of maturation. To better grasp the process of maturation, we have carried out cryo-EM reconstructions of immature Spondweni virus (SPOV), an emerging human flavivirus belonging to the same serogroup as ZIKV (~75% amino acid identity). By combining localized reconstruction and focused refinement, we were able to improve the resolution to 3.8 [A], yielding unprecedented insight into the immature form. The structure elucidates how, at neutral pH, polar interactions conceal the furin recognition site within trimeric envelope (E) protein spikes. Furthermore, we identify how a strictly conserved pH sensor anchors the precursor membrane (prM) protein to immature E. We reconstructed mature forms of SPONV and DENV to 2.6[A] and 3.1[A], respectively. Comparison with immature virus shows a conserved binding pocket for a lipid headgroup, which forms as a consequence of the rearrangement of amphipathic stem-helices of E. We propose a structural role for the pocket and suggest it stabilizes mature E. Taken together, our data suggest a compelling rationale for low-pH triggered conformational rearrangement in the Golgi, which occurs during flavivirus maturation.

microbiology↗

bioRxiv2020

Neutralization of SARS-CoV-2 by destruction of the prefusion Spike

There are as yet no licenced therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric Spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2 (ACE2), initiating conformational changes that drive membrane fusion. We find that monoclonal antibody CR3022 binds the RBD tightly, neutralising SARS-CoV-2 and report the crystal structure at 2.4 [A] of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilising, CR3022 epitope is inaccessible in the prefusion Spike, suggesting that CR3022 binding would facilitate conversion to the fusion-incompetent post-fusion state. Cryo-EM analysis confirms that incubation of Spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope may be useful therapeutically, possibly in synergy with an antibody blocking receptor attachment. HighlightsO_LICR3022 neutralises SARS-CoV-2 C_LIO_LINeutralisation is by destroying the prefusion SPIKE conformation C_LIO_LIThis antibody may have therapeutic potential alone or with one blocking receptor attachment C_LI

microbiology↗

bioRxiv2018

Parallel Evolution and the Emergence of Highly Pathogenic Avian Influenza A Viruses

Parallel molecular evolution and adaptation are important phenomena commonly observed in viruses. Here we exploit parallel molecular evolution to understand virulence evolution in avian influenza viruses (AIV). Highly-pathogenic AIVs independently evolve from low-pathogenic ancestors via acquisition of a polybasic cleavage sites (pCS). Why some AIV lineages but not others evolve in this way is unknown. We hypothesise that the parallel emergence of highly-pathogenic AIV may be facilitated by permissive or compensatory mutations occurring across the AIV genome. We combined phylogenetic, statistical and structural approaches to discover parallel mutations in AIV genomes associated with the highly-pathogenic phenotype. Parallel mutations were screened using a new statistical test of mutation-phenotype association and further evaluated in the contexts of positive selection and protein function. The mutational panel we present reveals new links between virulence evolution and other viral traits and raises the possibility of predicting aspects of AIV evolution.

evolutionary biology↗