Biology subjects

Da, L.-T.

Publications and source records attributed to Da, L.-T..

4 recordsLinked to original sources

bioRxiv2021

Altered O-glycosylation Level of SARS-CoV-2 Spike Protein by Host O-glycosyltransferase Strengthens Its Trimeric Structure

The trimeric spike protein (S) mediates host-cell entry and membrane fusion of SARS-CoV-2. S protein is highly glycosylated, whereas its O-glycosylation is still poorly understood. Herein, we site-specifically examine the O-glycosylation of S protein through a mass spectrometric approach with HCD-triggered-ETD model. We identify 15 high-confidence O-glycosites and at least 10 distinct O-glycan structures on S protein. Peptide microarray assays prove that human ppGalNAc-T6 actively participates in O-glycosylation of S protein. Importantly, the upregulation of ppGalNAc-T6 expression can profoundly enhance the O-glycosylation level by generating new O-glycosites and increasing both O-glycan heterogeneity and intensities. Further molecular dynamics simulations reveal that the O-glycosylation on the protomer-interface regions, which are mainly modified by ppGalNAc-T6, can potentially stabilize the trimeric S protein structure. Our work provides deep molecular insights of how viral infection harnesses the host O-glycosyltransferases to dynamically regulate the O-glycosylation level of the viral envelope protein responsible for membrane fusion.

biochemistry↗

bioRxiv2021

Mechanism of REST/NRSF Regulation of Clustered Protocadherin Alpha Genes

Repressor element-1 silencing transcription factor (REST) or neuron-restrictive silencer factor (NRSF) is a zinc-finger (ZF) containing transcriptional repressor that recognizes thousands of neuron-restrictive silencer elements (NRSEs) in mammalian genomes. How REST/NRSF regulates gene expression remains incompletely understood. Here, we investigate the binding pattern and regulation mechanism of REST/NRSF in the clustered protocadherin (PCDH) genes. We find that REST/NRSF directionally forms base-specific interactions with NRSEs via tandem ZFs in an anti-parallel manner but with striking conformational changes. In addition, REST/NRSF recruitment to the HS5-1 enhancer leads to the decrease of long-range enhancer-promoter interactions and downregulation of the clustered PCDH genes. Thus, REST/NRSF represses PCDH gene expression through directional binding to a repertoire of NRSEs within the distal enhancer and variable target genes.

molecular biology↗

bioRxiv2020

Allosteric regulation in CRISPR/Cas1-Cas2 protospacer acquisition mediated by DNA in association with Cas2

Cas1 and Cas2 are highly conserved proteins across CRISPR-Cas systems and play a significant role in protospacer acquisition. Here we study the protospacer (or ps) DNA binding, recognition, and response to cleavage on the protospacer-adjacent-motif complementary sequence or PAMc by Cas1-Cas2, implementing all-atom molecular dynamics simulations. First, we noticed that two active sites of Cas1&1 bind asymmetrically to two identical PAMc in the simulation. For psDNA containing only one PAMc to be recognized, it is then found that the non-PAMc association site remains destabilized until after the bound PAMc being cleaved. Thus, correlation appears to exist between the two active sites, which can be allosterically mediated by psDNA and Cas2&2 in bridging. To substantiate such findings, we further simulated Cas1-Cas2 in complex with synthesized psDNA sequences psL and psH, which have been measured with low and high efficiency in acquisition, respectively. Notably, such inter-site correlation becomes largely enhanced for Cas1-Cas2 in complex with psH, and remains low with psL. Hence, our studies demonstrate that PAMc recognition and cleavage in one active site of Cas1-Cas2 allosterically regulates non-PAMc association/reaction in the other site, and such allosteric regulation is mediated by non-catalytic Cas 2 and DNA protospacer in acquisition.

biophysics↗

bioRxiv2019

Understanding the molecular mechanism of umami recognition by T1R1-T1R3 using molecular dynamics simulations

Taste receptor T1R1-T1R3 can be activated by binding to several natural ligands, e.g., L-glutamate and 5-ribonucleotides etc., thereby stimulating the umami taste. The molecular mechanism of umami recognition at an atomic level, however, remains elusive. Here, using homology modeling, molecular docking and molecular dynamics (MD) simulations, we investigate the effects of five natural umami ligands on the structural dynamics of T1R1-T1R3. Our work identifies the key residues that are directly involved in recognizing the binding ligands. In addition, two adjacent binding sites in T1R1 are determined for substrate binding, and depending on the molecular size and chemical properties of the incoming ligand, one or both these binding sites can be occupied. More interestingly, the binding of varied ligands can lead to either closing or opening of T1R1, based on which, we further classify the five ligands into two groups. This different binding effects are likely associated with the distinct umami signals stimulated by various ligands. This work warrants new experimental assays to further validate the theoretical model and provides guidance to design more effective umami ligands.\n\nAuthor summaryUmami, as the fifth basic taste, is induced by umami substances from the natural food, such as L-glutamate, 5-ribonucleotides, and peptides etc. These umami substances are widely added to foods as flavor enhancers to promote food quality. However, although extensive experimental and theoretical studies have been devoted to revealing the recognition mechanisms of the taste receptor T1R1-T1R3 to the umami ligands, the detailed molecular mechanism is still unknown, largely due to the lack of the receptor structure. Here, using a new template structure different from the former theoretical studies, we constructed a more accurate homology model of T1R1-T1R3. Based on this receptor model, combined with molecular docking and MD simulations, we investigate how different ligands with varied molecular size and chemical groups might affect the dynamics of T1R1. Our work provides the structural basis for relating the dynamics of umami receptor induced by varied ligands to the resulting umami signal.

molecular biology↗