Biology subjects

Martinez-Carranza, M.

Publications and source records attributed to Martinez-Carranza, M..

8 recordsLinked to original sources

bioRxiv2024

Bacterial transcriptional repressor NrdR - a flexiblemultifactorial nucleotide sensor

NrdR is a bacterial transcriptional repressor consisting of a Zn-ribbon domain followed by an ATP-cone domain. Understanding its mechanism of action could aid the design of novel antibacterials. NrdR binds specifically to two "NrdR boxes" upstream of ribonucleotide reductase operons, of which Escherichia coli has three: nrdHIEF, nrdDG and nrdAB, where we identified a new box. We show that E. coli NrdR (EcoNrdR) has similar binding strength to all three sites when loaded with ATP plus dATP or equivalent diphosphate combinations. No other combination of nucleotides promotes binding to DNA. We present crystal structures of EcoNrdR-ATP-dATP and EcoNrdR-ADP-dATP, which are the first high-resolution crystal structures of an NrdR. We have also determined cryo-EM structures of DNA-bound EcoNrdR-ATP-dATP and novel filaments of EcoNrdR-ATP. Tetrameric forms of EcoNrdR involve alternating interactions between pairs of Zn-ribbon domains and ATP-cones. The structures reveal considerable flexibility in relative orientation of ATP-cones vs Zn-ribbon domains. The structure of DNA-bound EcoNrdR-ATP-dATP shows that significant conformational rearrangements between ATP-cones and Zn-ribbons accompany DNA binding while the ATP-cones retain the same relative orientation. In contrast, ATP-loaded EcoNrdR filaments show rearrangements of the ATP-cone pairs and sequester the DNA-binding residues of NrdR such that they are unable to bind to DNA. Our results, in combination with a previous structural and biochemical study, point to highly flexible EcoNrdR structures that when loaded with the correct nucleotides adapt to an optimal promoter binding conformation.

biophysics↗

bioRxiv2024

Communication between DNA polymerases and Replication Protein A within the archaeal replisome

Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. We demonstrated that archaeal RPA hosts a winged-helix domain (WH) that interacts with two key actors of the replisome: the DNA primase (PriSL) and the replicative DNA polymerase (PolD). Using an integrative structural biology approach, combining nuclear magnetic resonance, X-ray crystallography and cryo-electron microscopy, we unveiled how RPA interacts with PriSL and PolD through two distinct surfaces of the WH domain: an evolutionarily conserved interface and a novel binding site. Finally, RPA was shown to stimulate the activity of PriSL in a WH-dependent manner. This study provides a molecular understanding of the WH-mediated regulatory activity in conserved central replication factors such as RPA, which regulate genome maintenance in Archaea and Eukaryotes.

molecular biology↗

bioRxiv2023

Molecular basis for proofreading by the unique exonuclease domain of Family-D DNA polymerases

Replicative DNA polymerases duplicate entire genomes at high fidelity. This feature is shared among the three domains of life and is facilitated by their dual polymerase and exonuclease activities. Family D replicative DNA polymerases (PolD), found exclusively in Archaea, contain an unusual RNA polymerase-like catalytic core, and a unique Mre11-like proofreading active site. Here, we present cryo-EM structures of PolD trapped in a proofreading mode, revealing an unanticipated correction mechanism that extends the repertoire of protein domains known to be involved in DNA proofreading. Based on our experimental structures, mutants of PolD were designed and their contribution to mismatch bypass and exonuclease kinetics was determined. This study sheds light on the convergent evolution of structurally distinct families of DNA polymerases, and the domain acquisition and exchange mechanism that occurred during the evolution of the replisome in the three domains of life.

molecular biology↗

bioRxiv2023

A versatile synaptotagmin-1 nanobody provides perturbation-free live synaptic imaging and low linkage-error in super-resolution microscopy

Imaging of living synapses has relied for over two decades on the overexpression of synaptic proteins fused to fluorescent reporters. This strategy changes the stoichiometry of synaptic components and ultimately affects synapse physiology. To overcome these limitations, here we introduce a nanobody that binds the calcium sensor synaptotagmin-1 (NbSyt1). This nanobody functions in living neurons as an intrabody (iNbSyt1) and is minimally invasive, leaving synaptic transmission almost unaffected, as demonstrated by the crystal structure of the NbSyt1 bound to synaptotagmin-1 and by our physiological data. Its single-domain nature enables the generation of protein-based fluorescent reporters, as we showcase here by measuring spatially-localized presynaptic Ca2+ with an NbSyt1-jGCaMP8 chimera. Moreover, its small size makes the NbSyt1 ideal for various super-resolution imaging methods. Overall, NbSyt1 is a versatile binder that will enable imaging in cellular and molecular neuroscience at a higher precision than possible in the past, over multiple spatiotemporal scales.

neuroscience↗

bioRxiv2023

Structure and activity of botulinum neurotoxin X

Botulinum neurotoxins (BoNTs) are the most potent toxins known and are used to treat an increasing number of medical disorders. All BoNTs are naturally co-expressed with a protective partner protein (NTNH) with which they form a 300 kDa complex, to resist acidic and proteolytic attack from the digestive tract. We have previously identified a new botulinum neurotoxin serotype, BoNT/X, that has unique and therapeutically attractive properties. We present the cryo-EM structure of the BoNT/X-NTNH/X complex at 3.1 [A] resolution. Unexpectedly, the BoNT/X complex is stable and protease resistant at both neutral and acidic pH and disassembles only in alkaline conditions. Using the stabilizing effect of NTNH, we isolated BoNT/X and showed that it has very low potency both in vitro and in vivo. Given the high catalytic activity and translocation efficacy of BoNT/X, low activity of the full toxin is likely due to the receptor-binding domain, which presents weak ganglioside binding and exposed hydrophobic surfaces.

biochemistry↗

bioRxiv2022

DNA-binding mechanism and evolution of Replication Protein A

Replication Protein A (RPA) is a heterotrimeric single stranded DNA-binding protein with essential roles in DNA replication, recombination and repair, in both eukaryotic and archaeal cells. By using an integrative approach that combines three crystal structures, four cryo-EM structures in complex with single-stranded DNA (ssDNA) of different lengths, we extensively characterized RPA from Pyrococcus abyssi in different states. These structures show two essential features conserved in eukaryotes: a trimeric core and a module that promotes cooperative binding to ssDNA, as well as a newly identified archaeal-specific domain. These structures reveal for the first time how ssDNA is handed over from one RPA complex to the other, and uncover an unanticipated mechanism of self-association on ssDNA tracts. This work constitutes a significant step forward in the molecular understanding of the structure and DNA-binding mechanism of RPA, with far-reaching implications for the evolution of this primordial replication factor in Archaea and Eukarya.

biochemistry↗

bioRxiv2019

A rationally designed and highly versatile epitope tag for nanobody-based purification, detection and manipulation of proteins

Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a novel, rationally designed epitope tag that serves an exceptionally broad spectrum of applications in life sciences while outperforming established tags like the HA, FLAG or myc tags. The ALFA-tag forms a small and stable -helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We developed a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in-vivo detection of proteins. By solving the crystal structure of the complex we were able to design a nanobody mutant (NbALFAPE) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.

biochemistry↗

bioRxiv2018

Cotranslational folding of a pentarepeat β-helix protein

It is becoming increasingly clear that many proteins start to fold cotranslationally, before the entire polypeptide chain has been synthesized on the ribosome. One class of proteins that a priori would seem particularly prone to cotranslational folding is repeat proteins, i.e., proteins that are built from an array of nearly identical sequence repeats. However, while the folding of repeat proteins has been studied extensively in vitro with purified proteins, only a handful of studies have addressed the issue of cotranslational folding of repeat proteins. Here, we have determined the structure and studied the cotranslational folding of a {beta}-helix pentarepeat protein from the human pathogen Clostridium botulinum - a homolog of the Fluoroquinolone Resistance Protein MfpA - using an assay in which the SecM translational arrest peptide serves as a force sensor to detect folding events. We find that cotranslational folding of a segment corresponding to the first four of the eight {beta}-helix coils in the protein produces enough force to release ribosome stalling, and that folding starts when this unit is ~35 residues away from the P-site, near the distal end of the ribosome exit tunnel. An additional folding transition is seen when the whole PENT moiety emerges from the exit tunnel. The early cotranslational formation of a folded unit may be important to avoid misfolding events in vivo, and may reflect the minimal size of a stable {beta}-helix since it is structurally homologous to the smallest known {beta}-helix protein, a four-coil protein that is stable in solution.

molecular biology↗