Biology subjects

Perera, L.

Publications and source records attributed to Perera, L..

9 recordsLinked to original sources

bioRxiv2025

Structural and Computational Analysis of Pseudomonas Aeruginosa DNA Gyrase Reveals Molecular Characteristics That May Contribute to Ciprofloxacin Resistance

Pseudomonas aeruginosa is considered a priority pathogen by the World Health Organization due to its resistance to antibiotics. Isolates resistant to ciprofloxacin (CPFX), a bactericide commonly used against P. aeruginosa, usually carry the mutations T83I or D87N in the GyrA subunit of the DNA gyrase. Yet, the molecular mechanisms by which these mutations confer CPFX-resistance to P. aeruginosa are unknown. Here we solved the crystal structure of the P. aeruginosa gyrase catalytic cleavage core and used it to carry out molecular dynamic (MD) simulations of CPFX-gyrase binding in the wild-type as well as the T83I and the D87N mutant systems. Our results show that DNA plays the most prominent role in maintaining the CPFX-bound conformation, with no appreciable contributions from Thr83 or Asp87. Interestingly, we found a solvent cavity adjacent to these residues that may provide CPFX access to the active site. Interaction energy analysis using Umbrella Sampling indicates that Thr83 and Asp87 may influence CPFX trajectory during binding. In the mutant systems, the attractive potential decreases, which may hinder CPFX accessing the binding site. These results shed light on P. aeruginosa resistance to CPFX and may help provide a methodology to identify new therapeutic agents to target fluoroquinolone resistant bacteria. Graphical abstract caption O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/695517v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1c203f0org.highwire.dtl.DTLVardef@90aa78org.highwire.dtl.DTLVardef@1a7d286org.highwire.dtl.DTLVardef@ee8c7a_HPS_FORMAT_FIGEXP M_FIG C_FIG Molecular dynamic positioning of ciprofloxacin (CPFX) from the cavity site (green) to the bound inhibition site (magenta) suggests that mutations T83I and D87N, conferring CPFX resistance to the P. aeruginosa gyrase, may have an influence on the ability of CPFX to access the binding site. Protein is shown in cyan, DNA backbone in orange, and DNA bases in gradient from cyan to blue.

molecular biology↗

bioRxiv2024

Illuminating root-soil mechanics

Soil compaction and escalating global drought increase soil strength and stiffness. It remains unclear which plant root biomechanical mechanisms/traits enable growth in these harsh conditions. Here, we combine synchrotron X-ray computed tomography with spatially resolved X-ray diffraction to characterize the biomechanics of a replica root-soil system. We map the strain field around the root tip, finding strong agreement with finite element simulations, thereby demonstrating a promising new in-vivo measurement protocol.

biophysics↗

bioRxiv2024

Mouse α-synuclein fibrils are structurally and functionally distinct from human fibrils associated with Lewy body diseases

Short AbstractThe intricate process of -synuclein aggregation and fibrillization hold pivotal roles in Parkinsons disease (PD) and multiple system atrophy (MSA). While mouse -synuclein can fibrillize in vitro, whether these fibrils commonly used in research to induce this process or form can reproduce structures in the human brain remains unknown. Here we report the first atomic structure of mouse -synuclein fibrils, which was solved in parallel by two independent teams. The structure shows striking similarity to MSA-amplified and PD-associated E46K fibrils. However, mouse -synuclein fibrils display altered packing arrangements, reduced hydrophobicity, heightened fragmentation sensitivity, and evoke only weak immunological responses. Furthermore, mouse -synuclein fibrils exhibit exacerbated pathological spread in neurons and humanized -synuclein mice. These findings provide new insights into the structural underpinnings of -synuclein pathogenicity and emphasize a need to reassess the role of mouse -synuclein fibrils in the development of related diagnostic probes and therapeutic interventions.

neuroscience↗

bioRxiv2022

Structural Basis for pre-tRNA Recognition and Processing by the Human tRNA Splicing Endonuclease Complex

Across all walks of life, certain transfer RNA (tRNA) transcripts contain introns. Pre-tRNAs with introns require splicing to form the mature anticodon stem loop (ASL). In eukaryotes, tRNA splicing is initiated by the heterotetrameric tRNA splicing endonuclease (TSEN) complex. All TSEN subunits are essential and mutations within the complex are associated with a family of neurodevelopmental disorders known as pontocerebellar hypoplasia (PCH). The pathogenesis of PCH is poorly understood. Moreover, a lack of structures for any eukaryotic TSEN complex has hindered our understanding of tRNA recognition and processing. Here, we report Cryo-Electron Microscopy (cryo-EM) structures of the human TSEN*pre-tRNA complex, trapped in the pre-cleavage state, at near atomic resolution. These structures reveal the overall architecture of the complex, along with extensive tRNA binding interfaces within the complex. Although it shares structural homology with archaeal TSENs, the human TSEN complex contains additional features important for recognizing the acceptor stem and D-arm of the pre-tRNA. Our findings also establish the TSEN54 subunit as more than a simple molecular ruler; it functions as a pivotal scaffold for the pre-tRNA and the two endonuclease subunits, TSEN2 and TSEN34. Finally, the human TSEN structures enable detailed visualization of the molecular environments of PCH-causing missense mutations, providing crucial insight into the mechanism of eukaryotic pre-tRNA splicing and neurodevelopmental disease.

biochemistry↗

bioRxiv2021

The humanized nanobody RBD-1-2G tolerates the spike N501Y mutation to neutralize SARS-CoV-2

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

synthetic biology↗

bioRxiv2021

Characterization of SARS2 Nsp15 Nuclease Activity Reveals it's Mad About U

Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3 of uridines, it is unknown if Nsp15 has any additional substrate preferences. Here we used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp15 has a strong preference for purines 3 of the cleaved uridine. This work advances our understanding of how Nsp15 recognizes and processes viral RNA and will aid in the development of new anti-viral therapeutics.

biochemistry↗

bioRxiv2020

ISRIB blunts the integrated stress response by allosterically antagonising the inhibitory effect of phosphorylated eIF2 on eIF2B

The small molecule ISRIB antagonises the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(P). ISRIB and eIF2(P) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor (GEF) for eIF2. We have found that ISRIB-mediated acceleration of eIF2B activity in vitro is observed preferentially in the presence of eIF2(P) and is attenuated by mutations that desensitise eIF2B to the inhibitory effects of eIF2(P). ISRIBs efficacy as an ISR inhibitor in cells also depends on presence of eIF2(P). Cryo-EM showed that engagement of both eIF2B regulatory sites by two eIF2(P) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2 during active nucleotide exchange, thereby discouraging both binding events. In vitro, eIF2(P) and ISRIB reciprocally opposed each others binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR.

biochemistry↗

bioRxiv2020

Cryo-EM Structures of the SARS-CoV-2 Endoribonuclease Nsp15

New therapeutics are urgently needed to inhibit SARS-CoV-2, the virus responsible for the on-going Covid-19 pandemic. Nsp15, a uridine-specific endoribonuclease found in all coronaviruses, processes viral RNA to evade detection by RNA-activated host defense systems, making it a promising drug target. Previous work with SARS-CoV-1 established that Nsp15 is active as a hexamer, yet how Nsp15 recognizes and processes viral RNA remains unknown. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15. The UTP-bound cryo-EM reconstruction at 3.36 [A] resolution provides molecular details into how critical residues within the Nsp15 active site recognize uridine and facilitate catalysis of the phosphodiester bond, whereas the apo-states reveal active site conformational heterogeneity. We further demonstrate the specificity and mechanism of nuclease activity by analyzing Nsp15 products using mass spectrometry. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics.

biochemistry↗

bioRxiv2017

A class of GATA3 mutation reprograms the breast cancertranscriptional network through gain and loss of function

GATA3 is frequently mutated in breast cancer; these mutations are widely presumed to be loss of function. Here, we address molecular alterations downstream of a novel class of GATA3 mutations, revealing both gain and loss of function. Mutation of one allele of GATA3 led to loss of binding and decreased expression at a subset of genes, including Progesterone Receptor. At other loci, associated with epithelial to mesenchymal transition, gain of binding at a novel sequence motif correlated with increased gene expression. These results demonstrate that not all GATA3 mutations are equivalent and that these mutations impact breast cancer through gain and loss of function.

cell biology↗