Biology subjects

Garcia-Alai, M.

Publications and source records attributed to Garcia-Alai, M..

3 recordsLinked to original sources

bioRxiv2022

The Cryo-EM Structures of two Amphibian Antimicrobial Cross-β Amyloid Fibrils

The amyloid-antimicrobial link hypothesis is based on antimicrobial properties found in human amyloids involved in neurodegenerative and systemic diseases, along with amyloidal structural properties found in antimicrobial peptides (AMPs) across kingdoms of life. Supporting this hypothesis, we here determined the fibril structure of two AMPs from amphibians, uperin 3.5 and aurein 3.3, by cryogenic electron microscopy (cryo-EM), revealing amyloid cross-{beta} fibrils of mated {beta}-sheets at atomic resolution. Uperin 3.5 displayed substantial polymorphism with a protofilament of two mated {beta}-sheets. The determined structure was a polymorph showing a 3-blade symmetrical propeller of nine peptides per fibril layer including tight {beta}-sheet interfaces. This cross-{beta} cryo-EM structure complements the cross- fibril conformation previously determined by a crystal structure, substantiating a secondary structure switch mechanism of uperin 3.5. The aurein 3.3 arrangement consisted of six peptides per fibril layer, all showing kinked {beta}-sheets allowing a rounded compactness of the fibril. The kinked {beta}-sheets are similar to LARKS (Low-complexity, Amyloid-like, Reversible, Kinked segments) found in human functional amyloids. The amyloidal properties of antimicrobial peptides shed light on a mechanism of regulation of animicrobial activity involving self-assembly and fibril morphological variations. Moreover, the known endurance of amyloid structures can provide a template for the design of sturdy antimicrobials.

molecular biology↗

bioRxiv2021

Dissociation of β2m from MHC Class I Triggers Formation of Noncovalent, Transient Heavy Chain Dimers

At the plasma membrane of mammalian cells, major histocompatibility complex class I molecules (MHC-I) present antigenic peptides to cytotoxic T cells. Following the loss of the peptide and the light chain beta-2 microglobulin ({beta}2m), the resulting free heavy chains (FHCs) can associate into homotypic complexes in the plasma membrane. Here, we investigate the stoichiometry and dynamics of MHC-I FHCs assemblies by combining a micropattern assay with fluorescence recovery after photobleaching (FRAP) and with single molecule co-tracking. We identify non-covalent MHC-I FHC dimers mediated by the 3 domain as the prevalent species at the plasma membrane, leading a moderate decrease in the diffusion coefficient. MHC-I FHC dimers show increased tendency to cluster into higher order oligomers as concluded from an increased immobile fraction with higher single molecule co-localization. In vitro studies with isolated proteins in conjunction with molecular docking and dynamics simulations suggest that in the complexes, the 3 domain of one FHC binds to another FHC in a manner similar to the {beta}2m light chain. Significance StatementMHC class I molecules are cell surface transmembrane proteins with key functions in adaptive immunity against viral infections. The spatiotemporal organization of fully assembled MHC I at the cell surface and its function with respect to trans-interactions with T and NK cells has been studied in detail. By contrast, the consequences of peptide and {beta}2m dissociation yielding to formation of free heavy chains (FHC) have remained unclear. We have discovered that class I free heavy chains form distinct non-covalent dimers at the cell surface rather than non-specific clustering, and we have identified a dimerization interface mediated by the 3 domain. We propose that these non-covalent dimers are the basis of distinct signaling and endocytic sorting of MHC I FHC. This is to be explored in further work.

cell biology↗

bioRxiv2021

Deamidation drives molecular aging of the SARS-CoV-2 spike receptor-binding motif

The spike is the main protein component of the SARS-CoV-2 virion surface. The spike receptor binding motif mediates recognition of the hACE2 receptor, a critical infection step, and is the preferential target for spike-neutralizing antibodies. Post-translational modifications of the spike receptor binding motif can modulate viral infectivity and immune response. We studied the spike protein in search for asparagine deamidation, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, affecting both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike. Similar deamidation hotspots are frequently found at the spike receptor-binding motifs of related sarbecoviruses, at positions that are mutated in emerging variants and in escape mutants from neutralizing antibodies. Asparagine residues 481 and 501 from the receptor-binding motif deamidate with a half-time of 16.5 and 123 days at 37 {degrees}C, respectively. This process is significantly slowed down at 4 {degrees}C, pointing at a strong dependence of spike molecular aging on the environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the hACE2 receptor more than 3.5-fold. A model for deamidation of the full SARS-CoV-2 virion illustrates that deamidation of the spike receptor-binding motif leads to the accumulation in the virion surface of a chemically diverse spike population in a timescale of days. Our findings provide a mechanism for molecular aging of the spike, with significant consequences for understanding virus infectivity and vaccine development.

biochemistry↗