Biology subjects

Botello-Smith, W. M.

Publications and source records attributed to Botello-Smith, W. M..

6 recordsLinked to original sources

bioRxiv2021

Free energy and kinetics of cAMP permeation through connexin26 hemichannel with and without voltage

The connexin family is a diverse group of highly regulated non-{beta}-barrel wide-pore channels permeable to biological signaling molecules. Despite their critical roles in mediating selective molecular signaling in health and disease, the molecular basis of permeation through these pores remains unclear. Here, we report the thermodynamics and kinetics of binding and transport of a second messenger, adenosine-3,5-cyclophosphate (cAMP), through a connexin26 hemichannel. Inward and outward fluxes of cAMP were first obtained from 4 s simulations with voltages and multiple cAMPs in solution. The results are compared with the intrinsic potential of mean force (PMF) and the mean first passage times (MFPTs) of a single cAMP in the absence of voltage, obtained from a total of 16.5 s of multi-replica Voronoi-tessellated Markovian milestoning simulations. The computed transit times through the pore correspond well to existing experimental data. Both voltage simulations and milestoning simulations revealed two cAMP binding sites with binding constants and dissociation rates computed from PMF and MFPTs. The protein dipole inside the pore produces an asymmetric PMF, reflected in unequal cAMP MFPTs in each direction once within the pore. The free energy profiles under voltages derived from intrinsic PMF provided a unified understanding of directional transition rates with/without voltage, and revealed the unique role of channel polarity and the mobile electrolyte within a wide pore on the total free energy. In addition, we show how these factors influence the cAMP dipole vector during permeation, and how cAMP affects the local and non-local pore diameter in a position-dependent manner. Significance StatementConnexins are wide-pore channels permeable to cellular signaling molecules. They mediate molecular signaling crucial in physiology, pathology, and development; mutations in connexins cause human pathologies. However, the fundamental structural, thermodynamic, and kinetic determinants of molecular permeability properties are unknown. Using multiple molecular dynamics simulation techniques, we report, for the first time, an in-depth investigation of the free energy and the directional transition rates of an important biological signaling molecule, cAMP, through a connexin channel. We reveal the energetics and binding sites that determine the cAMP flux, and the effects of mobile ions and external electrical field on the process. The results provide a basis for understanding the unique features of molecular flux through connexins and other non-{beta}-barrel wide-pore channels.

biophysics↗

bioRxiv2020

A Piezo1 open state reveals a multi-fenestrated ion permeation pathway

Force-sensing Piezo channels are essential to many aspects of vertebrate physiology. Activation of Piezo1 is facilitated by the presence of negative membrane lipids in the inner leaflet, such as phosphatidylinositol-4,5-bisphosphate (PIP2). Here, to study how Piezo1 opens, we performed molecular dynamics simulations of Piezo1 in membranes flattened by the periodic boundary effect and with or without PIP2 lipids. The Piezo1 pore spontaneously opens in the asymmetrical bilayer but not in the symmetric membrane or when PIP2 lipids are neutralized. Electrophysiological characterization of putative PIP2-interacting Piezo1 residues suggests the contribution of multiple PIP2 binding sites. Our Piezo1 open state recapitulates ionic selectivity, unitary conductance and mutant phenotypes obtained from numerous experimental studies. Tracking ion diffusion through the open pore reveals the presence of intracellular and extracellular fenestrations, delineating a multi-fenestrated permeation pathway. This open state sheds light on the mechanisms of lipid modulation, permeation, and selectivity in a Piezo channel.

biophysics↗

bioRxiv2020

In silico prediction of ARB resistance: A first step in creating personalized ARB therapy

Angiotensin II type 1 receptor (AT1R) blockers (ARBs) are among the most prescribed drugs. However, ARB effectiveness varies widely, and some patients do not respond to ARB therapy. One reason for the variability between patients is non-synonymous single nucleotide polymorphisms (nsSNPs) within agtr1, the AT1R gene. There are over 100 nsSNPs in the AT1R; therefore, this study embarked on determining which nsSNPs may abrogate the binding of selective ARBs. The crystal structure of olmesartan-bound human AT1R (PDB:4ZUD) served as a template to create an inactive empty AT1R via molecular dynamics simulation (n = 3). All simulations resulted in a smaller ligand-binding pocket than 4ZUD due to the absence of olmesartan in the simulation yet remained inactive with little movement in the receptor core. A single frame representing the average stable AT1R was used as a template to thrice (n = 3) dock each ARB via AutoDock to obtain a predicted affinity from the weighted average of 100 docking simulations. The results were far from known values; thus, an optimization protocol was initiated, resulting in the predicted binding affinities within experimentally determined ranges (n = 6). The empty model AT1R was altered and minimized in Molecular Operating Environment software to represent 103 of the known human AT1R polymorphisms. Each of the eight ARBs was then docked, using the optimized parameters, to each polymorphic AT1R (n = 6). Although each nsSNP has little effect on global AT1R structure, most nsSNPs drastically alter a sub-set of ARBs affinity to the AT1R. Comparisons to previous binding studies suggest that the results have a 60% chance of predicting ARB resistance. Although more biochemical studies and refinement of the model are required to increase the accuracy of the prediction of ARB resistance, personalized ARB therapy based on agtr1 sequence could increase overall ARB effectiveness. Author SummaryThe term "personalized medicine" was coined at the turn of the century, but most medicines are currently prescribed based on disease categories and occasionally racial demographics, but not personalized attributes. In cardiovascular medicine, the personalization of medication is minimal; however, it is accepted that not all patients respond equally to common cardiovascular medications. Here we chose one prominent cardiovascular drug target, the angiotensin receptor, and, using computer modeling, created preliminary models of over 100 known alterations to the angiotensin receptor to determine if the alterations changed the ability of clinically used drugs to interact with the angiotensin receptor. The strength of interaction was compared to the unaltered angiotensin receptor, generating a map predicting which alteration affected each drug. It is expected that in the future, a patients receptors can be sequenced, and maps, such as the one presented here, can be used to select the optimum medication based on the patients genetics. Such a process would allow for the personalization of current medication therapy.

pharmacology and toxicology↗

bioRxiv2018

Using Current-flow Scheme to Capture Protein Allosteric Regulation

Protein dynamic network analysis provides a powerful tool for investigating protein allosteric regulation. We recently developed a current-flow betweenness scheme for protein network analysis and demonstrated that this method, i.e. using current-flow betweenness as edge weights, is more appropriate and more robust for investigating the signal transmission between two predefined protein residues or domains as compared with direct usage of correlation scores as edge weights. Here we seek to expand the current-flow scheme to study allosteric regulations involving protein-protein binding. Specifically, we investigated three gain-of-function mutations located at the binding interface of ALK2 (also known as ACVR1) kinase and its inhibitory protein FKBP12. We first searched for the optimal smoothing function for contact network construction and then calculated the subnetwork between FKBP12 protein and ALK2 ATP binding site using current-flow betweenness. By comparing the networks between the wild-type and three mutants, we have identified statistically significant changes in the protein-protein networks that are common among all three mutants that allosterically shift the kinase towards a catalytically competent configuration.

biophysics↗

bioRxiv2017

Polymodal Allosteric Regulation of Type 1 Serine/Threonine Kinase Receptors via a Conserved Electrostatic Lock

Type 1 Serine/Threonine Kinase Receptors (STKR1) transduce a wide spectrum of biological signals mediated by TGF-{beta} superfamily members. The STKR1 activity is tightly controlled by their regulatory glycine-serine rich (GS) domain adjacent to the kinase domain. Despite decades of studies, it remains unknown how physiological or pathological GS domain modifications are coupled to STKR1 kinase activity. Here, by performing molecular dynamics simulations and free energy calculation of Activin-Like Kinase 2 (ALK2), we found that GS domain phosphorylation, FKBP12 dissociation, and disease mutations all destabilize a D354-R375 salt-bridge, which normally acts as an electrostatic lock to prevent coordination of adenosine triphosphate (ATP) to the catalytic site. We developed a WAFEX-guided principal analysis and unraveled how phosphorylation destabilizes this highly conserved salt-bridge in temporal and physical space. Using current-flow betweenness scores, we identified an allosteric network of residue-residue contacts between the GS domain and the catalytic site that controls the formation and disruption of this salt bridge. Importantly, our novel network analysis approach revealed how certain disease-causing mutations bypass FKBP12-mediated kinase inhibition to produce leaky signaling in the absence of ligand. We further provide experimental evidence that this salt-bridge lock exists in other STKR1s, and acts as a general safety mechanism in STKR1 to prevent pathological leaky signaling. In summary, our study provides a compelling and unifying allosteric activation mechanism in STKR1 kinases that reconciles a large number of experimental studies and sheds light on a novel therapeutic avenue to target disease-related STKR1 mutants.\n\nAUTHOR SUMMARYKinases play central role in essential physiological process and are attractive therapeutic drug targets. One of the important kinase families is Type 1 Serine/Threonine Kinase Receptors (STKR1), which control gene expression in response to extracellular growth factors. The activities of STKR1 are tightly controlled by their regulatory domain, which is distant from the kinase catalytic site. The underlying molecular mechanism is elucidated here. We identified that formation or disruption of a highly conserved charge-charge interaction located near the ATP binding site, mediates the physiological inhibition or activation of STKR1. We find that the stability of this charge-charge interaction is remotely controlled by interactions propagated from the distant regulatory domain. Several disease-causing mutations are located at the regulatory domain. We demonstrate how those mutations bypass these endogenous STKR1 inhibition mechanisms to produce pathological phenotypes. This study provides a general activation mechanism in STKR1 kinases, thus may benefit understanding the molecular mechanism of diseases and drug development.

biophysics↗

bioRxiv2017

Chemical Gating of the Mechanosensitive Piezo1 Channel by Asymmetric Binding of its Agonist Yoda1

Piezo proteins are homotrimeric ion channels that play major roles in normal and pathological mechanotransduction signaling in mammalian organisms. Their pharmacological control hence represents a potential therapeutic avenue. Yoda1, a Piezo1-selective small molecule agonist, is the only known selective Piezo modulator. How Yoda1 selectively interacts with Piezo1 and opens its pore is unknown. Here, by engineering and characterizing chimeras, we identified a minimal region responsible for Yoda1 binding. This region is located at the interface between the pore and the putative mechanosensory domains in each subunit. By characterizing hybrid channels containing Yoda1-insensitive and Yoda1-sensitive monomers, we demonstrate that the presence of only one Yoda1-sensitive Piezo1 subunit is sufficient for chemical activation, implicating that the asymmetric binding of Yoda1 to a single subunit enables channel opening. These findings shed light onto the gating mechanisms of Piezo channels and will pave the way for the rationale design of new Piezo channels modulators.

biochemistry↗