Biology subjects

Ochoa-Lizarralde, B.

Publications and source records attributed to Ochoa-Lizarralde, B..

8 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

Probing the interaction between the WDR41 7CD loop of the C9ORF72 complex and purified PQLC2

The lysosomal cationic amino acid transceptor PQLC2 integrates transport with receptor-like signaling roles by recruiting the C9ORF72/SMCR8/WDR41 (CSW) complex in response to cellular cationic amino acid depletion. CSW recruitment depends on the interaction of PQLC2 with a disordered loop (7CD loop) extending from the WDR41 {beta}-propeller. Defining the molecular basis of this interaction is therefore essential for understanding how PQLC2 connects cellular amino acid availability to downstream signaling. Here, we used site-directed electron paramagnetic resonance (EPR) spectroscopy to characterize the interaction between purified PQLC2 and a synthetic WDR41 7CD loop peptide containing a site-specific nitroxide spin label. Using a consensus mutagenesis strategy, we stabilized detergent-purified PQLC2 and found that it assembles as a homotrimer, similar to other PQ-loop transporters. EPR experiments demonstrated the direct and specific association of the 7CD loop with PQLC2, and highlighted the key role of the the central region of the 7CD loop to establish this interaction via conserved aromatic residues. Spin labels introduced at different positions further provided local structural and dynamic information of the 7CD loop upon PQLC2 binding. The data supports a model in which the central region of the loop is anchored within the cytosolic cavity of PQLC2, whereas the flanking regions, which are dispensable for binding, retain substantial conformational flexibility. Together, we provide a direct biochemical evidence for PQLC2/WDR41 7CD loop recognition, a step forward towards understanding the transceptor mechanism of PQLC2.

biochemistry↗

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 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↗

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↗

bioRxiv2021

Orthoformimycin inhibits translation elongation by displacing the A-site tRNA and preventing peptide bond formation

The ribosome is a major target for antibiotics owing to its essential cellular role in protein synthesis. Structural analysis of ribosome-antibiotic complexes provides insight into the molecular basis for their inhibitory action and highlights possible avenues to improve their potential or overcome existing resistance mechanisms. Here we use X-ray crystallography and pre-steady state kinetics to detail the inhibitory mechanism of the antimicrobial on the large ribosomal subunit.

biochemistry↗

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↗

bioRxiv2018

The third-generation tetracycline, KBP-7072, exploits and reveals a new potential of the primary tetracycline binding pocket

Antibiotic resistance is a growing threat to human health requiring the discovery or development of new anti-infectives. As such, KBP-7072 is a novel tetracycline derivative that exhibits broad-spectrum activity against Gram-positive and -negative bacterial strains. To determine the mechanism of action of KBP-7072 and understand how its unique C9 extension can be used to combat the growing problem of antibiotic resistance we determined the structure of KBP-7072 bound to the bacterial 30S ribosomal subunit, the inhibitory target of typical tetracyclines. We show that KBP-7072 binds to the primary tetracycline binding site on the 30S ribosomal subunit consistent with it acting as a protein synthesis inhibitor that blocks A-site occupation. Moreover, the unique chemical nature of KBP-7072{acute}s C9 extension leads to a distinctive interaction pattern with the 30S subunit that distinguishes KBP-7072 from the third-generation tetracycline, Tigecycline, and thus expands the interaction potential of the primary tetracycline binding pocket.

biochemistry↗