The ‘hidden side’ of spin labelled oligonucleotides: Molecular dynamics study focusing on the EPR-silent components of base pairing

© 2021 Elsevier Inc. Nitroxide labels are combined with nucleic acid structures and are studied using electron paramagnetic resonance experiments (EPR). As X-ray/NMR structures are unavailable with the nitroxide labels, detailed residue level information, down to atomic resolution, about the effect of these nitroxide labels on local RNA structures is currently lacking. This information is critical to evaluate the choice of spin label. In this study, we compare and contrast the effect of TEMPO-based (NT) and rigid spin (Ç) labels (in both 2′-O methylated and not-methylated forms) on RNA duplexes. We also investigate sequence- dependent effects of NT label on RNA duplex along with the more complex G-quadruplex RNA. Distances measured from molecular dynamics simulations between the two spin labels are in agreement with the EPR experimental data. To understand the effect of labelled oligonucleotides on the structure, we studied the local base pair geometries and global structure in comparison with the unlabelled structures. Based on the structural analysis, we can conclude that TEMPO-based and Ç labels do not significantly perturb the base pair arrangements of the native oligonucleotide. When experimental structures for the spin labelled DNA/RNA molecules are not available, general framework offered by the current study can be used to provide information critical to the choice of spin labels to facilitate future EPR studies.Max Planck Society and Volkswagen Foundation grant 8394

PyCoM: a python library for large-scale analysis of residue-residue coevolution data

The version currently archived on this institutional repository is an accepted manuscript, the PDF version of the author’s final manuscript, as accepted for publication by the journal but prior to copyediting or typesetting. They can be cited using the author(s), article title, journal title, year of online publication, and DOI. They will be replaced by the final typeset articles, which may therefore contain changes. The DOI will remain the same throughout.Data availability The data underpinning this publication can be accessed from Brunel University London's data repository under CC BY license: Coevolution matrix database https://brunel.figshare.com/articles/dataset/PyCoM_ProCoM_Database_of_coevolu tion_matrices/23735613 and protein database https://brunel.figshare.com/articles/dataset/PyCoM_ProCoM_Curated_UniProt_pro tein_database/23733309 .Availability and implementation PyCoM code is freely available from https://github.com/scdantu/pycom and PyCoMdb and the Jupyter Notebook tutorials are freely available from https://pycom.brunel.ac.uk .Supplementary information: Supplementary data are available at Bioinformatics online at https://academic.oup.com/bioinformatics/advance-article/doi/10.1093/bioinformatics/btae166/7635577#supplementary-data .Motivation: Computational methods to detect correlated amino acid positions in proteins have become a valuable tool to predict intra and inter-residue protein contacts, protein structures, and effects of mutation on protein stability and function. While there are many tools and webservers to compute coevolution scoring matrices, there is no central repository of alignments and coevolution matrices for large-scale studies and pattern detection leveraging on structural and biological annotation already available in UniProt. Results: We present a Python library, PyCoM, which enables users to query and analyse coevolution matrices and sequence alignments of 457,622 proteins, selected from UniProtKB/Swiss-Prot database (length ≤ 500 residues), from a pre-compiled coevolution matrix database (PyCoMdb). PyCoM facilitates the development of statistical analyses of residue coevolution patterns using filters on structural and biological annotation from UniProtKB/Swiss-Prot, with simple access to PyCoMdb for both novice and advanced users, supporting Jupyter Notebooks, Python scripts, and a web API access. The resource is open source and will help in generating data-driven computational models and methods to study and understand protein structures, stability, function, and design.This project made use of time on HPC granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim (http://hecbiosim.ac.uk), supported by EPSRC (grant no. EP/R029407/1). Philipp Bibik was funded by Department of Computer Science, Brunel University London, summer internship programme

MDSubSampler: a posteriori sampling of important protein conformations from biomolecular simulations

Data availability: A sample of the trajectory data is included in the GitHub repository. The data underpinning this publication can be accessed from Brunel University London's data repository under CC BY license: https://doi.org/10.17633/rd.brunel.c.6620539 .Supplementary information: Supplementary data are available at Bioinformatics online at https://academic-oup-com.ezproxytest.brunel.ac.uk/bioinformatics/advance-article/doi/10.1093/bioinformatics/btad427/7221036?searchresult=1#supplementary-data .Copyright © The Author(s) 2023.. Motivation: Molecular dynamics (MD) simulations have become routine tools for the study of protein dynamics and function. Thanks to faster GPU-based algorithms, atomistic and coarse-grained simulations are being used to explore biological functions over the microsecond timescale, yielding terabytes of data spanning multiple trajectories, thereby extracting relevant protein conformations without losing important information is often challenging. Results: We present MDSubSampler, a Python library and toolkit for a posteriori subsampling of data from multiple trajectories. This toolkit provides access to uniform, random, stratified, weighted sampling and bootstrapping sampling methods. Sampling can be performed under the constraint of preserving the original distribution of relevant geometrical properties. Possible applications include simulations post-processing, noise reduction and structures selection for ensemble docking. Availability: MDSubSampler is freely available at https://github.com/alepandini/MDSubSampler, along with guidance on installation and tutorials on how it can be used.NO is supported by a scholarship from Brunel University London EPSRC DTP (grant no. EP/T518116/1). This project made use of time on HPC granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim (https://www.hecbiosim.ac.uk), supported by EPSRC (grant no. EP/X035603/1)

Structure-based enzyme engineering improves donor-substrate recognition of Arabidopsis thaliana Glycosyltransferases

Glycosylation of secondary metabolites involves plant UDP-dependent glycosyltransferases (UGTs). UGTs have shown promise as catalysts in the synthesis of glycosides for medical treatment. However, limited understanding at the molecular level due to insufficient biochemical and structural information has hindered potential applications of most of these UGTs. In the absence of experimental crystal structures, we employed advanced molecular modelling and simulations in conjunction with biochemical characterisation to design a workflow to study five Group H Arabidopsis thaliana (76E1, 76E2, 76E4, 76E5, 76D1) UGTs. Based on our rational structural manipulation and analysis, we identified key amino acids (P129 in 76D1; D374 in 76E2; K275 in 76E4), which when mutated improved donor-substrate recognition than wildtype UGTs. Molecular dynamics simulations and deep learning analysis identified structural differences, which drive substrate preferences. The design of these UGTs with broader substrate specificity may play important role in biotechnological and industrial applications. These findings can also serve as basis to study other plant UGTs and thereby advancing UGT enzyme engineering.Federal Scholarship Board/Presidential Special Scholarship Scheme for Innovation and Development (PRESSID), Nigeria; Sichuan Science and Technology Progra

Myricetin protects pancreatic β-cells from human islet amyloid polypeptide (hIAPP) induced cytotoxicity and restores islet function

The aberrant misfolding and self-assembly of human islet amyloid polypeptide (hIAPP)–a hormone that is co-secreted with insulin from pancreatic β-cells–into toxic oligomers, protofibrils and fibrils has been observed in type 2 diabetes mellitus (T2DM). The formation of these insoluble aggregates has been linked with the death and dysfunction of β-cells. Therefore, hIAPP aggregation has been identified as a therapeutic target for T2DM management. Several natural products are now being investigated for their potential to inhibit hIAPP aggregation and/or disaggregate preformed aggregates. In this study, we attempt to identify the anti-amyloidogenic potential of Myricetin (MYR)- a polyphenolic flavanoid, commonly found in fruits (like Syzygium cumini). Our results from biophysical studies indicated that MYR supplementation inhibits hIAPP aggregation and disaggregates preformed fibrils into non-toxic species. This protection was accompanied by inhibition of oxidative stress, reduction in lipid peroxidation and the associated membrane damage and restoration of mitochondrial membrane potential in INS-1E cells. MYR supplementation also reversed the loss of functionality in hIAPP exposed pancreatic islets via restoration of glucose-stimulated insulin secretion. Molecular dynamics simulation studies suggested that MYR molecules interact with the hIAPP pentameric fibril model at the amyloidogenic core region and thus prevents aggregation and distort the fibrils.Council of Scientific and industrial research, Government of India (RD/0111-CSIR000-016) and Indian Institute of Technology, Bombay (11IRCCSG003); Wadhwani Research Center of Bioengineering (RD/018/- DONWR04-001/); Ramalingaswami fellowship (BT/RLF/Re-entry/11/2012; Department of Biotechnology-DBT, Government of India); and University Grants Commission (UGC, Government of India F.4-5(18-FRP) (IV-Cycle)/2017(BSR)

Structure, Dynamics and Cellular Insight Into Novel Substrates of the Legionella pneumophila Type II Secretion System

Legionella pneumophila is a Gram-negative bacterium that is able to replicate within a broad range of aquatic protozoan hosts. L. pneumophila is also an opportunistic human pathogen that can infect macrophages and epithelia in the lung and lead to Legionnaires’ disease. The type II secretion system is a key virulence factor of L. pneumophila and is used to promote bacterial growth at low temperatures, regulate biofilm formation, modulate host responses to infection, facilitate bacterial penetration of mucin gels and is necessary for intracellular growth during the initial stages of infection. The L. pneumophila type II secretion system exports at least 25 substrates out of the bacterium and several of these, including NttA to NttG, contain unique amino acid sequences that are generally not observed outside of the Legionella genus. NttA, NttC, and NttD are required for infection of several amoebal species but it is unclear what influence other novel substrates have within their host. In this study, we show that NttE is required for optimal infection of Acanthamoeba castellanii and Vermamoeba vermiformis amoeba and is essential for the typical colony morphology of L. pneumophila. In addition, we report the atomic structures of NttA, NttC, and NttE and through a combined biophysical and biochemical hypothesis driven approach we propose novel functions for these substrates during infection. This work lays the foundation for future studies into the mechanistic understanding of novel type II substrate functions and how these relate to L. pneumophila ecology and disease.The United Kingdom Medical Research Council; The Wellcome Trust

Molecular dynamics simulations elucidate the mode of protein recognition by Skp1 and the F-box domain in the SCF complex

Polyubiquitination of the target protein by a ubiquitin transferring machinery is key to various cellular processes. E3 ligase Skp1-Cul1-F-box (SCF) is one such complex which plays crucial role in substrate recognition and transfer of the ubiquitin molecule. Previous computational studies have focused on S-phase kinase-associated protein 2 (Skp2), cullin, and RING-finger proteins of this complex, but the roles of the adapter protein Skp1 and F-box domain of Skp2 have not been determined. Using sub-microsecond molecular dynamics simulations of full-length Skp1, unbound Skp2, Skp2-Cks1 (Cks1: Cyclin-dependent kinases regulatory subunit 1), Skp1-Skp2, and Skp1-Skp2-Cks1 complexes, we have elucidated the function of Skp1 and the F-box domain of Skp2. We found that the L-16 loop of Skp1(,) which was deleted in previous X-ray crystallography studies, can offer additional stability to the ternary complex via its interactions with the C-terminal tail of Skp2. Moreover, Skp1 helices H6, H7, and H8 display vivid conformational flexibility when not bound to Skp2, suggesting that these helices can recognize and lock the F-box proteins. Furthermore, we observed that the F-box domain could rotate (5 degrees-129 degrees), and that the binding partner determined the degree of conformational flexibility. Finally, Skp1 and Skp2 were found to execute a domain motion in Skp1-Skp2 and Skp1-Skp2-Cks1 complexes that could decrease the distance between ubiquitination site of the substrate and the ubiquitin molecule by 3 nm. Thus, we propose that both the F-box domain of Skp2 and Skp1-Skp2 domain motions displaying preferential conformational control can together facilitate polyubiquitination of a wide variety of substrates. Proteins 2016; 84:159-171. (c) 2015 Wiley Periodicals, Inc

Backbone and side chain assignments of human cell cycle regulatory protein S-phase kinase-associated protein 1

Ubiquitination of proteins is required to regulate several cellular mechanisms in cells. Skp1-Cullin-1-F-box (SCF), the largest family of the RING E3 ligases, recognizes and carries out the poly-ubiquitination of many substrate proteins. SCF E3 ligase is a multi-component protein complex, and the human S-phase kinase-associated protein 1 (Skp1) is the adapter protein, which binds and presents the substrate binding protein F-box (FBP) to the rest of the E3 ligase. Several crystallographic studies have solved the partial structure of Skp1 in complex with various FBPs, but there is no structure of standalone Skp1. Understanding the conformational and structural properties of Skp1 with and without FBPs is required to understand the complete mechanism of poly-ubiquitination. Here, we report similar to 90 % backbone and 64 % side chain H-1, C-13, N-15 assignments of Skp1 protein using various double and triple resonance NMR experiments

A trade-off for covalent and intercalation binding modes: a case study for Copper (II) ions and singly modified DNA nucleoside

© 2019, The Author(s). Selective binding to nucleic acids and, more generally, to biopolymers, very often requires at a minimum the presence of specific functionalities and precise spatial arrangement. DNA can fold into defined 3D structures upon binding to metal centers and/or lanthanides. Binding efficiency can be boosted by modified nucleosides incorporated into DNA sequences. In this work the high selectivity of modified nucleosides towards copper (II) ions, when used in the monomeric form, is unexpectedly and drastically reduced upon being covalently attached to the DNA sequence in single-site scenario. Surprisingly, such selectivity is partially retained upon non-covalent (i.e. intercalation) mixture formed by native DNA duplex and a nucleoside in the monomeric form. Exploiting the electron spin properties of such different and rich binding mode scenarios, 1D/2D pulsed EPR experiments have been used and tailored to differentiate among the different modes. An unusual correlation of dispersion of hyperfine couplings and strength of the binding mode(s) is described

Mutation of Arg191 in FtsZ Impairs Cytokinetic Abscission of Bacillus subtilis Cells

FtsZ monomers assemble to form a dynamic Z-ring at the midcell position in bacteria that coordinates bacterial cell division. Antibacterial agents plumbagin. and SB-RA-2001 were found to bind to FtsZ and to inhibit Z-ring formation in bacteria. Docking analysis indicated similar binding regions for these two inhibitors on FtsZ, and residue R191 was involved in the binding interaction with both compounds. In this work, the importance of R191 in FtsZ assembly and in bacterial cell division was analyzed. R191A-FtsZ exhibited significantly poorer polymerization ability. Further, the mutant FtsZ could poison the assembly of wild-type FtsZ (WT-FtsZ). The expression of R191A-FtsZ in Bacillus subtilis strain PL2084 perturbed Z-ring formation and produced filamentous cells, indicating that the mutation hindered the division of,these cells. The results suggested that the R191A mutation is a dominant negative mutation of FtsZ. Molecular dynamics simulations of R191A-FtsZ and WT-FtsZ revealed a kink in helices H5 and H7 in the active site of R191A-FtsZ compared to that of WT-FtsZ, which is required for FtsZ assembly. The findings suggested that R191 is an important residue for FtsZ assembly, which Can be targeted for the design of FtsZ inhibitors