Biology subjects

de Martin Garrido, N.

Publications and source records attributed to de Martin Garrido, N..

7 recordsLinked to original sources

bioRxiv2026

Molecular basis of Cyclin B1-CDK1 recruitment by MAD1

The interaction between Cyclin B1-CDK1 and MAD1 recruits kinase activity to the nuclear pore complex and kinetochore corona to sustain spindle assembly checkpoint (SAC) signaling. Here, we characterize a short linear motif (SLiM) within MAD1 that mediates its interaction with Cyclin B1. This SLiM peptide is specific for B-type cyclins and lacks the canonical "RxL" cyclin-binding consensus sequence, suggesting a distinct mode of cyclin recognition. Using a combination of deep mutational scanning, structural analysis by cryogenic electron microscopy, and binding affinity measurements, we define the molecular basis of the Cyclin B1-MAD1 interaction. These approaches reveal that a short MAD1 peptide binds as an -helix to the RxL-binding hydrophobic patch of Cyclin B1. The interaction employs hydrophobic contacts that overlap with those used by canonical RxL motifs, but is distinguished by additional contacts and sequence features that confer preferential recognition by B-type cyclins while excluding other cyclin types. We also show that the MAD1 motif is sufficient to confer B-type cyclin-specific substrate phosphorylation in mitotic cells. Together, these findings reveal a non-canonical, B-type cyclin-specific helical binding mode that ensures selective Cyclin B1-CDK1 recruitment at two critical stages of SAC signaling, and suggest potential strategies for selective therapeutic targeting of this interaction.

molecular biology↗

bioRxiv2024

High-throughput discovery and deep characterization of cyclin-CDK docking motifs

Cyclin-CDKs are master regulators of cell division. In addition to directly activating the CDK, the cyclin subunit regulates CDK specificity by binding short peptide "docking" motifs in CDK substrates. Here, we measure the relative binding strength of [~]100,000 peptides to 11 human cyclins from five cyclin families (D, E, A, B and F). Using a quantitative intracellular binding assay and large-scale tiled peptide screening, we identified a range of non-canonical binders that unveil a broader than anticipated repertoire of cyclin docking motif types. Structural and saturation mutagenesis studies revealed distinct binding modes and sequence features that govern motif recognition, binding strength, and cyclin preference. Docking motifs vary from highly selective to pan-cyclin, thereby fine-tuning the timing of CDK phosphorylation during cell cycle progression. Overall, these findings provide an unprecedented depth of understanding about the rules encoding specificity and affinity within a group of related but distinct protein domains.

molecular biology↗

bioRxiv2023

On-grid purification of electron microscopy samples via a 3D-printed flow-cell

While recent advances in cryo-EM, coupled with single particle analysis, have the potential to allow structure determination in a near-native state from vanishingly few individual particles, this vision has yet to be realised in practise. Requirements for particle numbers that currently far exceed the theoretical lower limits, challenges with the practicalities of achieving high concentrations for difficult-to-produce samples, and inadequate sample-dependent imaging conditions, all result in significant bottlenecks preventing routine structure determination using cryo-EM. Therefore, considerable efforts are being made to circumvent these bottlenecks by developing affinity purification of samples on-grid; at once obviating the need to produce large amounts of protein, as well as more directly controlling the variable, and sample-dependent, process of grid preparation. In this proof-of-concept study, we demonstrate a further practical step towards this paradigm, developing a 3D-printable flow-cell device to allow on-grid affinity purification from raw inputs such as whole cell lysates, using graphene oxide-based affinity grids. Our flow-cell device can be interfaced directly with routinely-used laboratory equipment such as liquid chromatographs, or peristaltic pumps, fitted with standard chromatographic (1/16") connectors, and can be used to allow binding of samples to affinity grids in a controlled environment prior to the extensive washing required to remove impurities. Furthermore, by designing a device which can be 3D printed and coupled to routinely used laboratory equipment, we hope to increase the accessibility of the techniques presented herein to researchers working towards single-particle macromolecular structures.

biophysics↗

bioRxiv2021

Structure of the Bacteriophage PhiKZ non-Virion RNA Polymerase

Bacteriophage {Phi}KZ is the founding member of a family of massive bacterial viruses. It is considered to have therapeutic potential as its host, Pseudomonas aeruginosa, is an opportunistic, intrinsically antibiotic resistant, pathogen that kills tens of thousands worldwide each year. {Phi}KZ is an incredibly interesting virus, expressing many systems the host already possesses. On infection, it forms a "nucleus", erecting a barrier around its massive genome to exclude host restriction endonucleases and CRISPR-Cas systems. {Phi}KZ infection is independent of the host transcriptional apparatus. It expresses two different multi-subunit RNA polymerases (RNAPs): the virion RNAP (vRNAP) is injected with the viral DNA during infection to transcribe early genes, including those encoding the non-virion RNAP (nvRNAP), which transcribes all further genes. {Phi}KZ nvRNAP is formed by four polypeptides thought to represent homologues of the eubacterial {beta}/{beta}' subunits, and a fifth with unclear homology, but essential for transcription. We have resolved the structure of {Phi}KZ nvRNAP to 3.3 [A], shedding light on its assembly, homology, and the biological role of the fifth subunit: it is an embedded, integral member of the complex, with structural homology and a biochemical role implying that it has evolved from an ancestral homologue to {sigma}-factor.

biochemistry↗

bioRxiv2020

Direct information estimation from cryo-EM Movies with CARYON

While cryo-EM with modern direct electron detectors has proven incredibly powerful, becoming a dominant technique in structural biology, the analysis of cryo-EM images is significantly complicated by their exceptionally low signal-to-noise ratio, limiting the accuracy of the parameterisation of the physical models required for successful classification and reconstruction. Micrographs from modern direct electron detectors are typically collected as dose-fractionated multi-frame movies to allow the recording of separated individual electron impacts. These detectors improve electron detection and allow for both inter-frame motion correction, and dose-dependent image filtering, lessening the overall impact of effects deleterious to the recovery of high-resolution information. In this study we measured the information content at each spatial frequency in cryo-EM movies as it accrues during the course of an exposure. We show that, as well as correction for motion and radiation damage, the use of the information within movies allows substantially improved direct estimation of the remaining key image parameters required for accurate 3D reconstruction: the image CTF and spectral SNR. We are developing "CARYON" {insert contrived acronym here}, as a LAFTER-family filter for cryo-EM movies based upon such measurements. CARYON is intended to provide the best parameter estimation and filtration possible for a single complete, or large sub-section from a, movie micrograph without the use of a previously refined density. We demonstrate its utility in both single-particle and tomographic cryo-EM data processing.

biophysics↗

bioRxiv2020

Architecture of the Tuberous Sclerosis Protein Complex

The Tuberous Sclerosis Complex (TSC) protein complex (TSCC), comprising TSC1, TSC2, and TBC1D7, is widely recognised as a key integration hub for cell growth and intracellular stress signals upstream of the mammalian target of rapamycin complex 1 (mTORC1). The TSCC negatively regulates mTORC1 by acting as a GTPase-activating protein (GAP) towards the small GTPase Rheb. Both human TSC1 and TSC2 are important tumour suppressors, and mutations in them underlie the disease tuberous sclerosis. We used single-particle cryo-EM to reveal the organisation and architecture of the complete human TSCC. We show that TSCC forms an elongated scorpion-like structure, consisting of a central "body", with a "pincer" and a "tail" at the respective ends. The "body" is composed of a flexible TSC2 HEAT repeat dimer, along the inner surface of which runs the TSC1 coiled-coil backbone, breaking the symmetry of the dimer. Each end of the body is structurally distinct, representing the N- and C-termini of TSC1; a "pincer" is formed by the highly flexible N-terminal TSC1 core domains and a barbed "tail" makes up the TSC1 coiled-coil-TBC1D7 junction. The TSC2 GAP domain is found abutting the centre of the body on each side of the dimerisation interface, poised to bind a pair of Rheb molecules at a similar separation to the pair in activated mTORC1. Our architectural dissection reveals the mode of association and topology of the complex, casts light on the recruitment of Rheb to the TSCC, and also hints at functional higher order oligomerisation, which has previously been predicted to be important for Rheb-signalling suppression.

cell biology↗

bioRxiv2019

Unveiling the dimer/monomer propensities of Smad MH1-DNA complexes

R-Smads are effectors of the transforming growth factor {beta} (TGF{beta}) superfamily and along with Smad4 form trimers to interact with other transcription factors and with DNA. The 5GC-DNA complexes determined here by X-ray crystallography for Smad5 and Smad8 proteins corroborate that all MH1 domains bind SBE and 5GC sites similarly, although Smad2/3/4 MH1 domains bind DNA as monomers whereas Smad1/5/8 form helix-swapped dimers. To examine the relevance of the dimerization phenomenon and to exclude a possible crystallography-induced dimeric state, we studied these MH1 domains in solution. The results show that Smad5/8 domains populate dimers and monomers in equilibrium, whereas their Smad2/3/4 counterparts adopt monomeric conformations. We also found that swapping the loop1 sequence between Smad5 and Smad3 results in the Smad5 chimera-DNA complex crystallizing as a monomer, revealing that the loop1 sequence determines the monomer/dimer propensity of Smad MH1-domains. We propose that the distinct MH1-dimerization status of TGF{beta} and BMP activated Smads influences the interaction with specific loci genome-wide by distinct R-Smad and Smad4 complexes. SignificanceTGF{beta}- and BMP-activated R-Smads were believed to have different preferences with respect to the recognition of DNA motifs and to respond to specific activation inputs. However, recent results indicate that several types of R-Smads can be activated by similar receptors and that all Smads might recognize various DNA motifs. These results pose new questions as to why different types of R-Smads have been conserved for more than 500 million years if they could have a redundant function. They also raise questions as to how different Smad complexes recognize specific clusters of DNA motifs genome-wide. Here, using structural biology approaches, we elucidate some of the rules that help define the dimers of Smad-DNA complexes and propose how dimers and monomers could influence the composition of Smad complexes, as well as the recognition of specific cis-regulatory elements genome-wide. HighlightsR-Smads and Smad4 interact with 5GC and GTCT sites using a conserved binding mode. Functional differences of TGF{beta}- and BMP-activated R-Smads are not exclusively related to DNA specificity. Dimer/monomer propensities are detected in solution and in the absence of DNA. Loop1 sequence determines the propensity of R-Smads to form monomers or dimers in complexes with DNA. Author ContributionsL.R., Z.K., and T.G. designed and performed most experiments and coordinated collaborations with other authors. L.R. and E.A. cloned, expressed and purified all proteins, L.R., R.F., C.T., N.M., and J.C performed EMSA experiments. L.R., E.A., T.G., T.C., P.M.M., and M.J.M. performed the SAXS and NMR measurements and analyzed the data. P.M.M. analyzed the clustering of DNA motifs in ChIP-Seq data. Z.K. B.B. and R.P. screened crystallization conditions, collected X-ray data and determined the structures. Z.K., R.F. R.P., T.G., J.A.M., and M.J.M. analyzed the structures. All authors contributed ideas to the project. M.J.M. and R.P. supervised the project. M.J.M. wrote the manuscript with contributions from all other authors. The authors declare no conflict of interest. Data DepositionNMR assignments and chemical shifts have been deposited in the Biological Magnetic Resonance Data Bank, BMRB entry 27548, and the Small-angle scattering data and models have been deposited in SASBDB, entries SASDE32 (Smad5) and SASDE42 (Smad8). Densities and coordinates have been deposited in the Protein Data Bank, entries 6FZS (Smad5), 6FZT (Smad8), 6TBZ (Smad5_3 chimera), 6TCE (Smad5_gly mutant). This article contains supporting information.

molecular biology↗