Biology subjects

Leis, A.

Publications and source records attributed to Leis, A..

9 recordsLinked to original sources

bioRxiv2026

Cryo-EM structure of human LRRC15 reveals the basis of therapeutic antibody recognition

Leucine-rich repeat containing 15 (LRRC15) is a structural uncharacterised membrane protein, which is highly upregulated in cancer-associated fibroblasts and enriched in desmoplastic tumours including pancreatic, breast and head-and-neck cancers, where it is associated with therapy resistance. The LRRC15-targeting antibody-drug conjugate samrotamab vedotin has advanced into Phase I clinical evaluation, yet mechanistic understanding of how samrotamab engages its target has remained elusive in the absence of structural data. Here, using hydrogen-deuterium exchange mass spectrometry and cryo-electron microscopy, we define the samrotamab epitope on LRRC15 and determine the structure of the LRRC15-samrotamab complex to 2.6 [A] resolution - the first high-resolution structure of this receptor. Samrotamab engages a membrane-proximal epitope within the C-terminal leucine-rich repeat region, leaving the canonical concave surface fully exposed for potential interactions. The lateral binding mode of samrotamab and the open arc geometry of LRRC15 provide a structural rationale for why antibody occupancy may leave signalling-competent surfaces intact. Guided by these insights, we computationally designed minibinders targeting the concave surface, identifying multiple binders with nanomolar affinity. Together, our work establishes the first structural framework for LRRC15, defines the molecular basis of therapeutic antibody recognition, and identifies the concave surface as a promising target for next-generation LRRC15-directed therapeutics.

biochemistry↗

bioRxiv2026

Molecular basis for the role of Ripr in Plasmodium falciparum invasion of human erythrocytes

Plasmodium falciparum causes the majority of severe malaria, and merozoite invasion of erythrocytes is a vulnerable, antibody-accessible step of the blood-stage cycle. PfRipr is an essential component of the PCRCR invasion complex, yet the structural basis for antibody-mediated neutralisation remains unclear. Here, we map inhibitory and non-inhibitory epitopes across PfRipr and show that all potent inhibitors localise to the tail region (EGF6-8). Crystal structures reveal that inhibitory antibodies restrict the flexibility surrounding EGF7. Indeed, EGF7 buried surface area correlates strongly with inhibitory potency, identifying this domain as the principal invasion-inhibitory determinant. Pairwise antibody combinations revealed unexpected synergy, with non-inhibitory mAbs potentiating anti-Rh5 activity. Conditional deletion, sequence replacement or positional swapping of EGF6-8 abolished invasion, demonstrating that both sequence and spatial arrangement are indispensable. These data define EGF7 as a conserved, functionally essential vulnerability and provide a blueprint for rational EGF6-8 immunogen design capable of eliciting P. falciparum strain-transcending protection against blood-stage malaria.

biochemistry↗

bioRxiv2026

DNA binding by ATPase-adjacent domains stimulates SMCHD1 ATPase activity

SMCHD1 is an epigenetic regulator in which heterozygous variants are reported in facioscapulohumeral muscular dystrophy (FSHD), as well as Bosma arhinia microphthalmia syndrome (BAMS). While we have previously shown that SMCHD1 is able to interact with nucleic acids via its hinge domain, we have now identified a second DNA-binding site that is located C-terminal to the ATPase domain and formed by two domains: the Bromo-adjacent homology (BAH) and immunoglobulin-like 1 (IGL-1) domains. Here, we report their mode of DNA-interaction and we present the first high-resolution structure of the wild-type human SMCHD1 ATPase using site-directed mutagenesis and structural analysis via cryo-EM. We also reveal that DNA-binding at the BAH-IGL-1 domains stimulates the ATPase activity of full-length SMCHD1 in vitro, and demonstrate in a mouse model that ATP hydrolysis is essential for SMCHD1 function in vivo. Together, these findings establish bidentate DNA binding and DNA-stimulated ATP hydrolysis as central features of SMCHD1 function, providing new mechanistic insight into how SMCHD1 regulates gene silencing. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/704097v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@12b2fd5org.highwire.dtl.DTLVardef@5152c7org.highwire.dtl.DTLVardef@111c487org.highwire.dtl.DTLVardef@17d8d0d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

PartiNet is a dynamic adaptive neural network for high-performance particle picking in cryo-electron microscopy

Accurate, efficient and autonomous particle picking is a major bottleneck in high-resolution cryo-electron microscopy (cryo-EM). We introduce PartiNet, an Artificial Intelligence (AI)-based particle picker with size-agnostic detection and pre-trained models that eliminate manual parameter specification and dataset-specific training, pioneering dynamic neural network inference for single particle cryo-EM pipeline. Unlike static architectures, PartiNet employs a dynamic framework that adjusts network complexity in real-time based on perceived micrograph quality. This adaptive approach accelerates inference up to 7-fold compared to existing tools without sacrificing particle selection quality. Training on diverse protein datasets showed that PartiNet improves particle yields, enhances sampling of rare orientations, and is compatible with on-the-fly workflows. Comprehensive evaluation on benchmark datasets and validation on a new dataset of the chromatin remodeler MORC2 demonstrates superior precision and recall, with the ability to detect heterogeneous protein species, leading to more complete structural models and consistently higher-resolution reconstructions.

molecular biology↗

bioRxiv2025

Structural basis for Porcupine inhibition

Wnt signalling is essential for embryonic development and tissue homeostasis, and its dysregulation is associated with multiple types of cancer. Porcupine (PORCN), an endoplasmic reticulum (ER)-resident membrane-bound O-acyltransferase, catalyses the palmitoylation of all 19 human Wnts--a critical modification required for their secretion and activity. This central role makes PORCN an attractive therapeutic target for Wnt-driven cancers, with several inhibitors currently in clinical trials. Here, we present high-resolution cryo-electron microscopy structures of human PORCN in complex with the inhibitors C59 (2.4 [A]) and ETC159 (2.6 [A]), as well as in a ligand-free state (3.3 [A]). These structures reveal critical ordered water molecules that form a hydrogen-bonding network within the active site, mediating inhibitor binding. Our docking simulations of diverse PORCN inhibitors demonstrate that despite their different chemical scaffolds, these compounds adopt similar conformations within the acyl-CoA binding site and are also engaged through a conserved water molecule. Our findings provide a structural foundation for the rational design of next-generation PORCN inhibitors with improved pharmacological properties for cancer therapy.

biochemistry↗

bioRxiv2025

PTRAMP, CSS and Ripr form a conserved complex required for merozoite invasion of Plasmodium species into erythrocytes

Invasion of erythrocytes by members of the Plasmodium genus is an essential step of the parasite lifecycle, orchestrated by numerous host-parasite interactions. In P. falciparum Rh5, with PfCyRPA, PfRipr, PfCSS, and PfPTRAMP, forms the essential PCRCR complex which binds basigin on the erythrocyte surface. Rh5 is restricted to P. falciparum and its close relatives; however, PTRAMP, CSS and Ripr orthologs are present across the Plasmodium genus. We investigated PTRAMP, CSS and Ripr orthologs from three species to elucidate common features of the complex. Like P. falciparum, PTRAMP and CSS form a disulfide-linked heterodimer in both P. vivax and P. knowlesi with all three species forming a complex (PCR) with Ripr by binding its C-terminal region. Cross-reactive antibodies targeting the PCR complex differentially inhibit merozoite invasion. Cryo-EM visualization of the P. knowlesi PCR complex confirmed predicted models and revealed a core invasion scaffold in Plasmodium spp. with implications for vaccines targeting multiple species of malaria-causing parasites.

microbiology↗

bioRxiv2024

MORC2 phosphorylation fine tunes its DNA compaction activity

Variants in the poorly characterised oncoprotein, MORC2, a chromatin remodelling ATPase, lead to defects in epigenetic regulation and DNA damage response. The C-terminal domain (CTD) of MORC2, frequently phosphorylated in DNA damage, promotes cancer progression, but its role in chromatin remodelling remains unclear. Here, we report a molecular characterisation of full-length, phosphorylated MORC2, demonstrating its preference for binding open chromatin and functioning as a DNA sliding clamp. We identified a phosphate interacting motif within the CTD that dictates ATP hydrolysis rate and cooperative DNA binding. The DNA binding impacts several structural domains within the ATPase region. We provide the first visual proof that MORC2 induces chromatin remodelling through ATP hydrolysis-dependent DNA compaction, regulated by its phosphorylation state. These findings highlight phosphorylation of MORC2 CTD as a key modulator of chromatin remodelling, presenting it as a potential therapeutic target.

molecular biology↗

bioRxiv2023

Interaction of PINK1 with nucleotides and kinetin

PINK1 is a ubiquitin kinase that accumulates on damaged mitochondria to trigger mitophagy, and PINK1 loss-of-function mutations cause early onset Parkinsons disease. Nucleotide analogues such as kinetin triphosphate (KTP) have been suggested to enhance PINK1 activity and may represent a therapeutic strategy for the treatment of Parkinsons disease. Here, we investigate the interaction of PINK1 with nucleotides, including KTP. We establish a cryo-EM platform exploiting the previously observed dodecamer assembly of Pediculus humanus corporis (Ph) PINK1 to determine PINK1 structures bound to AMP-PNP and ADP, which reveal unexpected conformational changes in the kinase N-lobe to enable PINK1 to form a ubiquitin binding site. Strikingly, we find that KTP is unable to bind PhPINK1 or human (Hs) PINK1 due to a steric clash with the kinase gatekeeper residue. Mutation of the gatekeeper to Ala or Gly is required to enable PINK1 to bind and utilise KTP as a phosphate donor in ubiquitin phosphorylation and mitophagy. Indeed, HsPINK1 M318G can be used to conditionally uncouple PINK1 stabilisation and activity on mitochondria.

molecular biology↗

bioRxiv2019

Monoubiquitination by the Fanconi Anemia core complex locks FANCI:FANCD2 on DNA in filamentous arrays

FANCI:FANCD2 monoubiquitination is a critical event for replication fork stabilization by the Fanconi anemia (FA) DNA repair pathway. It has been proposed that at stalled replication forks, monoubiquitinated-FANCD2 serves to recruit DNA repair proteins that contain ubiquitin-binding motifs. Here we have reconstituted the FA pathway in vitro to study functional consequences of FANCI:FANCD2 monoubiquitination. We report that monoubiquitination does not promote any specific exogenous protein:protein interactions, but instead stabilizes FANCI:FANCD2 heterodimers on dsDNA. This locking of FANCI:FANCD2 complex on DNA requires monoubiquitination of only the FANCD2 subunit. We further show that purified monoubiquitinated FANCI:FANCD2 forms filament-like arrays on long dsDNA using electron microscopy. Our results reveal how monoubiquitinated FANCI:FANCD2 is activated upon DNA binding and present new insights to potentially modulate monoubiquitinated FANCI:FANCD2/DNA filaments in FA cells.

biochemistry↗