Biology subjects

Uson, I.

Publications and source records attributed to Uson, I..

9 recordsLinked to original sources

bioRxiv2024

The two crystal structures of unloaded and cargo-loaded Vacuolar Sorting Receptor 1 lumenal domain (VSR1-Nt) reveal two cargo binding sites and a mechanism of transport.

Vacuolar sorting receptors (VSRs) are type I membrane proteins crucial for seed germination and plant development. While VSR trafficking has been extensively studied, the mechanism of cargo binding and release by the N-terminal lumenal region is less understood. We have elucidated the crystal structures of unloaded and cargo-loaded forms of the lumenal region of VSR1 containing the protease-associated domain, the Central domain and epidermal growth factor-like repeats. Calcium coordination induces remodeling of the linkers between domains, triggering large conformational changes that expose two binding sites, one across the PA and Central domain and a second site in the Central domain. Our findings provide a mechanistic model for cargo binding in a calcium rich environment, where VSR is locked in a conformation exposing the cargo binding sites, while cargo release is favoured by lower calcium concentrations and trimer formation. These results advance our current knowledge on VSRs and will inform future studies on vacuolar trafficking and cargo binding/release.

plant biology↗

bioRxiv2023

Vibrio cholerae's ToxRS Bile Sensing System

Cholera represents a diarrheal disease caused by the Gram-negative bacterium Vibrio cholerae. Its environmental persistence causing recurring sudden outbreaks is enabled by V. choleraes rapid adaption to changing environments involving sensory proteins like ToxR and ToxS. Located at the inner membrane, ToxR and ToxS react to environmental stimuli like bile acid, thereby inducing survival strategies e.g. bile resistance and virulence regulation. Currently, transcription factor ToxR is described as main environmental sensor for bile acid, whose activity is enhanced by binding to ToxS. Here, the presented crystal structure of the sensory domains of ToxR and ToxS in combination with multiple bile acid interaction studies, reveals that a bile binding pocket of ToxS is only properly folded upon binding to ToxR. These findings support the previously suggested link between ToxRS and VtrAC-like co-component systems. Besides VtrAC, ToxRS is now the only experimentally determined structure within this recently defined superfamily, further emphasizing its significance. In-depth analysis of the ToxRS complex reveals its remarkable conservation across various Vibrio species, underlining the significance of conserved residues in the ToxS barrel and the more diverse ToxR sensory domain. Unraveling the intricate mechanisms governing ToxRSs environmental sensing capabilities, provides a promising tool for disruption of this vital interaction, ultimately inhibiting Vibrios survival and virulence. Our findings hold far-reaching implications for all Vibrio strains that rely on the ToxRS system as a shared sensory cornerstone for adapting to their surroundings.

biochemistry↗

bioRxiv2022

Model building in SHELXE

Density modification is a standard step to provide a route for routine structure solution by any experimental phasing method -with SAD and MAD being the most popular ones- as well as to extend fragments or incomplete models into a full solution. The effect of density modification on the starting maps from either source is illustrated in the case of SHELXE. The different modes in which the program can run are reviewed; these include less well-known uses such as reading external phase values and weights or phase distributions encoded in Hendrickson-Lattman coefficients. Typically in SHELXE, initial phases are calculated from experimental data, from a partial model or map, or from a combination of both sources. The initial phase set is improved and extended by density modification and, if the resolution of the data and the type of structure permits, poly-alanine tracing. The trace now includes an extension into the gamma position or hydrophobic and aromatic side chains if a sequence is provided, which is performed in every tracing cycle. Once a correlation coefficient over 30% between the structure factors calculated from such a trace and the native data indicates that the structure has been solved, in all model building cycles sequence is docked and side chains are fitted if the map supports it. The extensions to the tracing algorithm brought in to provide a complete model are discussed. The improvement in phasing performance is assessed using a set of tests. SynopsisSide chain tracing now completes model building in SHELXE to enhance density modification. All alternative SHELXE modes, using single or combined sources of starting phase information, are described. O_FD O_INLINEFIG[Formula 1]C_INLINEFIGM_FD(1)C_FD

biophysics↗

bioRxiv2022

Exploring generality of experimental conformational changes with AlphaFold predictions

Structural predictions have matched the accuracy of experimental structures in the case of close homologues, outperformed docking methods for multimeric complexes and helped sampling the conformational landscape of transporters and receptors. Such successes prompt the question whether predictions can be used to relate experimental structures in the context of available knowledge. LysR-type transcriptional regulators (LTTR) constitute the most common family of bacterial regulators. Intriguingly, their experimental structures are remarkably diverse. The active species, composed of flexible monomers dimerizing through their N- and C-terminal domains in a circular arrangement, differ across LTTR, due to intrinsic sequence differences or because crystals stabilize diverse snapshots of a common dynamic mechanism. We have used AlphaFold2 (AF) to interrogate the experimental AtzR structure in the context of predictions guided towards the different hetero-multimeric conformations known for other LTTR. Our approach drives AF prediction with the structure-based selection of the information input through sequence alignment and template conformation, linked to examination of the energy with PISA and interactions with ALEPH.

biophysics↗

bioRxiv2022

SPACA6 structure reveals a conserved superfamily of gamete fusion-associated proteins

SPACA6 is a sperm-expressed surface protein that is critical for gamete fusion during mammalian sexual reproduction. Despite this fundamental role, little is known about how SPACA6 specifically functions. We elucidated the crystal structure of SPACA6 at 2.2-[A] resolution, revealing a two-domain protein containing a four-helix bundle and Ig-like {beta}-sandwich connected via a quasi-flexible linker. Based on the structural analysis, we propose SPACA6 is a founding member of a superfamily of gamete fusion-associated proteins, herein dubbed the IST superfamily. The IST superfamily is defined structurally by its distorted four-helix bundle and a pair of disulfide-bonded CXXC motifs. A structure-based search of the AlphaFold human proteome identified more protein members to this superfamily; remarkably, many of these proteins are linked to gamete fusion. The SPACA6 structure and its connection to other IST-superfamily members provide a missing link in our knowledge of mammalian gamete fusion. Significance StatementSPACA6 is a human sperm protein vital for the fusion of gametes, though its exact function remains a mystery. We present the first solved structure of SPACA6: a two-domain fold comprised of an Ig-like domain and a distorted four-helix bundle. Dali searches of the PDB and AlphaFold reveal a family of structurally related proteins, several of which are also known to play a role in gamete fusion; as such, SPACA6 is a founding member of a conserved protein superfamily, dubbed the IST superfamily. Evolutionary analysis to ascertain functionally relevant structural elements in SPACA6 show a conservation of flexibility between the two domains and several conserved surfaces that could function as protein-protein interfaces.

developmental biology↗

bioRxiv2021

SEQUENCE SLIDER: integration of structural and genetic data to characterize isoforms from natural source

Proteins isolated from natural source can be composed of a mixture of isoforms with similar physicochemical properties that coexists in final steps of purification, toxins being prominent examples. Sequence composition is enforced throughout structural studies even when unsubstantiated. Herein, we propose a novel perspective to address the usually neglected heterogeneity of natural products by integrating biophysical, genetic and structural data in our program SEQUENCE SLIDER. The aim is to assess the evidence supporting chemical composition in structure determination. Locally, we interrogate the experimental map to establish which side chains are supported by the structural data and the genetic information relating sequence conservation is integrated in this statistic. Hence, we build a constrained peptide database, containing most probable sequences to interpret mass spectrometry data (MS). In parallel, we perform MS de novo sequencing with genomic-based algorithms that foresee point mutations. We calibrated SLIDER with Gallus gallus lysozyme, for which sequence is unequivocally established and numerous natural isoforms are reported. We used SLIDER to characterise a metalloproteinase and a phospholipase A2-like protein from the venom of Bothrops moojeni and a crotoxin from Crotalus durissus collilineatus. This integrated approach offers a more realistic structural descriptor to characterize macromolecules isolated from natural source. Key pointsO_LIThe method SEQUENCE SLIDER integrates biophysical, genetic and structural data to assign sequence. C_LIO_LIIt joins crystallography, mass spectrometry and phylogenetic data to characterize isoforms. C_LIO_LISequence heterogeneity of four proteins purified directly from snake venom was established. C_LI

bioinformatics↗

bioRxiv2021

Fragment-based ab initio phasing of peptidic nanocrystals by MicroED

Microcrystal electron diffraction (MicroED) is transforming the visualization of molecules from nanocrystals, rendering their three-dimensional atomic structures from previously unamenable samples. Peptidic structures determined by MicroED include naturally occurring peptides, synthetic protein fragments and peptide-based natural products. However, as a diffraction method, MicroED is beholden to the phase problem, and its de novo determination of structures remains a challenge. ARCIMBOLDO, an automated, fragment-based approach to structure determination. It eliminates the need for atomic resolution, instead enforcing stereochemical constraints through libraries of small model fragments, and discerning congruent motifs in solution space to ensure validation. This approach expands the reach of MicroED to presently inaccessible peptidic structures including segments of human amyloids, and yeast and mammalian prions, and portends a more general phasing solution while limiting model bias for a wider set of chemical structures.

biochemistry↗

bioRxiv2020

The Amphibian Antimicrobial Peptide Uperin 3.5 is a Cross-α/Cross-β Chameleon Functional Amyloid

Antimicrobial activity is being increasingly linked to amyloid fibril formation, suggesting physiological roles for some human amyloids, which have historically been viewed as strictly pathological agents. This work reports on formation of functional cross- amyloid fibrils of the amphibian antimicrobial peptide uperin 3.5 at atomic-resolution, an architecture initially discovered in the bacterial PSM3 cytotoxin. The fibrils of uperin 3.5 and PSM3 were comprised of parallel and anti-parallel helical sheets, respectively, recapitulating properties of {beta}-sheets. Uperin 3.5 helical fibril formation was largely induced by, and formed on, bacterial cells or membrane mimetics, and led to membrane damage and cell death. Uperin 3.5 demonstrated chameleon properties, with a secondary structure switch to cross-{beta} fibrils with reduced antibacterial activity in the absence of lipids or after heat shock. These findings suggest a regulation mechanism, which includes storage of inactive peptides as well as environmentally induced activation of uperin 3.5, via chameleon cross-/{beta} amyloid fibrils.

biochemistry↗

bioRxiv2018

A conserved mechanism for meiotic chromosome organization through self-assembly of a filamentous chromosome axis core

The meiotic chromosome axis plays key roles in meiotic chromosome organization and recombination, yet the underlying protein components of this structure are highly diverged. Here, we show that \"axis core proteins\" from budding yeast (Red1), mammals (SYCP2/SYCP3), and plants (ASY3/ASY4) are evolutionarily related and play equivalent roles in chromosome axis assembly. We first identify motifs in each complex that recruit meiotic HORMADs, the master regulators of meiotic recombination. We next find that axis core complexes form homotetrameric (Red1) or heterotetrameric (SYCP2:SYCP3 and ASY3:ASY4) coiled-coil assemblies that further oligomerize into micron-length filaments. Thus, the meiotic chromosome axis core in fungi, mammals, and plants shares a common molecular architecture and role in axis assembly and recombination control. We propose that the meiotic chromosome axis self-assembles through cooperative interactions between dynamic DNA loop-extruding cohesin complexes and the filamentous axis core, then serves as a platform for chromosome organization, recombination, and synaptonemal complex assembly.

biochemistry↗