Atg15 in Saccharomyces cerevisiae consists of two functionally distinct domains

Autophagy is a cellular degradation system widely conserved among eukaryotes. During autophagy, cytoplasmic materials fated for degradation are compartmentalized in double membrane–bound organelles called autophagosomes. After fusing with the vacuole, their inner membrane–bound structures are released into the vacuolar lumen to become autophagic bodies and eventually degraded by vacuolar hydrolases. Atg15 is a lipase essential for disintegration of autophagic body membranes and has a transmembrane domain at the N-terminus and a lipase domain at the C-terminus. However, the roles of both domains in vivo are not well understood. In this study, we found that the N-terminal domain alone can travel to the vacuole via the multivesicular body pathway, and that targeting of the C-terminal lipase domain to the vacuole is required for degradation of autophagic bodies. Moreover, we found that the C-terminal domain could disintegrate autophagic bodies when it was transported to the vacuole via the Pho8 pathway instead of the multivesicular body pathway. Finally, we identified H435 as one of the residues composing the putative catalytic triad, and W466 as an important residue for degradation of autophagic bodies. This study may provide a clue to understanding how the C-terminal lipase domain recognizes autophagic bodies to degrade them. [Media: see text] [Media: see text]

A critical residue in the α1M2–M3 linker regulating mammalian GABAA receptor pore gating by diazepam

Benzodiazepines (BZDs) are a class of widely prescribed psychotropic drugs that modulate activity of GABAA receptors (GABAARs), neurotransmitter-gated ion channels critical for synaptic transmission. However, the physical basis of this modulation is poorly understood. We explore the role of an important gating domain, the α1M2–M3 linker, in linkage between the BZD site and pore gate. To probe energetics of this coupling without complication from bound agonist, we use a gain of function mutant (α1L9'Tβ2γ2L) directly activated by BZDs. We identify a specific residue whose mutation (α1V279A) more than doubles the energetic contribution of the BZD positive modulator diazepam (DZ) to pore opening and also enhances DZ potentiation of GABA-evoked currents in a wild-type background. In contrast, other linker mutations have little effect on DZ efficiency, but generally impair unliganded pore opening. Our observations reveal an important residue regulating BZD-pore linkage, thereby shedding new light on the molecular mechanism of these drugs.

Inhibition of SARS-CoV-2 Main Protease: A Repurposing Study That Targets the Dimer Interface of the Protein

Coronavirus disease-2019 (COVID-19) was firstly reported in Wuhan, China, towards the end of 2019, and, unfortunately, within a short period of time, emerged as a pandemic. The spread and lethality rates of the COVID-19 have ignited studies that focus on the development of therapeutics for either treatment or prophylaxis purposes. In parallel, drug repurposing studies have also come into prominence. In this study, we aimed at having a holistic understanding of <br>conformational and dynamical changes induced by an experimentally characterized inhibitor on main protease (M-pro) which would enable the discovery of novel inhibitors. To this end, we performed molecular dynamics simulations using crystal structures of <i>apo</i> and α-ketoamide-13b-bound M-pro homodimer. Analysis of trajectories pertaining to <i>apo</i> M-pro revealed a new target site, which is located at the homodimer interface, next to the catalytic dyad. Thereafter, we performed ensemble-based virtual screening by exploiting the ZINC and DrugBank databases and identified three candidate molecules, namely eluxadoline, diosmin, and ZINC02948810 that could invoke local and global conformational rearrangements which were also elicited by α-ketoamide-13b on the catalytic dyad of M-pro. Furthermore, ZINC23881687 was also discerned as a promising candidate due to its interaction with catalytically important residues Glu166 and Ser1. Last but not least, we could find another candidate, namely ZINC20425029, whose mode of action was different. It modulated the dynamical properties of catalytically important residue, Ala285 rather than the catalytic dyad. As such, this study presents valuable findings that might be used in the development of novel therapeutics against SARS-CoV-2 M-pro. <br>

Inhibition of SARS-CoV-2 Main Protease: A Repurposing Study That Targets the Dimer Interface of the Protein

Coronavirus disease-2019 (COVID-19) was firstly reported in Wuhan, China, towards the end of 2019, and, unfortunately, within a short period of time, emerged as a pandemic. The spread and lethality rates of the COVID-19 have ignited studies that focus on the development of therapeutics for either treatment or prophylaxis purposes. In parallel, drug repurposing studies have also come into prominence. In this study, we aimed at having a holistic understanding of <br>conformational and dynamical changes induced by an experimentally characterized inhibitor on main protease (M-pro) which would enable the discovery of novel inhibitors. To this end, we performed molecular dynamics simulations using crystal structures of <i>apo</i> and α-ketoamide-13b-bound M-pro homodimer. Analysis of trajectories pertaining to <i>apo</i> M-pro revealed a new target site, which is located at the homodimer interface, next to the catalytic dyad. Thereafter, we performed ensemble-based virtual screening by exploiting the ZINC and DrugBank databases and identified three candidate molecules, namely eluxadoline, diosmin, and ZINC02948810 that could invoke local and global conformational rearrangements which were also elicited by α-ketoamide-13b on the catalytic dyad of M-pro. Furthermore, ZINC23881687 was also discerned as a promising candidate due to its interaction with catalytically important residues Glu166 and Ser1. Last but not least, we could find another candidate, namely ZINC20425029, whose mode of action was different. It modulated the dynamical properties of catalytically important residue, Ala285 rather than the catalytic dyad. As such, this study presents valuable findings that might be used in the development of novel therapeutics against SARS-CoV-2 M-pro. <br>

A critical residue in the α1M2-M3 linker regulating GABAA receptor pore gating by diazepam

Benzodiazepines (BZDs) are a class of widely prescribed psychotropic drugs. Their anxiolytic and sedative effects are conferred by modulating the activity of GABAA receptors (GABAARs), which are the primary inhibitory neurotransmitter receptors throughout the central nervous system. However, the physical mechanism by which BZDs exert their effects on the receptor is poorly understood. In particular, BZDs require coapplication with an agonist to effectively open the channel pore, making it difficult to dissect whether the drug has altered either agonist binding or channel gating as these two processes are intimately coupled. To isolate effects on gating we used a spontaneously active gain of function mutant (α1L9’Tβ2γ2L) that is directly gated by BZDs alone in the absence of agonist. In the α1L9’T background we explored effects of alanine substitutions throughout the α1M2-M3 linker on modulation of the channel pore by the BZD positive modulator diazepam (DZ). The M2-M3 linker is known to be an important element for channel activation. Linker mutations generally impaired unliganded pore opening, indicating that side chain interactions are important for channel gating in the absence of bound agonist. All but one mutation had no effect on the transduction of chemical energy from DZ binding to pore gating. Strikingly, α1V279A doubles DZ’s energetic contribution to gating, whereas larger side chains at this site do not. In a wild-type background α1V279A enhances DZ-potentiation of currents evoked by saturating GABA, consistent with a direct effect on the pore closed/open equilibrium. Our observations identify an important residue regulating coupling between the BZD site and the pore gate, thereby shedding new light on the molecular mechanism of a frequently prescribed class of psychotropic drugs.

Ratio of hydrophobic-hydrophilic and positive-negative residues at lipid-water-interface influences surface expression and channel-gating of TRPV1

During evolution, TRPV1 has lost, retained or selected certain residues at Lipid-Water-Interface (LWI) and formed specific patterns there. The ratio of “hydrophobic-hydrophilic” and “positive-negative charged” residues at the inner LWI remains conserved throughout vertebrate evolution and play important role in regulating TRPV1 trafficking, localization and functions. Arg575 is an important residue as Arg575Asp mutant has reduced Capsaicin-sensitivity, surface expression, colocalization with lipid-raft markers, cell area, and increased cell lethality. This lethality is due to the disruption of the ratio between positive-negative charges there. Such lethality can be rescued by either using TRPV1-specfic inhibitor 5’-IRTX or by restoring the positive-negative charge ratio at that position, i.e. by introducing Asp576Arg mutation in Arg575Asp backbone. We propose that Arg575Asp mutant confers TRPV1 in a “constitutive-open-like” condition. These findings have broader implication in understanding the molecular basis of thermo-gating and channel-gating and the microenvironments involved in such process that goes erratic in different diseases.

Dynamical important residue network (DIRN): network inference via conformational change

Motivation Protein residue interaction network has emerged as a useful strategy to understand the complex relationship between protein structures and functions and how functions are regulated. In a residue interaction network, every residue is used to define a network node, adding noises in network post-analysis and increasing computational burden. In addition, dynamical information is often necessary in deciphering biological functions. Results We developed a robust and efficient protein residue interaction network method, termed dynamical important residue network, by combining both structural and dynamical information. A major departure from previous approaches is our attempt to identify important residues most important for functional regulation before a network is constructed, leading to a much simpler network with the important residues as its nodes. The important residues are identified by monitoring structural data from ensemble molecular dynamics simulations of proteins in different functional states. Our tests show that the new method performs well with overall higher sensitivity than existing approaches in identifying important residues and interactions in tested proteins, so it can be used in studies of protein functions to provide useful hypotheses in identifying key residues and interactions. Supplementary information Supplementary data are available at Bioinformatics online.

An efficient combination of BEST and NUS methods in multidimensional NMR spectroscopy for high throughput analysis of proteins

Application of NUS along with BEST NMR experiments has been demonstrated for obtaining the important residue-specific atomic level backbone chemical shift values in short durations of time.