Biology subjects

Garcia-Alai, M. M.

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

10 recordsLinked to original sources

bioRxiv2026

Domain-Specific Agonist Binding Affinities Explain Structural and Functional Regulation of TRPM2

TRPM2 is a Ca{superscript 2}-permeable cation channel activated by ADP-ribose (ADPR) and oxidative stress, yet the relative contributions of its two nucleotide-binding domains, MHR1/2 and NUDT9H, remain incompletely understood. Here, we quantitatively determine the affinities of the isolated human TRPM2 MHR1/2 and NUDT9H domains for ADPR, 2-deoxy-dADPR (dADPR), and 8-Br-cADPR using biophysical approaches. The MHR1/2 domain binds ADPR with high affinity (Kd {approx} 0.5 {micro}M), whereas the NUDT9H domain displays substantially lower affinity (Kd {approx} 192 {micro}M), revealing a difference of nearly three orders of magnitude. Mutational analysis demonstrates that alterations in MHR1/2 strongly affect ligand binding and channel activation, while mutations within NUDT9H that markedly reduce ligand affinity exert only modest effects on gating. In parallel, we quantify intracellular ADPR concentrations in resting and hydrogen peroxide-stimulated cells and find that they remain well below the affinity required for substantial NUDT9H occupancy. Together, our findings indicate that high-affinity binding to the MHR1/2 domain is sufficient to drive TRPM2 activation under physiological conditions, whereas the NUDT9H domain likely contributes to maintaining the structural integrity of the channel rather than directly mediating ligand-dependent activation. These results provide a quantitative framework for understanding ligand-dependent TRPM2 regulation in cells.

biophysics↗

bioRxiv2025

Clathrin adaptors drive phase separation in endocytosis and trafficking

Liquid-liquid phase separation (LLPS) underlies the formation of biomolecular condensates that organize cellular processes, including endocytosis and membrane trafficking. Although LLPS has been implicated in clathrin-mediated pathways, the specific contribution of adaptor proteins to condensate formation and function remains unclear. Here we show that two yeast adaptors, Ent5 and Sla2, undergo LLPS in vitro and that this property correlates with their distinct roles in membrane trafficking and condensate recruitment in vivo. Ent5 condensation is driven by a dynamic helix within its disordered region that acts as a molecular "sticker." Deletion of this helix disrupts membrane-associated condensation and leads to altered cargo trafficking dynamics in vivo, with Ent5-dependent transport events becoming delayed. In contrast, Sla2 behaves both as a driver and as a client of LLPS, with its coiled-coil (CC) region mediating condensation. Together, these findings reveal that endocytic adaptors can promote condensate formation through distinct structural features, thereby coupling clathrin binding and membrane association through phase separation.

biophysics↗

bioRxiv2025

Tectonic setting shapes microbial biosynthetic potential across global geothermal environments

Microbial communities in geothermal environments constitute an underexplored reservoir of biosynthetic gene clusters with significant biotechnological potential. Here, we investigate the secondary metabolite potential of 219 microbial communities across marine and continental geothermal field sites, encompassing broad environmental gradients in temperature (4.7{degrees}C to 93.5{degrees}C), pH (0.85 to 10.3), and tectonic setting, including volcanic arcs, backarcs, divergent margins at on-axis mid-ocean ridges, post-subduction extensional arcs, and paleo-convergent intraplate plume systems. We identified 9,019 putative new biosynthetic gene cluster families, mostly lacking similarity to known biosynthetic gene clusters. Volcanic arc systems consistently exhibit the highest diversity of biosynthetic repertoires, whereas intraplate plume systems showed a greater representation of terpene-associated gene cluster families. In contrast, divergent margin systems were primarily characterized by nonribosomal peptide synthetases and ribosomally synthesized and post-translationally modified peptides pathways, together accounting for a large fraction of their predicted biosynthetic diversity. These findings suggest that tectonic context could be associated with large-scale patterns in microbial biosynthetic potential and provide a geobiological framework for guiding natural product discovery in geothermal ecosystems.

microbiology↗

bioRxiv2025

Seasonal biogeochemical variations in a modern microbialite reef under early Earth-like conditions

Microbialites are organosedimentary structures dating to the Precambrian that serve as archives of Earths environmental evolution. Today, they persist in only a few environments markedly different from those in which they first arose. Here, we report a modern microbialite reef in Laguna Pozo Bravo (Puna region, Argentina), exposed to high radiation, low oxygen pressure, and volcanic inputs reminiscent of early Earth. Through physicochemical, mineralogical, spectroscopic, electron microscopy, and metagenomic analyses, we identified diverse microbial communities with metabolic capacities that induce mineralisation. Seasonal environmental fluctuations drive cyclical changes in community composition, producing potential mineralisation patterns. Our findings suggest that carbon fixation and the metabolic drivers of alkalinity in microbialites evolved over time. Moreover, the variability in prokaryotic compositions among modern microbialites demonstrates that carbonate precipitation is governed by metabolic potential rather than taxonomy, reinforcing their role as dynamic records of environmental conditions.

microbiology↗

bioRxiv2025

ChiraKit, an online tool for the analysis of circular dichroism spectroscopy data

Circular dichroism (CD) spectroscopy is an established biophysical technique to study chiral molecules. CD allows investigating conformational changes under varying experimental conditions and has been used to understand secondary structure, folding and binding of proteins and nucleic acids. Here, we present ChiraKit, a user-friendly, online, open-source tool to process raw CD data and perform advanced analysis. ChiraKit features include the calculation of protein secondary structure with the SELCON3 and SESCA algorithms, estimation of peptide helicity using the helix-ensemble model, the fitting of thermal/chemical unfolding or user-defined models, and the decomposition of spectra through singular value decomposition (SVD) or principal component analysis (PCA). ChiraKit can be accessed at https://spc.embl-hamburg.de/.

biophysics↗

bioRxiv2024

Sla2 is a core interaction hub for Clathrin Light Chain and the Pan1/End3/Sla1Complex

The interaction network of Sla2, a vital adaptor protein in the endocytic mid-coat, undergoes constant rearrangement incorporating or replacing interacting proteins over time. Sla2 serves as a scaffold linking the membrane to the actin cytoskeleton, with this role modulated by Clathrin Light Chain (CLC), which inhibits Sla2s function under certain conditions. We show that Sla2 has two independent binding sites for CLC: one previously described in homologs of Fungi (Sla2) and Metazoa (Hip1R), and a second found only in Fungi. We present the structural model of the Sla2 actin-binding domains in the context of regulatory structural domains by electron cryo-microscopy. We provide an interaction map of Sla2 and the regulatory proteins Sla1 and Pan1, predicted by AI modelling and confirmed by molecular biophysics techniques. Pan1 competes with CLC for the conserved binding site on Sla2. These results enhance the mapping of crucial interactions at endocytic checkpoints and highlight the divergence between Metazoa and Fungi in this vital process. TeaserSla2 forms complexes with three regulatory proteins in the endocytic pit, two of which compete for the same site

molecular biology↗

bioRxiv2024

Subtleties in Clathrin Heavy Chain Binding Boxes provide selectivity among Adaptor Proteins of Budding Yeast

Clathrin, forming the triskelion network, orchestrates highly regulated cellular processes facilitating cargo internalization and trafficking in eukaryotes, with its N-terminal domain (NTD) pivotal for adaptor protein (AP) interactions. The NTD contains up to four AP-binding sites, and their roles in preferential occupancy by APs have not been addressed. Here, employing a combination of integrative biophysical and structural approaches together with in vivo functional experiments, we investigated the binding hierarchy and selectivity of adaptors for clathrin, aiming to understand the evolutionary conservation of redundant APs and their specialized roles in endocytosis and cellular trafficking mechanisms. We found that yeast epsin Ent5 displayed the highest affinity for clathrin, indicating its significant role in cellular trafficking processes. Epsins Ent1 and Ent2, which are crucial for endocytosis but described to have redundant functions, revealed distinct binding patterns; Ent1 demonstrated stronger interactions with clathrin than Ent2, explaining its functional divergence towards actin binding. Despite both having actin anchoring domains, since Ent1 is actually more stably recruited by clathrin, it would provide a better actin anchoring function. These results offer molecular insights into AP selectivity, suggesting they competitively bind clathrin while also targeting different clathrin sites.

molecular biology↗

bioRxiv2023

Raynals, an online tool for the analysis of dynamic light scattering

Dynamic light scattering (DLS) is routinely employed to assess the homogeneity and size distribution profile of samples containing microscopic particles in suspension or solubilised polymers. In this work, we introduce Raynals, an user-friendly software for the analysis of single-angle DLS data that uses the Tikhonov-Phillips regularisation. Performance is evaluated on simulated and experimental data, generated by different DLS instruments, for several proteins and gold nanoparticles. DLS data can be easily misinterpreted and the simulation tools available in Raynals allow understanding of the limitations of the measurement and its resolution. It has been designed as a tool to address quality control of biological samples, during sample preparation and optimisation, and it helps in the detection of aggregates showing the influence of large particles. Last, Raynals provides flexibility in the way the data is presented, allows exporting publication-quality figures, it is free for academic use, and can be accessed online on the eSPC data analysis platform at spc.embl-hamburg.de.

biophysics↗

bioRxiv2021

Mapping the peptide binding groove of MHC class I

An essential element of adaptive immunity is the selective binding of peptide antigens by major histocompatibility complex (MHC) class I proteins and their presentation to cytotoxic T lymphocytes on the cell surface. Using native mass spectrometry, we here analyze the binding of peptides to an empty disulfide-stabilized HLA-A*02:01 molecule. This novel approach allows us to examine the binding properties of diverse peptides. The unique stability of our MHC class I even enables us to determine the binding affinity of complexes, which are suboptimally loaded with truncated or charge-reduced peptides. Notably, a unique erucamide adduct decouples affinity analysis from peptide identity alleviating issues usually attributed to clustering during electrospray ionization. We discovered that two anchor positions at the binding surface between MHC and peptide can be stabilized independently and further identify the contribution of other peptidic amino acids on the binding. We propose this as an alternative, likely universally applicable method to artificial prediction tools to estimate the binding strength of peptides to MHC class I complexes quickly and efficiently. This newly described MHC class I-peptide binding affinity quantitation represents a much needed orthogonal, independent approach to existing computational affinity predictions and has the potential to eliminate binding affinity biases and thus accelerate drug discovery in infectious diseases, autoimmunity, vaccine design, and cancer immunotherapy.

biophysics↗

bioRxiv2020

Structure of the endocytic adaptor complex reveals the basis for efficient membrane anchoring during clathrin-mediated endocytosis

During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process, middle coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryoEM structure of a 16-mer complex of membrane binding domains from Sla2 and Ent1 that anchors to the plasma membrane. Detailed mutagenesis in vitro and in vivo of the tetramer interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate the biological relevance of these interactions. Finally, time-resolved experiments in solution suggest that adaptors have evolved to achieve a fast subsecond timescale assembly in the presence of PIP2. Together, these findings provide a molecular understanding of an essential piece for the molecular puzzle of clathrin-coated sites.

biophysics↗