Biology subjects

Ruiz, F. M.

Publications and source records attributed to Ruiz, F. M..

4 recordsLinked to original sources

bioRxiv2025

Determinants of Natural Killer Cell-Mediated Antibody Dependent Cellular Cytotoxicity in SARS-CoV-2 Antibodies

A growing body of evidence underscores the role of antibody-dependent cellular cytotoxicity (ADCC) in antiviral immunity. Yet, the mechanisms underlying the ability of certain antibodies (Abs) to mediate potent ADCC activity remain poorly characterized - in particular the contribution of features within the antigen binding Fab region remain largely unexplored. In this study, we leveraged a collection of 142 SARS-CoV2 monoclonal Abs to systematically dissect the determinants of ADCC activity. We analyzed their epitope domain target, binding characteristics, neutralization potency, somatic hypermutation (SHM) and CDR3 length to determine the contribution of these features to ADCC activity. We found that ADCC activity is primarily shaped by epitope target-particularly targeting of the S2 domain of the Spike glycoprotein. ADCC potency was not associated with the degree of SHM or neutralization. Notably, ADCC activity was not correlated with binding affinity and moderate binding to antigen was sufficient for ADCC activity. By integrating these analyses, we provide a comprehensive framework for understanding the molecular and functional determinants of ADCC. Together, these findings offer novel insights into the mechanisms that underpin ADCC functions, with implications for vaccine design and therapeutic Abs development. Author SummaryWhile antibodies are best known for stopping viruses from entering cells, they also activate other arms of the immune system. One important function is their ability to signal immune cells to destroy virus-infected targets, a process called antibody-dependent cellular cytotoxicity (ADCC). In this study, we examined 142 antibodies directed against the spike protein of the virus that causes COVID-19. We compared their binding patterns, neutralizing ability, level of maturation, and other features to determine what drives their capacity to trigger ADCC. We found that the region of the spike protein targeted by the antibody was the main factor shaping this activity, with antibodies that recognized the S2 region being particularly effective. In contrast, the amount of antibody maturation, the strength of binding, or the ability to neutralize the virus did not predict immune cell activation. Our findings show that antibodies can contribute to antiviral defense through distinct mechanisms and provide new insights for guiding vaccine and antibody therapy design.

immunology↗

bioRxiv2024

Cryo-EM uncovers a sequential mechanism for RNA polymerase I pausing and stalling at abasic DNA lesions

RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) to produce the rRNA precursor, which accounts for up to 60% of the total transcriptional activity in growing cells. Pol I monitors rDNA integrity and influences cell survival, but little is known about how this enzyme processes abasic DNA lesions. Here, we report electron cryo-microscopy (cryo-EM) structures of Pol I at different stages of stalling at abasic sites, supported by in vitro transcription studies. Our results show that templating abasic sites can slow nucleotide addition by base sandwiching between the RNA 3-end and the Pol I bridge helix. However, the presence of a templating abasic site induces opening of the Pol I cleft for either enzyme dissociation from DNA or for access of A12-Ct into the active site to stimulate RNA cleavage. Nucleotide addition opposite the lesion induces an early translocation intermediate that is different from previously-described RNA polymerase paused states, as DNA bases in the hybrid tilt to form hydrogen bonds with the newly-added RNA base. While in this state nucleotide addition is strongly disfavoured, intrinsic Pol I RNA cleavage activity acts as a failsafe mechanism to minimize lesion bypass. Our results uncover a two-step mechanism leading to persistent Pol I stalling after nucleotide addition opposite Ap sites, which is distinct from arrest by CPD lesions and from Pol II blockage at Ap sites.

molecular biology↗

bioRxiv2023

The cryo-EM structure of trypanosome 3-methylcrotonyl-CoAcarboxylase provides mechanistic and dynamic insights into enzymatic function

3-methylcrotonyl-CoA carboxylase (MCC) catalyzes the two-step, biotin-dependent production of 3-methylglutaconyl-CoA, an essential intermediate in leucine catabolism. Given its critical metabolic role, deficiencies in this enzyme associate with organic aciduria, while its overexpression is linked to tumor development. MCC is a dodecameric enzyme composed of six copies of each - and {beta}-subunit. We present the cryo-EM structure of the endogenous MCC holoenzyme from Trypanosoma brucei in its soluble, non-filamentous state at 2.5 [A] resolution. We unambiguously observe the position of biotin, covalently-bound to the BBCP domain of -subunits and occupying a novel binding pocket next to the active site of a neighboring {beta}-subunit dimer. Moreover, flexibility of key residues at the /- and /{beta}-subunit interfaces enables pivoting of -subunit trimers to sequentially approach the otherwise distant active sites for the two steps in MCC catalysis. Our results provide a structural framework to understand the enzymatic mechanism of eukaryotic MCCs and assist drug discovery against trypanosome infections.

molecular biology↗

bioRxiv2021

Mechanism of FtsZ assembly dynamics revealed by filament structures in different nucleotide states

Treadmilling protein filaments perform essential cellular functions by growing from one end while shrinking from the other, driven by nucleotide hydrolysis. Bacterial cell division relies on the primitive tubulin homolog FtsZ, a target for antibiotic discovery that assembles into single treadmilling filaments that hydrolyse GTP at an active site formed upon subunit association. We determined high-resolution filament structures of FtsZ from the pathogen Staphylococcus aureus in complex with different nucleotide analogues and cations, including mimetics of the ground and transition states of catalysis. Together with mutational and biochemical analyses, our structures reveal interactions made by the GTP {gamma}-phosphate and Mg2+ at the subunit interface, a K+ ion stabilizing loop T7 for co-catalysis, new roles of key residues at the active site and a nearby crosstalk area, and rearrangements of a dynamic water shell bridging adjacent subunits upon GTP hydrolysis. We propose a mechanistic model that integrates nucleotide hydrolysis signalling with assembly-associated conformational changes and filament treadmilling. Equivalent assembly mechanisms may apply to more complex tubulin and actin cytomotive filaments that share analogous features with FtsZ.

biophysics↗