Biology subjects

Lopez-Sagaseta, J.

Publications and source records attributed to Lopez-Sagaseta, J..

9 recordsLinked to original sources

bioRxiv2026

Structure-function profiling identifies determinants of TCR-T cell therapeutic efficacy

Glypican-3 (GPC3) is a promising target in adoptive T-cell therapy for hepatocellular carcinoma (HCC). TCR-A and TCR-B are two recently identified GPC3-specific TCRs recognizing the same HLA-A*02:01-restricted epitope but displaying markedly different therapeutic efficacy. To define the determinants of productive TCR-antigen recognition, we integrated high-resolution structures of both binary pMHC and ternary TCR:pMHC complexes, biolayer interferometry, peptide mutagenesis, target-cell conjugation assays, repetitive antigen challenge (RAC) and in vivo models. Structurally, conformational permissiveness of the immunodominant GPC3(522-530) peptide within HLA-A*02:01's groove is central for productive TCR docking, with TCR-B presenting slower association but prolonged dwell time. Functional divergence emerged during target-cell engagement and amplified under RAC conditions, where TCR-B sustained cytotoxic activity while TCR-A progressively lost function. This superior functional endurance translated into complete tumor eradication and durable responses in vivo. These findings provide a mechanistic basis and a conceptual framework for the selection and optimization of TCRs for adoptive immunotherapy.

immunology↗

bioRxiv2026

Structural presentation of amyloid β by HLA

HLA-DR-restricted T-cell reactivity to amyloid {beta} (A{beta}) has been associated with Alzheimers disease (AD), but structural evidence for HLA presentation of A{beta}-derived peptides remains elusive. We present the crystal structure of the A{beta}1-15 fragment bound to HLA-DR1, providing, to the best of our knowledge, the first experimental structure of an Alzheimers A{beta} peptide bound to an HLA molecule. The molecular architecture of this complex defines a peptide:MHC interaction dictated by engagement of A{beta}1-15 peptide central core with further involvement of N- and C-terminal peptide flanks. The structure reveals that DR{beta}1 Arg70, a polymorphic position, directly binds P4 and P5 through polar contacts, providing a rationale for HLA-DRB allelic bias underpinning accommodation of A{beta}1-15. We also describe the A{beta}1-15:HLA-DRB1 surface topology, informing a candidate binding surface for potential T-cell recognition. Collectively, these findings contribute a structural framework for further research in the context of A{beta}-specific CD4+ T-cell autoreactivity in AD.

neuroscience↗

bioRxiv2025

Structure of Factor VII Gla domain bound to EPCR

The precise molecular determinants that underlie the binding of activated factor VII (FVIIa) to the endothelial cell protein C receptor (EPCR) have long remained unresolved. We report here the crystal structure of factor VII Gla domain bound to human EPCR, revealing a binding geometry in essence indistinguishable from that of the PC-EPCR interface and confirming absolute structural competition for EPCR binding. Relative to previous FVII structures, we observe receptor-dependent repositioning of the Gla {omega} loop coupled to a selective arrangement in which metal ion at position four adopts a Ca2+-compatible and EPCR-restricted coordination state. These findings resolve a long-standing gap and inform the structural basis for the binding of EPCR to FVIIa, a current therapeutic intervention in hemophilia.

biochemistry↗

bioRxiv2025

Robust TCR production for the structural study of TCR-pMHC complexes

A precise comprehension of how T cell receptors (TCRs) engage their antigens is pivotal for advancing basic research and T cell immunotherapy in cancer. While TCR refolding from inclusion bodies has greatly facilitated X-ray studies over the past decades, the procedures remain labor-intensive and can yield poorly. We have developed a simplified strategy for efficient production of soluble TCRs in CHO cells which, coupled with the removal of N-glycosylation, enable structural studies of TCR-pMHC complexes. An equivalent of just 20 ml of cell culture delivered sufficient deglycosylated TCR (dgTCR) to screen, upon complexation with a cognate pMHC, over 350 crystallization conditions and obtain a high-resolution dataset. This approach illustrates an effective alternative for TCR production to support studies devoted to research and development of TCRs.

immunology↗

bioRxiv2024

Severe deviation in protein fold prediction by advanced AI: a case study

Artificial intelligence (AI) and deep learning are making groundbreaking strides in protein structure prediction. AlphaFold is remarkable in this arena for its outstanding accuracy in modelling proteins fold based solely on their amino acid sequences. In spite of these remarkable advances, experimental structure determination remains critical. Here we report severe deviations (>30 [A]) between the experimental structure of a two-domain protein and its equivalent AI-prediction. These observations are particularly relevant to the relative orientation of the domains within the global protein scaffold. Significant divergence between experimental structures and AI-predicted models echoes the presence of unusual conformations, insufficient training data and high complexity in protein folding that can ultimately lead to current limitations in protein structure prediction.

biochemistry↗

bioRxiv2024

Unusual traits shape the architecture of the Ig ancestor molecule

Understanding the ancestral Ig domains molecular structure and tracing the evolution of Ig-like proteins are fundamental components missing from our comprehension of their evolutionary trajectory and function. We have elucidated the molecular structures of two Ig-like proteins from the evolutionary most ancestral phylum, Porifera, revealing previously unidentified Ig-domain features that highlight the concomitant presence of a novel Ig "Early Variable" (EV)-set in tandem with a C1-set domain. The latter, to our knowledge, has never been reported before in non-vertebrates. The IgV and IgC1 sets and their combination into functional Ig-like receptors are part of the adaptive immune system in higher vertebrates, which allows for highly specific immune responses. These observations in the ancient Porifera phylum could indicate the presence of primitive forms of adaptive immunity or a foundational immune strategy that has been conserved through evolution. By unveiling important clues into the molecular configuration of ancestral Ig domains, these findings challenge and expand our understanding of how immunity has evolved within its current landscape.

evolutionary biology↗

bioRxiv2021

Structure of EPCR in a non-canonical conformation

Structural motion and conformational flexibility are often linked to biological functions of proteins. Whether the endothelial protein C receptor (EPCR), like other molecules, is vulnerable to folding transitions or might adopt alternative conformations remains unknown. The current understanding points to a rigid molecular structure suitable for binding of its ligands, like the anticoagulant protein C, or the CIDR1 domains of Plasmodium falciparum. In this study, we have identified a novel conformation of EPCR, captured by X-ray diffraction analyses, whereby Tyr154 shows a dramatically altered structural arrangement, likely incompatible with protein C binding. Biolayer interferometry analysis confirms previous results supporting a critical role for this position in protein C binding. Importantly, the conformational change has no apparent effect in the bound lipid. We conclude these findings reveal a site of conformational vulnerability in EPCR and inform a highly malleable region that could modulate EPCR functions.

biochemistry↗

bioRxiv2021

A single de novo substitution in SARS-CoV-2 spike informs enhanced adherence to human ACE2.

SARS-CoV-2 initiates colonization of host cells by binding to cell membrane ACE2 receptor. This binding is mediated by the viral spike receptor binding domain (RBD). The COVID-19 pandemic has brought devastating consequences at a clinical, social and economical levels. Therefore, anticipation of potential novel SARS-causing species or SARS-CoV-2 variants with enhanced binding to ACE2 is key in the prevention of future threats to come. We have characterized a de novo single substitution, Q498Y, in SARS-CoV-2 RBD that confers stronger adherence to ACE2. While the SARS-CoV-2 {beta} variant, which includes three simultaneous amino acid replacements, induces a 4-fold stronger affinity, a single Q498Y substitution results in 2.5-fold tighter binding, compared to the Wuhan-Hu-1 SARS-CoV-2 2019 strain. Additionally, we crystallized RBDQ498Y complexed with ACE2 and provide here the structural basis for this enhanced affinity. These studies inform a rationale for prevention of potential SARS-causing viruses to come.

immunology↗

bioRxiv2021

A novel α/β T-cell subpopulation defined by recognition of EPCR

T-cell self-recognition of antigen presenting molecules is led by antigen-dependent or independent mechanisms. The endothelial protein C receptor (EPCR) shares remarkable similarity with CD1d, including a lipid binding cavity. We have identified EPCR-specific /{beta} T-cells in the peripheral blood of healthy donors. The average frequency in the CD3+ leukocyte pool is comparable to other autoreactive T-cell subsets that specifically bind MHC-like receptors. Alteration of the EPCR lipid cargo, revealed by X-ray diffraction studies, points to a prevalent, yet not exclusive, lipid-independent self-recognition. In addition, we solve the EPCR lipidome, and detect species not yet described as EPCR ligands. These studies report, for the first time, novel recognition by circulating /{beta} T-cells and provide grounds for EPCR and lipid mediated T-cell restriction.

immunology↗