Biology subjects

Ahmed, W. S.

Publications and source records attributed to Ahmed, W. S..

5 recordsLinked to original sources

bioRxiv2024

Coevolving Residues Distant from the Ligand Binding Site are Involved in GAF Domain Function

Ligand binding to GAF domains regulates the activity of associated catalytic domains in a wide variety of proteins. For instance, cGMP binding to the GAFa domain of phosphodiesterase 5 (PDE5) activates the cGMP-hydrolyzing catalytic domain in the protein. However, the residues involved and the mechanism of GAF domain function are not entirely clear. Here, combining computational and experimental analysis, we show that two highly coevolving residues distant from the ligand binding site play a critical role in GAF domain allostery. Specifically, Statistical Coupling Analysis (SCA) of GAF domain sequences revealed the highest coevolution score for residues L267 and F295. Molecular dynamics (MD) simulations of both apo and holo forms of the wild type and mutant (L267A and F295A) PDE5 GAFa domains revealed significant alterations in structural dynamics and interaction with cGMP. Incorporation of the mutations in a Bioluminescence Resonance Energy Transfer (BRET)-based biosensor, which reports a ligand-induced conformational change, revealed a change in the conformation of the GAF domain and an increase in the EC50 of cGMP-induced conformational change. Similar results were obtained regarding cGMP-induced conformational change in the full-length PDE5 and in the fluorescence of the GAF domain fluorescent protein, miRFP670nano3. Finally, structural analysis of conformers observed in MD simulations revealed a possible mechanism underlying the impact of mutations of these two coevolving residues in the PDE5 GAFa domain. Our results provide insight into the role of distant, coevolving residues in GAF domain allostery, and may aid in understanding evolution of allostery in proteins.

molecular biology↗

bioRxiv2023

A Slow but Steady NanoLuc: R162A mutation results in a decreased, but stable, NanoLuc activity

NanoLuc (NLuc) luciferase has found extensive application in designing a range of biological assays including gene expression analysis, protein-protein interaction and protein conformational changes due to its enhanced brightness and small size. However, questions related to its mechanism of interaction with the substrate, furimazine, as well as bioluminescence activity remains elusive. Here, we combined molecular dynamics (MD) simulation and mutational analysis to show that the R162A mutation results in a decreased but stable bioluminescence activity of NLuc in vitro. Specifically, we performed multiple, all-atom, explicit solvent MD simulations of the apo and furimazine-docked (holo) NLuc structures revealing differential dynamics of the protein in the absence and presence of the ligand. Further, analysis of trajectories for hydrogen bonds (H-bonds) formed between NLuc and furimazine revealed substantial H-bond interaction between R162 and Q32 residues. Mutation of the two residues in NLuc revealed a decreased but stable activity of the R162A, but not Q32A, mutant NLuc in in vitro assays performed with furimazine. In addition to highlighting the role of the R162 residue in NLuc activity, we believe that the mutant NLuc will find wide application in designing in vitro assays requiring extended monitoring of NLuc bioluminescence activity.

biophysics↗

bioRxiv2022

SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to downregulate MHC-I surface expression

Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small protein {beta}2-microglobulin ({beta}2m), bind peptides in the endoplasmic reticulum that are generated by the cytosolic turnover of cellular proteins. In virus-infected cells these peptides may include those derived from viral proteins. Peptide-MHC-I complexes then traffic through the secretory pathway and are displayed at the cell surface where those containing viral peptides can be detected by CD8+ T lymphocytes that kill infected cells. Many viruses enhance their in vivo survival by encoding genes that downregulate MHC-I expression to avoid CD8+ T cell recognition. Here we report that two accessory proteins encoded by SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic, downregulate MHC-I expression using distinct mechanisms. One, ORF3a, a viroporin, reduces global trafficking of proteins, including MHC-I, through the secretory pathway. The second, ORF7a, interacts specifically with the MHC-I heavy chain, acting as a molecular mimic of {beta}2m to inhibit its association. This slows the exit of properly assembled MHC-I molecules from the endoplasmic reticulum. We demonstrate that ORF7a reduces antigen presentation by the human MHC-I allele HLA-A*02:01. Thus, both ORF3a and ORF7a act post-translationally in the secretory pathway to lower surface MHC-I expression, with ORF7a exhibiting a novel and specific mechanism that allows immune evasion by SARS-CoV-2. Significance StatementViruses may down-regulate MHC class I expression on infected cells to avoid elimination by cytotoxic T cells. We report that the accessory proteins ORF7a and ORF3a of SARS-CoV-2 mediate this function and delineate the two distinct mechanisms involved. While ORF3a inhibits global protein trafficking to the cell surface, ORF7a acts specifically on MHC-I by competing with {beta}2m for binding to the MHC-I heavy chain. This is the first account of molecular mimicry of {beta}2m as a viral mechanism of MHC-I down-regulation to facilitate immune evasion.

immunology↗

bioRxiv2022

A Genetically Encoded BRET-based SARS-CoV-2 Mpro Protease Activity Sensor

The SARS-CoV-2 main protease, Mpro, is critical for its replication and is an appealing target for designing anti-SARS-CoV-2 agents. In this regard, a number of assays have been developed based on its cleavage sequence preferences to monitor its activity. These include the usage of Fluorescence Resonance Energy Transfer (FRET)-based substrates in vitro and a FlipGFP reporter, one which fluoresces after Mpro-mediated cleavage, in live cells. Here, we have engineered a pair of genetically encoded, Bioluminescence Resonance Energy Transfer (BRET)-based sensors for detecting SARS-CoV-2 Mpro proteolytic activity in living host cells as well as in vitro assays. The sensors were generated by sandwiching Mpro N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and nanoLuc proteins. Co-expression of the sensor with the Mpro in live cells resulted in its cleavage in a dose- and time-dependent manner while mutation of the critical C145 residue (C145A) in Mpro completely abrogated the sensor cleavage. Importantly, the BRET-based sensors displayed increased sensitivities and specificities as compared to the recently developed FlipGFP-based Mpro sensor. Additionally, the sensors recapitulated the inhibition of Mpro by the well-characterized pharmacological agent GC376. Further, in vitro assays with the BRET-based Mpro sensors revealed a molecular crowding-mediated increase in the rate of Mpro activity and a decrease in the inhibitory potential of GC376. The sensor developed here will find direct utility in studies related to drug discovery targeting the SARS-CoV-2 Mpro and functional genomics application to determine the effect of sequence variation in Mpro.

biophysics↗

bioRxiv2020

Assessment of research ethics education offerings of pharmacy master programs: a qualitative content analysis

The importance of research ethics (RE) training has led academic and funding institutions to require that students, trainees, and faculty obtain such training at various stages of their careers. Despite the increasing awareness of the value RE education offers, this training requirement is absent in Jordan. We aimed to assess RE education offerings of pharmacy master programs in Jordan and compare with the top-ranked pharmacy graduate programs globally. Therefore, a list of universities that offer research-based pharmacy master programs was created. Each program was evaluated for the inclusion of RE education. A qualitative content analysis approach based on inductive reasoning and latent analysis was followed to analyze the data. Results of the study showed a lack of appropriate RE education for graduate-level pharmacy programs in Jordan with only 40% of the programs partially discuss selected topics related to RE. Regarding pharmacy graduate programs globally, 10% offer a standalone RE course, 40% offer some discussions related to RE, another 10% do not offer RE education in any form, and the remaining 40% of the programs were difficult to assess due to lack of sufficient information available online. Based on the findings of this study, training in RE is tends to be lacking in pharmacy graduate programs in Jordan and globally, with a greater lack in Jordan than globally. There is a need to incorporate formal RE education into programs that do not offer this type of instruction. Programs that formally touch on some aspects of RE need to expand the scope of topics to include more RE-related themes. Integrating a standalone RE course into pharmacy graduate programs is highly encouraged.

scientific communication and education↗