Biology subjects

Arranz, R.

Publications and source records attributed to Arranz, R..

6 recordsLinked to original sources

bioRxiv2025

Activation Mechanism and Structural Assembly of the Mycobacterium tuberculosis ClpP1P2 Protease and Its Associated ATPases.

Supramolecular assemblies are integral to cellular biochemical processes, relying on their dynamic nature to fulfill essential functions. The protease ClpP1P2, paired with ATPase partners ClpC1 or ClpX, is vital for the survival of Mycobacterium tuberculosis (Mtb). While the ClpP1P2 complex requires activation by specific N-blocked dipeptides (e.g., Z-Leu-Leu) to exhibit proteolytic activity in vitro, the mechanism of in vivo activation remains unclear. In this study, we identified novel activators that enabled the structural determination of the ClpC1P1P2 complex, providing insights into its assembly. Furthermore, we discovered that trehalose - a key metabolite and molecular crowding agent in Mtb, significantly enhances the activity of both ClpC1P1P2 and ClpXP1P2 complexes without the need for activating peptides. Analytical ultracentrifugation revealed that trehalose promotes the formation of these active complexes, mimicking intracellular conditions. These findings propose a new model of Clp system activation in vivo, offering promising avenues for therapeutic targeting in tuberculosis treatment. Significance StatementThe proteolytic complex formed by the essential proteins ClpP1 and ClpP2, along with their specific ATP-dependent activators ClpX and ClpC1, has emerged as a highly attractive target for anti-tuberculosis drug development. While previous studies have shown that ClpP1P2 can be activated in vitro by small peptide activators, its in vivo activation mechanism remains unclear. In this study, we identify novel activators and demonstrate that trehalose, a key metabolite in Mycobacterium tuberculosis, enhances ClpC1P1P2 and ClpXP1P2 activity without the need for activating peptides. These findings propose a new model for Clp system activation in Mycobacterium tuberculosis, advancing our understanding of its regulation and potential as a therapeutic target.

biochemistry↗

bioRxiv2024

The action mechanism of actinoporins revealed through the structure of pore-forming intermediates

Pore-forming proteins exemplify the transformative potential of biological molecules. Initially produced in a monomeric, water-soluble form, they spontaneously assemble into multimeric integral membrane proteins in the presence of suitable target lipids. Their functions include roles in apoptosis, cell signaling, immunity, as well as attack and defense systems between different organisms. This latter group encompasses actinoporins, a family of pore-forming toxins from sea anemones that kill target cells by perforating their plasma membrane. Here, we have determined the structures of two such toxins, fragaceatoxin C and sticholysin II, in a membrane environment using cryogenic electron microscopy. The structures reveal how dozens of lipid molecules interact in an orderly manner, forming an intrinsic part of the pore. We have also isolated different pore-forming intermediates, where only a fraction of the constituent monomers is incorporated, exhibiting non-closed, arc-shaped structures. Based on these structures we propose a mechanism of action where the sequential assembly of toxin monomers onto the membrane, accompanied by conformational changes, triggers pore formation and membrane perforation. Our results contribute to a better understanding of the transforming capacity of these pore-forming proteins, which are becoming increasingly important for their diverse biotechnological applications.

biochemistry↗

bioRxiv2023

A Novel Monoclonal Antibody Targeting a Large Surface of the Receptor Binding Motif Shows Pan-neutralizing SARS-CoV-2 Activity Including BQ.1.1 Variant

In the present study we report the functional and structural characterization of 17T2, a new highly potent pan-neutralizing SARS-CoV-2 human monoclonal antibody (mAb) isolated from a convalescent COVID-19 individual infected during the first wave of the COVID-19 pandemic. 17T2 is a class 1 VH1-58/{kappa}3-20 antibody, derived from a receptor binding domain (RBD)-specific IgA memory B cell and developed as a human recombinant IgG1. Functional characterization revealed that 17T2 mAb has a high and exceptionally broad neutralizing activity against all SARS-CoV-2 spike variants tested, including BQ.1.1. Moreover, 17T2 mAb has in vivo prophylactic activity against Omicron BA.1.1 infection in K18-hACE2 transgenic mice. 3D reconstruction from cryogenic-electron microscopy (cryo-EM) showed that 17T2 binds the Omicron BA.1 spike protein with the RBD domains in "up" position and recognizes an epitope overlapping with the receptor binding motif, as it is the case for other structurally similar neutralizing mAbs, including S2E12. Yet, unlike S2E12, 17T2 retained its high neutralizing activity against all Omicron sublineages tested, probably due to a larger contact area with the RBD, which could confer a higher resilience to spike mutations. These results highlight the impact of small structural antibody changes on neutralizing performance and identify 17T2 mAb as a potential candidate for future therapeutic and prophylactic interventions.

immunology↗

bioRxiv2022

Structural basis and dynamics of Chikungunya alphavirus RNA capping by the nsP1 capping pores

Alphaviruses are emerging positive stranded RNA virus which replicate and transcribe their genomes in membranous organelles formed in the cell cytoplasm. The non-structural protein 1 (nsP1) is responsible for RNA capping and the gating of replication organelles by assembling into monotopic membrane-associated dodecameric pores (Jones R. et al. Nature 2021). The capping path is unique for Alphavirus; beginning with the N7 methylation of a GTP molecule, followed by the covalent linkage of a m7GMP group to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate RNA (Ahola T. et al. PNAS 1995). Here we provide structural snapshots of different stages of the reaction pathway showing how nsP1 pores recognize the substrates of the methyl-transfer reaction, GTP and SAM, how it reaches a metastable post-methylation state with SAH and m7GTP in the active site, the subsequent covalent transfer of m7GMP to nsP1 and post-reaction conformational changes triggering the opening of the pore. In addition, we biochemically characterize the capping reaction, demonstrating specificity for the RNA substrate and the reversibility of the cap transfer resulting in decapping activity and the release of intermediates of the reaction. Our data identify the molecular determinants allowing each pathway transition, provide explanation for the need for the SAM methyl donor all along the pathway and new clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together our results set new ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals. Significance statementHere we present biochemical and structural characterization of the capping pathway carried out by the Chikungunya virus nsP1 capping pores. We provide five Cryo-EM structures representative of the different steps of the reaction. These structures reveal the molecular determinants and dynamics associated with the alphavirus capping process. In addition, we biochemically show the RNA capping specificity and the reversibility of the reaction which allow nsP1 to cap and decap RNAs and to release intermediates of the reaction. These data provide a new biochemical clues on the enzymatic activity of nsP1 capping pores and a new structural landscape that will be instrumental for the design of effective antivirals targeting the viral RNA capping for blocking the infection.

molecular biology↗

bioRxiv2022

A fijivirus major viroplasm protein shows RNA-stimulated ATPase activity by adopting pentameric and hexameric assemblies of dimers

The fijivirus Mal de Rio Cuarto virus (MRCV) causes a devastating maize disease. Its non-structural protein P9-1, which shows ATPase and RNA binding activities, is the major component of the intracellular viroplasm where virus replication takes place. Here, we established that the 24 C-terminal residues (C-arm) of P9-1 are required for the formation of viroplasm-like structures (VLS) in vivo and for the protein multimerization in vitro. Employing an integrative structural approach, we found that the C-arm is dispensable for P9-1 dimer assembly, but essential for the formation of doughnut-shaped pentamers and hexamers of dimers (decamers and dodecamers). Both assemblies, larger than those reported for other reoviruses, contain disordered loops oriented towards the inner pore of the structures, where RNA binding sites and conditional proteasome-mediated degradation signals (PEST) were predicted. In vitro assays demonstrated that ssRNA binding is favored towards P9-1 (do)decamers over the dimeric {Delta}C-arm version. In addition, although both P9-1 and P9-1{Delta}C-arm catalyze the hydrolysis of ATP with similar activity values, an RNA-stimulated ATPase activity was only observed in the full-length protein, indicating a C-arm-mediated interaction between the ATP catalytic site and the allosteric RNA binding sites in the (do)decameric assemblies. Computational studies revealed a stronger preference of phosphate moieties to the decamer in the pore and the C-arm regions, suggesting that the allosteric communication between the ATP and RNA binding sites is favored with this protein arrangement. Overall, our work reveals the structural versatility of a major viroplasm protein providing unprecedented insights into fijivirus viroplasm assembly and function and establishes the structural basis for the development of antiviral strategies against the Mal de Rio Cuarto crop disease.

biochemistry↗

bioRxiv2020

Mitochondrial cytochrome c liberates the nucleophosmin-sequestered ARF tumor suppressor in the nucleolus

The alternative reading frame (ARF) protein is crucial in the cellular response to oncogenic stress, being likewise the second most frequently inactivated gene in a wide spectrum of human cancers. ARF is usually sequestered in the nucleolus by the well-known oncogenic nucleophosmin (NPM) protein and is liberated in response to cell damage to exhibit its tumor-suppressor ability. However, the mechanism underlying ARF activation is unknown. Here we show that mitochondria-to-nucleus translocation of cytochrome c upon DNA damage leads to the break-off of the NPM-ARF ensemble and subsequent release of ARF from the nucleoli. Our structural and subcellular data support a molecular model in which the hemeprotein triggers the extended-to-compact conformation of NPM and competes with ARF for binding to NPM.

biophysics↗