Biology subjects

Montoya, G.

Publications and source records attributed to Montoya, G..

At least 19 recordsLinked to original sources

bioRxiv2026

A DNA-scaffolded Retron Ring Mediates Antiphage Immunity

Retrons are a diverse family of bacterial defence systems that couple reverse transcriptase (RT) activity to antiphage immunity by producing multicopy single-stranded DNA (msDNA). Yet, the molecular mechanisms by which most systems detect infection and execute immunity remain unknown. Here, we define the structure, immune sensing and effector mechanisms of retron type IX, composed of an RT, a non-coding RNA, a tandem KH-HTH subunit and a HEPN RNase effector. Retron IX confers robust protection against diverse bacteriophages. Cryo-EM and biochemical analyses reveal a supramolecular ring assembly composed of 8-10 repeating modules, spanning up to 230 [A] in diameter. Within this assembly, the msDNA scaffolds the outer rim inhibiting the HEPN ribonuclease in a catalytically poised state, with the active site occluded within the inner layer. We identify a phage-encoded PD-(D/E)XK nuclease as the immune trigger that cleaves the exposed msDNA stem-loop, leading to ring disassembly and HEPN effector release. Upon activation, the HEPN ribonucleases cleave tRNAs indiscriminately, inducing dormancy and blocking phage propagation. Our findings unveil how ring-shaped higher-order assembly controls enzymatic activities while allowing signal-specific activation and establish the ncRNA as an evolvable scaffold underlying the remarkable structural and defensive diversity of bacterial retron immune complexes.

microbiology↗

bioRxiv2026

Development and Characterisation of a Versatile Single-Domain Antibody Specific for M1-linked Ubiquitin Chains

Ubiquitin signalling is mediated by structurally distinct polyubiquitin chains that encode discrete cellular functions. Progress in deciphering this ubiquitin code, particularly for the less abundant atypical chain types, has been hindered by limited availability of versatile chain type-specific affinity reagents. Here, we demonstrate that human single-domain antibodies (sdAbs) provide a versatile scaffold for the generation of ubiquitin linkage-specific binders. Using phage display and synthetic human sdAb libraries, we identified 2A6, an sdAb that specifically recognises methionine-1 (M1)-linked ubiquitin chains. To our knowledge, 2A6 represents the first reported sdAb with specificity for a defined homotypic ubiquitin chain linkage. 2A6 bound M1-linked ubiquitin chains with nanomolar affinity and was specific for M1-linked chains at the level of both diubiquitin and long polyubiquitin chains. AlphaFold3 modelling, supported by saturation mutagenesis, predicted that 2A6 recognises the proximal and distal ubiquitin moieties together with the region near the M1 linkage. Functionally, 2A6 enabled specific detection and enrichment of M1-linked ubiquitin across multiple applications, including ELISA, immunoblotting, immunoprecipitation under semi-denaturing conditions, substrate ubiquitination analysis, and immunofluorescence microscopy. The sdAb can be readily produced in E. coli from a single expression plasmid, providing a tractable, cost-effective and versatile reagent for investigating M1-linked ubiquitin signalling. Our work establishes sdAbs as a versatile scaffold for ubiquitin linkage-specific affinity reagents, providing a framework for the development of analogous binders specifically targeting additional ubiquitin linkages or architectures.

biochemistry↗

bioRxiv2026

The HSP90-CDC37 Chaperone System Orchestrates RAF1 Kinase Activation Through a Pre-Dimerization Mechanism

RAF kinases activate MEK in the RAS-MAPK signaling pathway, and changes in RAF kinase signaling have been linked to tumor formation. RAF1 requires the HSP90-CDC37 chaperone system for proper activation, but how the HSP90-CDC37 chaperone system regulates RAF kinase maturation remains enigmatic. We present novel cryo-EM structures of previously uncharacterized RAF1 chaperone complexes, including a 2:2:2 RAF1-HSP90-CDC37 complex (RRHCC), intermediate assemblies (RHCC), and a RAF1-HSP90-CDC37-p23 complex (RHCp23). These reveal an asymmetric stepwise folding mechanism unique among HSP90 kinase clients in which one RAF1 threads through the HSP90 lumen while another is captured in a "casting mold" formed by CDC37 and HSP90 that stabilizes the partially folded C helix of RAF1. The RHCp23 structure shows how p23 cooperates with CDC37 to regulate ATP hydrolysis and client release. The HSP90-CDC37 system supports pre-dimerization of RAF1 and BRAFV600E homodimers and RAF1 heterodimers, a mechanism unique to RAF among kinase clients of HSP90. Phosphoproteomics reveals selective activating phosphorylations within RRHCC. These RAF isoform complexes differentially activate MEK signaling and cell proliferation, establishing HSP90-CDC37 as not just a passive stabilizer but an active regulator of RAF signaling with therapeutic implications.

biochemistry↗

bioRxiv2025

The SPATA5-SPATA5L1-CINP-C1ORF109 complex is essential for cytoplasmic pre-60S ribosome maturation

The ribosome is a universally conserved and essential protein complex, but its biogenesis in mammals is more complex than in single-celled eukaryotes. To explore this added complexity, we conducted a protein-protein interaction screen in human cells. This led to the identification of the eumetazoan-specific SPATA5-SPATA5L1-CINP-C1ORF109 (55LCC) complex as a key regulator of ribosome biogenesis. Structural analyses using cryo-EM and X-ray crystallography defined the architecture of 55LCC. Functional studies following acute depletion revealed that each component is essential for pre-60S maturation. Swapping endogenous 55LCC components with mutant versions pinpointed critical functional interactions and showed that SPATA5s ATPase activity is more important than SPATA5L1s. Our findings support that SPATA5 evolved from the solitary yeast ATPase Drg1 into the multiprotein 55LCC complex in metazoans. This work provides insights into the complexity of ribosome biogenesis and lays the foundation for deeper exploration of 55LCCs role in pre-60S maturation.

biochemistry↗

bioRxiv2025

Reverse transcribed ssDNA derepresses translation of a retron antiviral protein

Retrons are bacterial immune systems that prevent the spread of phages by initiating a toxic response within infected hosts. All previously characterized retrons produce high levels of multicopy single-stranded DNA (msDNA) in the cell by reverse transcription, which acts as an antitoxin in the absence of phage infection. However, we describe here a non-canonical mechanism for Type VI retrons, which do not produce detectable msDNA in the absence of phage, yet still provide phage defense. Focusing primarily on Retron-Vpa2, a Type VI retron from Vibrio parahaemolyticus, we show broad defense against phages and identify triggers of the system within phage recombination systems. Within the Retron-Vpa2 operon, we find a highly enriched, structured transcript that we term a hybrid RNA (hyRNA), which contains both the retrons reverse transcription template and a translationally repressed toxic effector coding sequence. We find that phage infection induces the accumulation of high levels of msDNA and that this msDNA is necessary for derepressing translation of the antiviral toxin. These findings present key biological and mechanistic insights into a distinct group of retrons while highlighting the diversity of systems that participate in bacterial immunity.

microbiology↗

bioRxiv2023

VCF1 is an unconventional p97/VCP cofactor promoting recognition of ubiquitylated p97-UFD1-NPL4 substrates

The hexameric AAA+ ATPase p97/VCP functions as an essential mediator of ubiquitin-dependent cellular processes, extracting ubiquitylated proteins from macromolecular complexes or membranes by catalyzing their unfolding. p97 is directed to ubiquitylated client proteins via multiple cofactors, most of which interact with the p97 N-domain. Here, we discovered that FAM104A, a protein of unknown function that we named VCF1 (VCP/p97 Cofactor FAM104 1), acts as a novel p97 cofactor in human cells. Detailed structure-function studies revealed that VCF1 directly binds p97 via a conserved novel -helical motif that recognizes the p97 N-domain with unusually high affinity, exceeding that of other cofactors. We show that VCF1 engages in joint p97 complex formation with the heterodimeric primary p97 cofactor UFD1-NPL4 and promotes p97-UFD1-NPL4-dependent proteasomal degradation of ubiquitylated substrates in cells. Mechanistically, VCF1 indirectly stimulates UFD1-NPL4 interactions with ubiquitin conjugates via its binding to p97 but has no intrinsic affinity for ubiquitin. Collectively, our findings establish VCF1 as an unconventional p97 cofactor that promotes p97-dependent protein turnover by facilitating p97-UFD1-NPL4 recruitment to ubiquitylated targets.

molecular biology↗

bioRxiv2023

Detuning of the Ribosome Conformational Landscape Promotes Antibiotic Resistance and Collateral Sensitivity

Around 50% of the current antibiotic arsenal targets the ribosome, thus resistance to ribosome-targeting antibiotics poses severe challenges to antimicrobial treatments. Here, we characterize a 12-nucleotide deletion in the rplF gene encoding the uL6 ribosomal protein, which was identified in a tobramycin-resistant strain of Pseudomonas aeruginosa isolated from a cystic fibrosis patient. To understand this resistance, we determined 87 cryo-EM structures of wild-type and mutant ribosomes characterizing their conformational landscape. Our analysis reveals how detuning of the ribosome dynamics alters its rotational movement circumventing tobramycin inhibition. The mutation compromises the 50S assembly, triggering structural instability and inducing a different rotational dynamic of the 70S. We found 4 new binding sites of tobramycin, one of them exclusive of the mutant, where the binding of the antibiotic acts as an allosteric activator skipping inhibition. Our data also illustrate how chloramphenicol stabilizes the mutant ribosome, enhancing inhibition and thereby leading to collateral sensitivity.

biophysics↗

bioRxiv2022

DAXX adds a de novo H3.3K9me3 deposition pathway to the histone chaperone network

A multitude of histone chaperones are required to protect histones after their biosynthesis until DNA deposition. They cooperate through the formation of co-chaperone complexes, but the crosstalk between nucleosome assembly pathways remains enigmatic. Using explorative interactomics approaches, we characterize the organization of the histone H3-H4 chaperones network and define the interplay between histone chaperone systems. We identify and validate several novel histone dependent complexes and predict the structure of the ASF1 and SPT2 co-chaperone complex, expanding the role of ASF1 in histone dynamics. We show that DAXX acts separately from the rest of the network, recruiting heterochromatin factors and promoting lysine 9 tri-methylation of new histone H3.3 prior to deposition onto DNA. With its functionality, DAXX provides a molecular mechanism for de novo heterochromatin assembly. Collectively, our findings provide a new framework for understanding how cells orchestrate histone supply and comply with chromatin dynamics throughout the cell cycle.

molecular biology↗

bioRxiv2022

Structure of the TnsB transposase-DNA complex of type V-K CRISPR-associated transposon

CRISPR-associated transposons (CASTs) represent unique mobile genetic elements that co-opted CRISPR-Cas immune systems for RNA-guided DNA transposition. Type V-K CAST is composed by Cas12k, TniQ, TnsC and TnsB. Here, we present the 2.46 [A] cryoelectron microscopy structure of the Scytonema hofmannii CAST TnsB transposase in complex with the strand transfer DNA in a post-catalytic state. The shTnsB strand transfer complex maintains the intertwined architecture of the MuA phage transpososome. However, the building of the assembly depends on different local interactions. The protein-DNA complex forms a pseudo-symmetrical assembly in which the 4 protomers of shTnsB adopt two different conformations. The recognition of the transposon ends is accomplished by two small helical domains. The two protomers involved in the strand transfer reaction display a catalytically competent active site composed by three acidic residues (DDE), while the other two, which play a key role in the complex architecture, show catalytic pockets where the DDE residues are not properly positioned for cleavage. Quantification of in vivo transposition assays of mutants in key DNA binding residues, reveals that the lack of specificity generally decreases activity, but it could increase transposition in some cases. Our structure sheds light on the strand transfer reaction of the DDE DNA transposases and offers new insights into RNA-guided transposition in CAST systems.

biochemistry↗

bioRxiv2022

Structure of the RAF1-HSP90-CDC37 complex reveals the basis of RAF1 regulation

RAF kinases are RAS-activated enzymes that initiate signalling through the MAPK cascade to control cellular proliferation, differentiation, and survival. Here, we describe the structure of the full-length RAF1 protein in complex with HSP90 and CDC37 obtained by cryo-electron microscopy. The reconstruction reveals a RAF1 kinase with an unfolded N-lobe separated from its C-lobe. The hydrophobic core of the N-lobe is trapped in the HSP90 dimer while CDC37 wraps around the chaperone and interacts with the N- and C-lobes of the kinase. The structure indicates how CDC37 can discriminate between the different members of the RAF family. Our structural analysis also reveals that the folded RAF1 assembles with 14-3-3 dimers, suggesting that after folding follows a similar activation as B-RAF. Finally, disruption of the interaction between CDC37 and the DFG segment of RAF1 unveils potential vulnerabilities to attempt the pharmacological degradation of RAF1 for therapeutic purposes.

biochemistry↗

bioRxiv2022

Evolutionary Origin of Vertebrate OCT4/POU5 Functions in Supporting Pluripotency

The support of pluripotent cells over time is an essential feature of development. In eutherian embryos, pluripotency is maintained from naive states in peri-implantation to primed pluripotency at gastrulation. To understand how these states emerged, we reconstruct the evolutionary trajectory of the Pou5 gene family, which contains the central pluripotency factor OCT4. By coupling evolutionary sequence analysis with functional studies in mouse Embryonic Stem Cells (ESCs), we found that the ability of POU5 proteins to support pluripotency originated in the gnathostome lineage, prior to the generation of two paralogues, Pou5f1 and Pou5f3 via gene duplication. In osteichthyans, retaining both genes, the paralogues differ in their support of naive and primed pluripotency. This specialization of these duplicates enables the diversification of function in self-renewal and differentiation. By integrating sequence evolution, ESC phenotypes, developmental contexts and structural modelling, we pinpoint OCT4 regions sufficient for naive pluripotency and describe their adaptation over evolutionary time.

developmental biology↗

bioRxiv2021

Cellular toxicity of iHAP1 and DT-061 does not occur through PP2A-B56 targeting

PP2A is an abundant phosphoprotein phosphatase that acts as a tumor suppressor. For this reason, compounds able to activate PP2A are attractive anticancer agents. The small molecule compounds iHAP1 and DT-061 have recently been reported by Leonard et al. (2020) and Morita et al. (2020) in Cell to selectively stabilize specific PP2A-B56 complexes which mediate cell killing. Here, we show that this is not the case and question key findings in these papers. Through genome wide CRISPR-Cas9 screens, we uncover the biological pathways targeted by these compounds. We find that iHAP1 directly blocks microtubule assembly both in vitro and in vivo and thus acts as a microtubule poison. In contrast, DT-061 disrupts both the Golgi apparatus and the endoplasmic reticulum and we directly visualize DT-061 in cytoplasmic granules that co-localize with Golgi markers. Our work demonstrates that iHAP1 and DT-061 cannot be used for dissecting PP2A-B56 biology.

cell biology↗

bioRxiv2021

Structure of the mini-RNA-guided endonuclease CRISPR-Cas{Phi}3

CRISPR-Cas{Phi} is a novel family of miniaturized RNA-guided endonucleases from phages 1,2. These novel ribonucleoproteins (RNPs) provide a compact scaffold gathering all key activities of a genome editing tool2. Here, we provide the first structural insight into Cas{Phi} singular DNA targeting and cleavage mechanism by determining the cryoEM structure of Cas{Phi}3 with the triple strand R-loop generated after DNA cleavage. The structure reveals the unique machinery for target unwinding to form the crRNA-DNA hybrid and cleaving the target DNA. The protospacer adjacent motif (PAM) is recognised by the target strand (T-strand) and non-target strand (NT-strand) PAM interacting domains (TPID and NPID). Unwinding occurs after insertion of the conserved 1 helix disrupting the dsDNA, thus facilitating the crRNA-DNA hybrid formation. The NT-strand is funnelled towards the RuvC catalytic site, while a long helix of TPID separates the displaced NT-strand and the crRNA-DNA hybrid avoiding DNA re-annealing. The crRNA-DNA hybrid is directed to the stop (STP) domain that splits the hybrid guiding the T-strand towards the RuvC active site. The conserved RuvC insertion of the Cas{Phi} family is extended along the hybrid, interacting with the phosphate backbone of the crRNA. A cluster of hydrophobic residues anchors the RuvC insertion in a cavity of the STP domain. The assembly of the hybrid promotes the shortening of the RuvC insertion, thus pulling the STP towards the RuvC active site to activate catalysis. These findings illustrate why Cas{Phi} unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. Site-directed mutagenesis in key residues support Cas{Phi}3 target DNA and non-specific ssDNA cutting mechanism. Our analysis provides new avenues to redesign the compact CRISPR-Cas{Phi} nucleases for genome editing.

biochemistry↗

bioRxiv2021

Cas12a is a dynamic and precise RNA-guided nuclease without off-target activity on {lambda}-DNA

Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we combined optical tweezers with fluorescence to monitor Cas12a binding onto {lambda}-DNA, providing insight into its DNA binding and cleavage mechanisms. At low forces Cas12a binds DNA specifically with two off-target sites, while at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. Despite the multiple binding events, cleavage is only observed on the target site at low forces, when the DNA is flexible. Activity assays show that the preferential off-target sites are not cleaved, and the {lambda}-DNA is severed at the target site. This precision is also observed in Cas12a variants where the specific dsDNA and the unspecific ssDNA cleavage are dissociated or nick the target DNA. We propose that Cas12a and its variants are precise endonucleases that efficiently scan the DNA for its target but only cleave the selected site in the {lambda}-DNA.

biophysics↗

bioRxiv2020

A highly conserved binding pocket on PP2A-B56 is required for shugoshin binding and cohesion protection

The shugoshin proteins are universal protectors of centromeric cohesin during mitosis and meiosis. The binding of human Sgo1 to the PP2A-B56 phosphatase through a coiled coil (CC) region is believed to mediate cohesion protection during mitosis. Here we undertook a structure function analysis of the PP2A-B56-Sgo1 complex, revealing unanticipated aspects of complex formation and function. We establish that a highly conserved pocket of the B56 regulatory subunit is required for Sgo1 binding and cohesion protection. Consistent with this, we show that Sgo1 blocks the binding of PP2A-B56 substrates containing a canonical B56 binding motif. Surprisingly, we identify B56 and Sgo1 mutants that prevent complex formation yet support cohesion protection and normal mitotic progression. This suggests that Sgo1 and PP2A-B56 have cohesion protection activity independently of complex formation. Collectively our work provides important insight into cohesion protection during mitosis.

cell biology↗

bioRxiv2020

DeepFRET: Rapid and automated single molecule FRET data classification using deep learning

Single molecule Forster Resonance energy transfer (smFRET) is a mature and adaptable method for studying the structure of biomolecules and integrating their dynamics into structural biology. The development of high throughput methodologies and the growth of commercial instrumentation have outpaced the development of rapid, standardized, and fully automated methodologies to objectively analyze the wealth of produced data. Here we present DeepFRET, an automated standalone solution based on deep learning, where the only crucial human intervention in transiting from raw microscope images to histogram of biomolecule behavior, is a user-adjustable quality threshold. Integrating all standard features of smFRET analysis, DeepFRET will consequently output common kinetic information metrics for biomolecules. We validated the utility of DeepFRET by performing quantitative analysis on simulated, ground truth, data and real smFRET data. The accuracy of classification by DeepFRET outperformed human operators and current commonly used hard threshold and reached >95% precision accuracy only requiring a fraction of the time (<1% as compared to human operators) on ground truth data. Its flawless and rapid operation on real data demonstrates its wide applicability. This level of classification was achieved without any preprocessing or parameter setting by human operators, demonstrating DeepFRETs capacity to objectively quantify biomolecular dynamics. The provided a standalone executable based on open source code capitalises on the widespread adaptation of machine learning and may contribute to the effort of benchmarking smFRET for structural biology insights.

biophysics↗

bioRxiv2020

Structures of the Cmr-βComplex Reveal the Regulation of the Immunity Mechanism of Type III-B CRISPR-Cas

Cmr-{beta} is a Type III-B CRISPR-Cas complex that upon target RNA recognition unleashes a multifaceted immune response against invading genetic elements, including ssDNA cleavage, cyclic oligoadenylate synthesis, and also a unique UA-specific ssRNA hydrolysis by the Cmr2 subunit. Here, we present the structure-function relationship of Cmr-{beta} unveiling how binding of the target RNA regulates the Cmr2 activities. CryoEM analysis revealed the unique subunit architecture of Cmr-{beta} and captured the complex in different conformational stages of the immune response, including the non-cognate and cognate target-RNA bound complexes. The binding of the target RNA induces a conformational change of Cmr2, which together with the complementation between the 5-handle in the crRNA and the 3-antitag of the target RNA, activate different configurations in a unique loop of the Cmr3 subunit, which acts as an allosteric sensor signaling the self vs. non-self recognition. These findings highlight the diverse defense strategies of Type III complexes.

biochemistry↗

bioRxiv2018

Contribution of common and rare variants to bipolar disorder susceptibility in extended pedigrees from population isolates

Current evidence from case/control studies indicates that genetic risk for psychiatric disorders derives primarily from numerous common variants, each with a small phenotypic impact. The literature describing apparent segregation of bipolar disorder (BP) in numerous multigenerational pedigrees suggests that, in such families, large-effect inherited variants might play a greater role. To evaluate this hypothesis, we conducted genetic analyses in 26 Colombian (CO) and Costa Rican (CR) pedigrees ascertained for BP1, the most severe and heritable form of BP. In these pedigrees, we performed microarray SNP genotyping of 856 individuals and high-coverage whole-genome sequencing of 454 individuals. Compared to their unaffected relatives, BP1 individuals had higher polygenic risk scores estimated from SNPs associated with BP discovered in independent genome-wide association studies, and also displayed a higher burden of rare deleterious single nucleotide variants (SNVs) and rare copy number variants (CNVs) in genes likely to be relevant to BP1. Parametric and non-parametric linkage analyses identified 15 BP1 linkage peaks, encompassing about 100 genes, although we observed no significant segregation pattern for any particular rare SNVs and CNVs. These results suggest that even in extended pedigrees, genetic risk for BP appears to derive mainly from small to moderate effect rare and common variants.

genetics↗