Biology subjects

Lopez-Alonso, J. P.

Publications and source records attributed to Lopez-Alonso, J. P..

11 recordsLinked to original sources

bioRxiv2026

Structural basis of selective phospholipid recognition and local membrane deformation by CTL1

The physiological function of the human choline transporter-like protein CTL1 (SLC44A1) remains elusive despite its implication in key pathophysiological processes. Here we combine cryo-electron microscopy, native mass spectrometry, lipidomics, molecular dynamics simulations and functional assays to characterise the molecular basis of phospholipid recognition by CTL1 and explore its potential molecular role. High-resolution structures of CTL1 in both monomeric and dimeric states reveal a broad amphipathic cavity and an extracellular domain that accommodate multiple phospholipid species, identifying phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and phosphatidic acid as endogenous CTL1 ligands. Molecular dynamics simulations show spontaneous phospholipid recruitment into these conserved binding sites accompanied by membrane thinning, curvature and local phospholipid reorganisation, with membrane deformation being enhanced upon dimerisation. Functional analyses in mammalian cells and in reconstituted proteoliposomes failed to detect CTL1-mediated choline transport, supporting a function distinct from canonical choline uptake. Together, our findings suggest that CTL1 acts as a phospholipid-binding protein capable of locally deforming the membrane and provide a structural framework linking phospholipid recognition to membrane organisation. These results offer new insights into potential mechanisms underlying the altered phospholipid homeostasis associated with CTL1 dysfunction in neurological diseases and cancer.

biophysics↗

bioRxiv2026

Structure and mechanism of the human TMEM260 O-mannosyltransferase

Protein O-linked mannose (O-Man) glycosylation is essential for mammalian development, and mutations in its biosynthetic glycosyltransferases cause severe muscular, neurological and cardiac disorders. Despite its biological and clinical importance, the structural basis of mammalian O-Man biosynthesis has remained unknown. Here we report cryo-electron microscopy (cryo-EM) structures of human TMEM260, an endoplasmic reticulum glycosyltransferase that selectively catalyzes O-mannosylation of semaphorin plexin receptors and receptor tyrosine kinases cMET and RON, key regulators of cell guidance and migration. Structures of TMEM260 in a ternary complex with its natural donor dolichyl-phosphate-{beta}-mannose (Dol-P-Man) and an acceptor peptide derived from plexin-B2, together with binary complexes with Dol-P-Man or a synthetic donor analogue, capture physiologically relevant, substrate-loaded states and reveal the structural basis of O-Man transfer. We identify a conserved O-mannosylation sequon that underlies acceptor specificity and show that TMEM260 modifies extended polypeptide substrates, consistent with a co-translational glycosylation mechanism. These findings establish the molecular mechanism of a mammalian O-mannosyltransferase required for the maturation of physiologically critical receptors and provide a structural framework for interpreting TMEM260-associated congenital malformations.

biophysics↗

bioRxiv2025

Structural remodeling of the mitochondrial protein biogenesis machinery under proteostatic stress

Cells have evolved organelle-specific responses to maintain protein homeostasis (proteostasis). During proteostatic stress, mitochondria downregulate translation and enhance protein folding, yet the underlying mechanisms remain poorly defined. Here, we employed cryo-electron tomography to observe the structural consequences of mitochondrial proteostatic stress within human cells. We detected protein aggregates within the mitochondrial matrix, accompanied by a marked remodeling of cristae architecture. Concomitantly, the number of mitochondrial ribosome complexes was significantly reduced. Mitochondrial Hsp60 (mHsp60), a key protein folding machine, underwent major conformational changes to favor complexes with its co-chaperone mHsp10. We visualized the interactions of mHsp60 with native substrate proteins, and determined in vitro mHsp60 cryo- EM structures enabling nucleotide state assignment of the in situ structures. These data converge on a model of the mHsp60 functional cycle and its essential role in mitochondrial proteostasis. More broadly, our findings reveal structural mechanisms governing mitochondrial protein biosynthesis and their remodeling under proteostatic stress.

cell biology↗

bioRxiv2025

Structural basis for ATP-driven double-ring assembly of the human mitochondrial Hsp60 chaperonin

The ATP-driven mHsp60:mHsp10 chaperonin system assists protein folding within the mitochondrial matrix of human cells. Substrate protein folding has been proposed to occur through interconnected single- and double-ring pathways. In the absence of nucleotide, mHsp60 exists in equilibrium between free protomers and heptameric single rings, while the formation of double rings requires ATP. Here, we present cryo-electron microscopy structures of mHsp60 in the apo state, bound to ATP, and bound to ATP in complex with the cochaperonin mHsp10. ATP binding to single-ring apo mHsp607 triggers coordinated conformational changes in the intermediate and apical domains, resulting in a highly dynamic apical region within the ring. Extensive inter-subunit rearrangements flatten the equatorial surface of each ring, thereby enabling inter-ring contacts that stitch the rings together to form double-ring mHsp6014. Collectively, these structures define the structural basis of ATP-driven double-ring assembly of a human mitochondrial chaperonin responsible for maintaining mitochondrial protein homeostasis.

biophysics↗

bioRxiv2025

Structural insights into a conserved mechanism of choline translocation through CHT

The essential nutrient choline is critical for cellular homeostasis across all domains of life. In humans, choline uptake in cholinergic neurons for its recycling into acetylcholine is mediated by the high-affinity Na-dependent transporter SLC5A7 (also known as CHT1). Prokaryotes also depend on choline as an osmo-protectant and as metabolite, raising the possibility that bacteria also possess choline transporters akin to CHT1. Here, we present a bacterial Na+-dependent choline transporter (sfCHT) with high sequence identity to CHT1. sfCHT transport activity can be blocked by the choline transport inhibitor hemicholinium-3. Cryo-EM structures of Na+- and choline-bound sfCHT reveal a 14 transmembrane helix topology with a LeuT-fold architecture and Na+ coordination geometry similar to CHT1. Captured in an inward-facing conformation, in sfCHT choline is found at a site near the cytoplasmic side. Computational analysis and transport assays reveal local conformational changes along a choline translocation pathway to the cytosolic site. Transport assays with CHT1 variants, carrying substitutions at conserved residues along the proposed translocation pathway in sfCHT, reveal a conserved mechanism of choline transport between the bacterial and human choline transporters.

biophysics↗

bioRxiv2025

Structural Basis for the Assembly of Amyloid Fibrils by the Master Cell-Signaling Regulator Human Receptor-Interacting Protein Kinase 1

Amyloid fibrils, typically associated with neurodegenerative diseases, also play critical roles as functional assemblies in biological processes. The RIP homotypic interaction motifs (RHIMs) in receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3) are essential for necroptosis, orchestrating the formation of amyloid-like fibrils that assemble into necrosomes. These supramolecular complexes propagate cell death signals and activate effectors like MLKL. While the structures of human RIPK3 (hRIPK3) homomeric fibrils and RIPK1-RIPK3 heteromeric fibrils have been resolved, the atomic structure of human RIPK1 (hRIPK1) homomeric fibrils has remained elusive. Here, we present a high-resolution structure of hRIPK1 RHIM-mediated amyloid fibrils, determined using an integrative approach combining cryoprobe-detected solid-state nuclear magnetic resonance spectroscopy and cryo-electron microscopy. The fibrils adopt an N-shaped amyloid fold consisting of three {beta}-sheets stabilized by the conserved IQIG RHIM motif through hydrophobic interactions and hydrogen bonding. A key hydrogen bond between N545 and G542 closes the {beta}2-{beta}3 loop, resulting in denser side-chain packing compared to hRIPK3 homomeric fibrils. This structural feature likely contributes to the compact architecture of hRIPK1 fibrils, in contrast to the more relaxed S-shaped fold observed in hRIPK3. These findings provide structural insights into how hRIPK1 homomeric fibrils nucleate hRIPK3 recruitment and fibrillization during necroptosis, offering broader perspectives on the molecular principles governing RHIM-mediated amyloid assembly and functional amyloids.

biophysics↗

bioRxiv2024

Tertiary and quaternary structure remodeling by occupancy of the substrate binding pocket in a large glutamate dehydrogenase

Glutamate dehydrogenases (GDHs) catalyze the oxidative deamination of L-glutamate to 2-oxoglutarate using NAD(P)+ as a cofactor. The large type of GDHs (L-GDHs) displays a dynamic homotetrameric architecture that alternates between open and closed states. However, the catalytic mechanism and the functional relevance of the large conformational changes in L-GDHs remain poorly understood. Here, we use cryo-EM to investigate the structure and the conformational landscape of the mycobacterial L-GDH composed of 180 kDa subunits (mL-GDH180) when incubated with L-glutamate and NAD+. Classification of the heterogeneous population of tetramers reveals opening-closing motions and sorting of individual subunits resolves the occupancy of the cofactor and substrate binding pockets. Cryo-EM maps show that ligand binding to the glutamate binding pocket is accompanied by structural changes in a region approximately two nanometers away from the active site, leading to the formation of a previously undetected interaction between the catalytic domains of neighboring subunits in mL-GDH180 closed tetrameric states. Our findings indicate that the occupancy of the substrate binding site of mL-GDH180 is linked to a remodeling of both the tertiary and quaternary structure of the enzyme. STATEMENT FOR A BROADER AUDIENCEThis work reveals how the binding of L-glutamate and NAD+ reshapes the architecture of a large glutamate dehydrogenase, linking active site occupancy to long-range structural remodeling. By capturing previously unseen conformational transitions with cryo-electron microscopy, we provide insights into the molecular logic of enzyme function in mycobacteria. These findings establish a framework to understand how structural plasticity supports metabolic control.

molecular biology↗

bioRxiv2024

A binding site for the antibiotic GE81112 in the ribosomal mRNA channel

The initiation phase is the rate-limiting step of protein synthesis (translation) and is finely regulated, making it an important drug target. In bacteria, initiation is guided by three initiation factors and involves positioning the start site on the messenger RNA within the P-site on the small ribosomal subunit (30S), where it is decoded by the initiator tRNA. This process can be efficiently inhibited by GE81112, a natural hydrophilic, noncyclic, nonribosomal tetrapeptide. It is found in nature in three structural variants (A, B and B1 with molecular masses of 643-658 Da). Previous biochemical and structural characterisation of GE81112 indicates that the primary mechanism of action of this antibiotic is to (1) prevent the initiator tRNA from binding correctly to the P-site and (2) block conformational rearrangements in initiation factor IF3, resulting in an unlocked 30S preIC state. In this study, using cryoEM, we have determined the binding site of GE81112 in initiation complexes (3.2-3.7[A]) and on empty ribosomes (2.09 [A]). This binding site is within the mRNA channel (E-site) but remote from the binding site of the initiation factors and initiator tRNA. This suggests that it acts allosterically to prevent the initiator tRNA from being locked into place. The binding mode is consistent with previous biochemical studies and recent work identifying the key pharmacophores of GE81112.

microbiology↗

bioRxiv2023

A synthetic CRISPR-Cas nuclease with expanded enzymatic activities

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonucleases have revolutionized biotechnology for their potential as programmable genome editors. Yet, most natural nucleases and their variants have limitations. Here, we report a fully synthetic CRISPR-associated (Cas) nuclease (-synCas) designed by Ancestral Sequence Reconstruction (ASR) that displays a set of robust and distinct targeting properties, not found in any other known CRISPR-Cas Class 2 system. We show that -synCas is a PAMless nuclease able to catalyse RNA-guided, specific cleavage of dsDNA, ssDNA and ssRNA. The synthetic enzyme is also capable of sequence-nonspecific degradation of dsDNA, ssDNA and ssRNA following activation by complementary dsDNA, ssDNA and ssRNA targets. Furthermore, -synCas exhibits a robust genome editing activity in human cells and bacteria. Cryo-electron microscopy structures of -synCas ternary and quaternary complexes provide a framework to understand the structural basis for its expanded enzymatic activities. The capability for programmable multimodal targeting of virtually any nucleic acid sequence distinguishes -synCas as a promising new tool to extend current CRISPR-based technologies.

molecular biology↗

bioRxiv2020

3D architecture and structural flexibility revealed in the subfamily of large glutamate dehydrogenases by a mycobacterial enzyme

Glutamate dehydrogenases (GDHs) are widespread metabolic enzymes that play key roles in nitrogen homeostasis. Large glutamate dehydrogenases composed of 180 kDa subunits (L-GDHs180) contain long N- and C-terminal segments flanking the catalytic core. Despite the relevance of L-GDHs180 in bacterial physiology, the lack of structural data for these enzymes has limited the progress of functional studies. Here we show that the mycobacterial L-GDH180 (mL-GDH180) adopts a quaternary structure that is radically different from that of related low molecular weight enzymes. Intersubunit contacts in mL-GDH180 involve a C-terminal domain that we propose as a new fold and a flexible N-terminal segment comprising ACT-like and PAS-type domains that could act as metabolic sensors for allosteric regulation. These findings uncover unique aspects of the structure-function relationship in the subfamily of L-GDHs.

microbiology↗

bioRxiv2020

A conserved rRNA switch is central to decoding site maturation on the small ribosomal subunit

While a structural description of the molecular mechanisms guiding ribosome assembly in eukaryotic systems is emerging, bacteria employ an unrelated core set of assembly factors for which high-resolution structural information is still missing. To address this, we used single-particle cryo-EM to visualize the effects of bacterial ribosome assembly factors RimP, RbfA, RsmA, and RsgA on the conformational landscape of the 30S ribosomal subunit and obtained eight snapshots representing late steps in the folding of the decoding center. Analysis of these structures identifies a conserved secondary structure switch in the 16S rRNA central to decoding site maturation, and suggests both a sequential order of action and molecular mechanisms for the assembly factors in coordinating and controlling this switch. Structural and mechanistic parallels between bacterial and eukaryotic systems indicate common folding features inherent to all ribosomes.

microbiology↗