Biology subjects

Salas-Estrada, L.

Publications and source records attributed to Salas-Estrada, L..

5 recordsLinked to original sources

bioRxiv2024

Burkholderia cenocepacia epigenetic regulator M.BceJIV simultaneously engages two DNA recognition sequences for methylation

Burkholderia cenocepacia is an opportunistic and infective bacterium containing an orphan DNA methyltransferase (M.BceJIV) with roles in regulating gene expression and motility of the bacterium. M.BceJIV recognizes a GTWWAC motif (where W can be an adenine or a thymine) and methylates the N6 of the adenine at the fifth base position (GTWWAC). Here, we present a high-resolution crystal structure of M.BceJIV/DNA/sinefungin ternary complex and allied biochemical, computational, and thermodynamic analyses. Remarkably, the structure shows not one, but two DNA substrates bound to the M.BceJIV dimer, wherein each monomer contributes to the recognition of two recognition sequences. This unexpected mode of DNA binding and methylation has not been observed previously and sets a new precedent for a DNA methyltransferase. We also show that methylation at two recognition sequences occurs independently, and that GTWWAC motifs are enriched in intergenic regions of a strain of B. cenocepacias genome. We further computationally assess the interactions underlying the affinities of different ligands (SAM, SAH, and sinefungin) for M.BceJIV, as a step towards developing selective inhibitors for limiting B. cenocepacia infection.

biophysics↗

bioRxiv2023

Structural basis of allosteric modulation ofmetabotropic glutamate receptor activation and desensitization

The metabotropic glutamate receptors (mGluRs) are neuromodulatory family C G protein coupled receptors which assemble as dimers and allosterically couple extracellular ligand binding domains (LBDs) to transmembrane domains (TMDs) to drive intracellular signaling. Pharmacologically, mGluRs can be targeted either at the LBDs by glutamate and synthetic orthosteric compounds or at the TMDs by allosteric modulators. Despite the potential of allosteric TMD-targeting compounds as therapeutics, an understanding of the functional and structural basis of their effects on mGluRs is limited. Here we use a battery of approaches to dissect the distinct functional and structural effects of orthosteric versus allosteric ligands. We find using electrophysiological and live cell imaging assays that both agonists and positive allosteric modulators (PAMs) can drive activation and desensitization of mGluRs. The effects of PAMs are pleiotropic, including both the ability to boost the maximal response to orthosteric agonists and to serve independently as desensitization-biased agonists across mGluR subtypes. Conformational sensors reveal PAM-driven inter-subunit re-arrangements at both the LBD and TMD. Motivated by this, we determine cryo-electron microscopy structures of mGluR3 in the presence of either an agonist or antagonist alone or in combination with a PAM. These structures reveal PAM-driven re-shaping of intra- and inter-subunit conformations and provide evidence for a rolling TMD dimer interface activation pathway that controls G protein and beta-arrestin coupling. Highlights-Agonists and PAMs drive mGluR activation, desensitization, and endocytosis -PAMs are desensitization-biased and synergistic with agonists -Four combinatorial ligand conditions reveal an ensemble of full-length mGluR structures with novel interfaces -Activation and desensitization involve rolling TMD interfaces which are re-shaped by PAM

biophysics↗

bioRxiv2023

De Novo Design of κ-Opioid Receptor Antagonists Using a Generative Deep Learning Framework

Likely effective pharmacological interventions for the treatment of opioid addiction include attempts to attenuate brain reward deficits during periods of abstinence. Pharmacological blockade of the {kappa}-opioid receptor (KOR) has been shown to abolish brain reward deficits in rodents during withdrawal, as well as to reduce the escalation of opioid use in rats with extended access to opioids. Although KOR antagonists represent promising candidates for the treatment of opioid addiction, very few potent selective KOR antagonists are known to date and most of them exhibit significant safety concerns. Here, we used a generative deep learning framework for the de novo design of chemotypes with putative KOR antagonistic activity. Molecules generated by models trained with this framework were prioritized for chemical synthesis based on their predicted optimal interactions with the receptor. Our models and proposed training protocol were experimentally validated by binding and functional assays.

bioinformatics↗

bioRxiv2022

Gαs slow conformational transition upon GTP binding and a novel Gαs regulator

G proteins are major signaling partners for G protein-coupled receptors (GPCRs). Although stepwise structural changes during GPCR-G protein complex formation and guanosine diphosphate (GDP) release have been reported, no information is available with regard to guanosine triphosphate (GTP) binding. Here, we used a novel Bayesian integrative modeling framework that combines data from hydrogen-deuterium exchange mass spectrometry, tryptophan-induced fluorescence quenching, and metadynamics simulations to derive a kinetic model and atomic-level characterization of stepwise conformational changes incurred by the {beta}2-adrenergic receptor ({beta}2AR)-Gs complex after GDP release and GTP binding. Our data suggest rapid GTP binding and GTP-induced dissociation of Gs from {beta}2AR and G{beta}{gamma}, as opposed to a slow closing of the Gs -helical domain (AHD). Yeast-two-hybrid screening using Gs AHD as bait identified melanoma-associated antigen D2 (MAGE D2) as a novel AHD-binding protein, which was also shown to accelerate the GTP-induced closing of the Gs AHD.

molecular biology↗

bioRxiv2021

Differences in interactions between transmembrane domains tune the activation of metabotropic glutamate receptors

The metabotropic glutamate receptors (mGluRs) form a family of neuromodulatory G protein-coupled receptors that contain both a seven-helix transmembrane domain (TMD) and a large extracellular ligand-binding domain (LBD) which enables stable dimerization. While numerous studies have revealed variability across subtypes in the initial activation steps at the level of LBD dimers, an understanding of inter-TMD interaction and rearrangement remains limited. Here we use a combination of single molecule fluorescence, molecular dynamics, functional assays, and conformational sensors to reveal that distinct TMD assembly properties drive differences between mGluR subtypes. We uncover a variable region within transmembrane helix 4 (TM4) that contributes to homo- and heterodimerization in a subtype-specific manner and tunes orthosteric, allosteric and basal activation. We also confirm a critical role for a conserved inter-TM6 interface in stabilizing the active state during orthosteric or allosteric activation. Together this study informs a working model of inter-TMD rearrangement that drives mGluR function.

neuroscience↗