Biology subjects

Mondragon, A.

Publications and source records attributed to Mondragon, A..

9 recordsLinked to original sources

bioRxiv2026

Analysis of the heterogenous structural states of the hexameric ATPase PilU of the Type IV pili from Vibrio cholerae

Type IV pili (T4P) mediate surface motility, host interactions, and DNA uptake through cycles of extension and retraction. While the primary retraction ATPase PilT has been extensively characterized, its homolog PilU remains less well understood despite being demonstrated as a PilT-dependent retraction ATPase. Here, we determined six PilU structures by cryo-electron microscopy and X-ray crystallography. The structures reveal a homohexameric assembly stabilized by interactions between the C-terminal catalytic domain of one subunit and the N-terminal PAS-like domain of a neighboring subunit. PilU adopts multiple conformational states, exhibiting digerent combinations of open and closed interfaces even in the absence of nucleotide. Comparison with PilT highlights structural features that likely underlie PilUs weak ATPase activity and its dependence on PilT for function. Together, these findings provide a structural framework for understanding PilUs role within the T4P retraction machinery.

molecular biology↗

bioRxiv2024

Multi-modal comparison of molecular programs driving nurse cell death and clearance in Drosophila melanogaster oogenesis

The death and clearance of nurse cells is a consequential milestone in Drosophila melanogaster oogenesis. In preparation for oviposition, the germline-derived nurse cells bequeath to the developing oocyte all their cytoplasmic contents and undergo programmed cell death. The death of the nurse cells is controlled non-autonomously and is precipitated by epithelial follicle cells of somatic origin acquiring a squamous morphology and acidifying the nurse cells externally. Alternatively, stressors such as starvation can induce the death of nurse cells earlier in mid-oogenesis, manifesting apoptosis signatures, followed by their engulfment by epithelial follicle cells. To identify and contrast the molecular pathways underlying these morphologically and genetically distinct cell death paradigms, both mediated by follicle cells, we compared their genome-wide transcriptional, translational, and secretion profiles before and after differentiating to acquire a phagocytic capability, as well as during well-fed and nutrient-deprived conditions. By coupling the GAL4-UAS system to Translating Ribosome Affinity Purification (TRAP-seq) and proximity labeling (HRP-KDEL) followed by Liquid Chromatography tandem mass-spectrometry, we performed high-throughput screens to identify pathways selectively activated or repressed by follicle cells to employ nurse cell-clearance routines contextually and preferentially. We also integrated two publicly available single-cell RNAseq atlases of the Drosophila ovary to define the transcriptomic profiles of follicle cells. In this report, we describe the genes and major pathways identified in the screens and the striking consequences to Drosophila melanogaster oogenesis caused by RNAi perturbation of prioritized candidates. To the best of our knowledge, our study is the first of its kind to comprehensively characterize two distinct apoptotic and non-apoptotic cell death paradigms in the same multi-cellular system. Beyond molecular differences in cell death, our investigation may also provide insights into how key systemic trade-offs are made between survival and reproduction when faced with physiological stress.

genetics↗

bioRxiv2024

NRP1-expressing myeloid cells promote arteriogenesis in the ischemic limb

Peripheral arterial disease can cause limb threatening blood flow restriction, requiring amputation in up to a third of patients despite contemporary treatments. Stimulating new blood vessel growth in ischemic limbs using growth factors such as VEGF-A or supplying mixed cell populations via autologous transplantation has been investigated as a therapy for surgically intractable cases, but to date has proven ineffective in randomised controlled clinical studies. To identify more potent pro-arteriogenic cell populations, we have investigated VEGF-A responsive myeloid cell populations in a mouse model of severe hindlimb ischemia. We found that NRP1 ablation from monocytes and macrophages impaired arteriogenesis in the adductor muscle and flow recovery in the injured limb. Vice versa, the exogenous delivery of NRP1-expressing (but not NRP1-negative) macrophages enhanced arteriogenesis in the adductor and flow recovery in the ischemic limb in a VEGF signalling-dependent manner. Further, patient-derived NRP1-expressing monocytes promoted vascular morphogenesis ex vivo. As the levels of circulating NRP1-expressing monocytes were raised in patients with limb ischemia, the therapeutic delivery of autologous NRP1-expressing monocytes could be explored as a treatment for critical limb ischemia.

pathology↗

bioRxiv2024

Translational T-box riboswitches bind tRNA by modulating conformational flexibility

T-box riboswitches, paradigmatic non-coding RNA elements involved in genetic regulation in most Gram-positive bacteria, are adept at monitoring amino acid metabolism through direct interactions with specific tRNAs. T-box riboswitches assess tRNA aminoacylation status, subsequently regulating the transcription or translation of downstream genes involved in amino acid metabolism. Here we present single-molecule FRET studies of the Mycobacterium tuberculosis IleS T-box riboswitch, a model of T-box translational regulation. The data supports a two-step binding model where the tRNA anticodon is recognized first, followed by interactions with the NCCA sequence. Specifically, after anticodon recognition, tRNA in the partially bound state can transiently dock into the discriminator domain, resembling the fully bound state, even in the absence of the tRNA NCCA-discriminator interactions. Establishment of the NCCA-discriminator interactions significantly stabilizes the fully bound state. Collectively, the data suggests higher conformational flexibility in translation-regulating T-box riboswitches, compared to transcription-regulating ones, and supports a conformational selection model for NCCA recognition. Furthermore, it was found that the conserved RAG sequence is pivotal in maintaining specific interactions with the tRNA NCCA sequence by preventing sampling of an aberrant conformational state, while Stem IIA/B-linker interactions impact the conformational dynamics and the stability of both the partially bound and fully bound states. The present study provides a critical kinetic basis for how specific sequences and structural elements in T-box riboswitches enable the binding efficiency and specificity required to achieve gene regulation.

biophysics↗

bioRxiv2021

Structures of topoisomerase V in complex with DNA reveal unusual DNA binding mode and novel relaxation mechanism

Topoisomerase V is a unique topoisomerase that combines DNA repair and topoisomerase activities. The enzyme has an unusual arrangement, with a small topoisomerase domain followed by 12 tandem (HhH)2 domains, which include three AP lyase repair domains. The unusual architecture of this enzyme bears no resemblance to any other known topoisomerase. Here we present structures of topoisomerase V in complex with DNA. The structures show that the (HhH)2 domains wrap around the DNA and in this manner appear to act as a processivity factor. There is a conformational change in the protein to expose the topoisomerase active site. The DNA bends sharply to enter the active site, which melts the DNA and probably facilitates relaxation. The structures show a DNA binding mode not observed before and provide information on the way this unusual topoisomerase relaxes DNA.

biophysics↗

bioRxiv2018

Structural Organization of a Type III-A CRISPR Effector Subcomplex Determined by X-ray Crystallography and Cryo-EM

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins provide an immune-like response in many prokaryotes against extraneous nucleic acids. CRISPR-Cas systems are classified into different classes and types. Class 1 CRISPR-Cas systems form multi-protein effector complexes that includes a guide RNA (crRNA) used to identify the target for destruction. Here we present crystal structures of Staphylococcus epidermidis Type III-A CRISPR subunits Csm2 and Csm3 and a 5.2 [A] resolution single-particle cryo-electron microscopy (cryo-EM) reconstruction of an effector subcomplex including the crRNA. The structures help to clarify the quaternary architecture of Type III-A effector complexes, as well as to provide details on crRNA binding, target RNA binding and cleavage, and intermolecular interactions essential for effector complex assembly. The structures allow a better understanding of the organization of Type III-A CRISPR effector complexes as well as highlighting the overall similarities and differences with other Class 1 effector complexes.

biophysics↗

bioRxiv2018

CryoEM structures of open dimers of Gyrase A in complex with DNA illuminate mechanism of strand passage

Gyrase is a unique type IIA topoisomerase that uses ATP hydrolysis to maintain the negatively supercoiled state of bacterial DNA. In order to perform its function, gyrase undergoes a sequence of conformational changes that consist of concerted gate openings, DNA cleavage, and DNA strand passage events. Structures where the transported DNA molecule (T-segment) is trapped by the A subunit have not been observed. Here we present the cryoEM structures of two oligomeric complexes of open gyrase A dimers and DNA. The protein subunits in these complexes were solved to 4 [A] and 5.16 [A] resolution. One of the complexes traps a linear DNA molecule, a putative T-segment, which interacts with the open gyrase A dimers in two states, representing steps either prior to or after passage through the DNA-gate. The structures locate the T-segment in important intermediate conformations of the catalytic cycle and provide insights into gyrase-DNA interactions and mechanism.

biophysics↗

bioRxiv2018

Two-step binding kinetics of tRNAGly by the glyQS T-box riboswitch and its regulation by T-box structural elements

T-box riboswitches are cis-regulatory RNA elements that regulate mRNAs encoding for aminoacyl tRNA synthetases or proteins involved in amino acid biosynthesis and transport. Rather than using small molecules as their ligands, as do most riboswitches, T-box riboswitches uniquely bind tRNA and sense their aminoacylated state. Whereas the anticodon and elbow regions of the tRNA interact with Stem I, located in the 5 portion of the T-box, sensing of the aminoacylation state involves direct binding of the NCCA sequence at the tRNA 3 end to the anti-terminator sequence located in the 3 portion of the T-box. However, the kinetic trajectory that describes how each of these interactions are established temporally during tRNA binding remains unclear. Using singlemolecule fluorescence resonance energy transfer (smFRET), we demonstrate that tRNA binds to the riboswitch in a two-step process, first with anticodon recognition followed by NCCA binding, with the second step accompanied by an inward motion of the 3 portion of the T-box riboswitch relative to Stem I. By using site-specific mutants, we further show that the T-loop region of the T-box significantly contributes to the first binding step, and that the K-turn region of the T-box influences both binding steps, but with a more dramatic effect on the second binding step. Our results set up a kinetic framework describing tRNA binding by T-box riboswitches and highlight the important roles of several T-box structural elements in regulating each binding step.\n\nSIGNIFICANCEBacteria commonly use riboswitches, cis-regulatory RNA elements, to regulate the transcription or translation of the mRNAs upon sensing signals. Unlike small molecule binding riboswitches, T-box riboswitches bind tRNA and sense their aminoacylated state. T-box modular structural elements that recognize different parts of a tRNA have been identified, however, how each of these interactions is established temporally during tRNA binding remains unclear. Our study reveals that tRNA binds to the riboswitch in a two-step mechanism, with anticodon recognition first, followed by binding to the NCCA sequence at the 3 end of the tRNA with concomitant conformational changes in the T-box. Our results also highlight the importance of the modular structural elements of the T-box in each of the binding steps.

biophysics↗

bioRxiv2017

Nucleation of multiple buckled structures in intertwined DNA double helices

We study the statistical-mechanical properties of intertwined double-helical DNAs (DNA braids). In magnetic tweezers experiments we find that torsionally-stressed stretched braids supercoil via an abrupt buckling transition, which is associated with nucleation of a braid end loop, and that the buckled braid is characterized by proliferation of multiple domains. Differences between the mechanics of DNA braids and supercoiled single DNAs can be understood as an effect of increased bulkiness in the structure of the former. The experimental results are in accord with the predictions of a previously-described statistical-mechanical model.

biophysics↗