ResearchPad - full‐length-papers https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Crystal structure of Nsp15 endoribonuclease NendoU from SARS‐CoV‐2]]> https://www.researchpad.co/article/elastic_article_16592 Severe Acute Respiratory Syndrome coronavirus 2 (SARS‐CoV‐2) is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS‐CoVs and Middle East Respiratory Syndrome coronavirus (MERS‐CoVs), the detailed information about SARS‐CoV‐2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies, and antivirals. We applied high‐throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS‐CoV‐2 proteins and structures. Here we report two high‐resolution crystal structures of endoribonuclease Nsp15/NendoU. We compare these structures with previously reported homologs from SARS and MERS coronaviruses.

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<![CDATA[Comparison of backbone dynamics of the p50 dimerization domain of NFκB in the homodimeric transcription factor NFκB1 and in its heterodimeric complex with RelA (p65)]]> https://www.researchpad.co/article/N1b66a352-f261-4cf8-97ec-48bbea122886

Abstract

The nuclear factor of kappa light polypeptide gene enhancer in B‐cells (NFκB) transcription factors play a critical role in human immune response. The family includes homodimers and heterodimers of five component proteins, which mediate different transcriptional responses and bind preferentially to different DNA sequences. Crystal structures of DNA complexes show that the dimers of the Rel‐homology regions are structurally very similar. Differing DNA sequence preference together with structural similarity suggests that the dimers may differ in their dynamics. In this study, we present the first near‐complete 15N, 13Cα/β, and HN backbone resonance assignments of two dimers of the dimerization domain (DD) of the NFκB1 (p50) protein (residues 241–351): the homodimer of two p50 domains and a heterodimer of the p50 DD with the p65 DD. As expected, the two dimers behave very similarly, with chemical shift differences between them largely concentrated in the dimer interface and attributable to specific differences in the amino acid sequences of p50 and p65. A comparison of the picosecond‐nanosecond dynamics of the homo‐ and heterodimers also shows that the environment of p50 is similar, with an overall slightly reduced correlation time for the homodimer compared to the heterodimer, consistent with its slightly smaller molecular weight. These results demonstrate that NMR spectroscopy can be used to explore subtle changes in structure and dynamics that have the potential to give insights into differences in specificity that can be exploited in the design of new therapeutic agents.

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<![CDATA[Thermal stability of single‐domain antibodies estimated by molecular dynamics simulations]]> https://www.researchpad.co/article/N2b3189a8-da13-430a-9a2b-534088c4551f

Abstract

Single‐domain antibodies (sdAbs) function like regular antibodies, however, consist of only one domain. Because of their low molecular weight, sdAbs have advantages with respect to production and delivery to their targets and for applications such as antibody drugs and biosensors. Thus, sdAbs with high thermal stability are required. In this work, we chose seven sdAbs, which have a wide range of melting temperature (T m) values and known structures. We applied molecular dynamics (MD) simulations to estimate their relative stability and compared them with the experimental data. High‐temperature MD simulations at 400 K and 500 K were executed with simulations at 300 K as a control. The fraction of native atomic contacts, Q, measured for the 400 K simulations showed a fairly good correlation with the T m values. Interestingly, when the residues were classified by their hydrophobicity and size, the Q values of hydrophilic residues exhibited an even better correlation, suggesting that stabilization is correlated with favorable interactions of hydrophilic residues. Measuring the Q value on a per‐residue level enabled us to identify residues that contribute significantly to the instability and thus demonstrating how our analysis can be used in a mutant case study.

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<![CDATA[The structural and biochemical impacts of monomerizing human acetylcholinesterase]]> https://www.researchpad.co/article/N54d2aff9-d01e-4725-aa3d-b8adbe39231b

Abstract

Serving a critical role in neurotransmission, human acetylcholinesterase (hAChE) is the target of organophosphate nerve agents. Hence, there is an active interest in studying the mechanism of inhibition and recovery of enzymatic activity, which could lead to better countermeasures against nerve agents. As hAChE is found in different oligomeric assemblies, certain approaches to studying it have been problematic. Herein, we examine the biochemical and structural impact of monomerizing hAChE by using two mutations: L380R/F535K. The activities of monomeric hAChE L380R/F535K and dimeric hAChE were determined to be comparable utilizing a modified Ellman's assay. To investigate the influence of subunit–subunit interactions on the structure of hAChE, a 2.1 Å X‐ray crystallographic structure was determined. Apart from minor shifts along the dimer interface, the overall structure of the hAChE L380R/F535K mutant is similar to that of dimeric hAChE. To probe whether the plasticity of the active site was overtly impacted by monomerizing hAChE, the kinetic constants of (PR/S) − VX (ethyl({2‐[bis(propan‐2‐yl)amino]ethyl}sulfanyl)(methyl)phosphinate) inhibition and subsequent rescue of hAChE L380R/F535K activity with HI‐6 (1‐(2′‐hydroxyiminomethyl‐1′‐pyridinium)‐3‐(4′‐carbamoyl‐1‐pyridinium)) were determined and found to be comparable to those of dimeric hAChE. Thus, hAChE L380R/F535K could be used as a substitute for dimeric hAChE when experimentally probing the ability of the hAChE active site to accommodate future nerve agent threats or judge the ability of new therapeutics to access the active site.

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