The functional motions and related key residues behind the uncoating of coxsackievirus A16

TMADH (trimethylamine dehydrogenase) is a complex iron-sulphur flavoprotein that forms a soluble electron-transfer complex with ETF (electron-transferring flavoprotein). The mechanism of electron transfer between TMADH and ETF has been studied using stopped-flow kinetic and mutagenesis methods, and more recently by X-ray crystallography. Potentiometric methods have also been used to identify key residues involved in the stabilization of the flavin radical semiquinone species in ETF. These studies have demonstrated a key role for 'conformational sampling' in the electron-transfer complex, facilitated by two-site contact of ETF with TMADH. Exploration of three-dimensional space in the complex allows the FAD of ETF to find conformations compatible with enhanced electronic coupling with the 4Fe-4S centre of TMADH. This mechanism of electron transfer provides for a more robust and accessible design principle for interprotein electron transfer compared with simpler models that invoke the collision of redox partners followed by electron transfer. The structure of the TMADH-ETF complex confirms the role of key residues in electron transfer and molecular assembly, originally suggested from detailed kinetic studies in wild-type and mutant complexes, and from molecular modelling.

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Stability and Folding of the Unusually Stable Hemoglobin from Synechocystis is Subtly Optimized and Dependent on the Key Heme Pocket Residues.

Protein and Peptide Letters ◽

10.2174/0929866527666200613220245 ◽

2020 ◽

Vol 27 ◽

Author(s):

Sheetal Uppal ◽

Mohd. Asim Khan ◽

Suman Kundu

Keyword(s):

Molten Globule ◽

Life Forms ◽

Model System ◽

Heme Pocket ◽

The Past ◽

Specific Manner ◽

Delivery Vehicles ◽

The Stability ◽

Key Residues ◽

Synechocystis Sp

Aims: The aim of our study is to understand the biophysical traits that govern the stability and folding of Synechocystis hemoglobin, a unique cyanobacterial globin that displays unusual traits not observed in any of the other globins discovered so far. Background: For the past few decades, classical hemoglobins such as vertebrate hemoglobin and myoglobin have been extensively studied to unravel the stability and folding mechanisms of hemoglobins. However, the expanding wealth of hemoglobins identified in all life forms with novel properties, like heme coordination chemistry and globin fold, have added complexity and challenges to the understanding of hemoglobin stability, which has not been adequately addressed. Here, we explored the unique truncated and hexacoordinate hemoglobin from the freshwater cyanobacterium Synechocystis sp. PCC 6803 known as “Synechocystis hemoglobin (SynHb)”. The “three histidines” linkages to heme are novel to this cyanobacterial hemoglobin. Objective: Mutational studies were employed to decipher the residues within the heme pocket that dictate the stability and folding of SynHb. Methods: Site-directed mutants of SynHb were generated and analyzed using a repertoire of spectroscopic and calorimetric tools. Result: The results revealed that the heme was stably associated to the protein under all denaturing conditions with His117 playing the anchoring role. The studies also highlighted the possibility of existence of a “molten globule” like intermediate at acidic pH in this exceptionally thermostable globin. His117 and other key residues in the heme pocket play an indispensable role in imparting significant polypeptide stability. Conclusion: Synechocystis hemoglobin presents an important model system for investigations of protein folding and stability in general. The heme pocket residues influenced the folding and stability of SynHb in a very subtle and specific manner and may have been optimized to make this Hb the most stable known as of date. Other: The knowledge gained hereby about the influence of heme pocket amino acid side chains on stability and expression is currently being utilized to improve the stability of recombinant human Hbs for efficient use as oxygen delivery vehicles.

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Biochemical study of fibrinolytic protease from Euphausia superba possessing multifunctional serine protease activity

Protein and Peptide Letters ◽

10.2174/0929866527666201112123714 ◽

2020 ◽

Vol 27 ◽

Author(s):

Guo-Ying Qian ◽

Gyutae Lim ◽

Shang-Jun Yin ◽

Jun-Mo Yang ◽

Jinhyuk Lee ◽

...

Keyword(s):

Defense Mechanisms ◽

Fluorescence Spectra ◽

Biochemical Study ◽

Euphausia Superba ◽

Md Simulations ◽

Time Interval ◽

Serine Residue ◽

Catalytic Function ◽

Serine Protease Activity ◽

Key Residues

Background: Background: Fibrinolytic protease from Euphausia superba (EFP) was isolated. Objective: Biochemical distinctions, regulation of the catalytic function, and the key residues of EFP were investigated. Methods: The serial inhibition kinetic evaluations coupled with measurements of fluorescence spectra in the presence of 4- (2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF) was conducted. The computational molecular dynamics (MD) simulations were also applied for a comparative study. Results: The enzyme behaved as a monomeric protein with a molecular mass of about 28.6 kD with Km BApNA = 0.629 ± 0.02 mM and kcat/Km BApNA = 7.08 s-1 /mM. The real-time interval measurements revealed that the inactivation was a first-order reaction, with the kinetic processes shifting from a monophase to a biphase. Measurements of fluorescence spectra showed that serine residue modification by AEBSF directly caused conspicuous changes of the tertiary structures and exposed hydrophobic surfaces. Some osmolytes were applied to find protective roles. These results confirmed that the active region of EFP is more flexible than the overall enzyme molecule and serine, as the key residue, is associated with the regional unfolding of EFP in addition to its catalytic role. The MD simulations were supportive to the kinetics data. Conclusion: Our study indicated that EFP has an essential serine residue for its catalyst function and associated folding behaviors. Also, the functional role of osmolytes such as proline and glycine that may play a role in defense mechanisms from environmental adaptation in a krill’s body was suggested.

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Computer-aided structural and molecular insights into the mechanisms by which Pseudouridimycin (PUM) disrupts cleft extension in bacterial RNA polymerase to block DNA entry and exit

Letters in Drug Design & Discovery ◽

10.2174/1570180817999201123165144 ◽

2020 ◽

Vol 17 ◽

Author(s):

Ali H. Rabbad ◽

Fisayo A. Olotu ◽

Mahmoud E. Soliman

Keyword(s):

Rna Polymerase ◽

Bacterial Infections ◽

Entry And Exit ◽

Dynamic Binding ◽

Binding Behavior ◽

Bacterial Rna ◽

Nucleotide Addition ◽

Switch Regions ◽

Cleft Extension ◽

Key Residues

Background: The ability of Pseudouridimycin (PUM) to occupy the nucleotide addition site of bacterial RNA Polymerase (RNAP) underlies its inhibitory potency as previously reported. PUM has gained high research interest as a broad-spectrum nucleoside analog that has demonstrated exciting potentials in treating drug-resistant bacterial infections. Objective: Herein, we identified, for the first time, a novel complementary mechanism by which PUM elicits its inhibitory effects on bacterial RNAP. Methods: The dynamic binding behavior of PUM to bacterial RNAP was studied using various dynamic analyses approaches. Results and Discussion: Findings revealed that in addition to occupying the nucleotide addition site, PUM also interrupts the unimpeded entry and exit of DNA by reducing the mechanistic extension of the RNAP cleft and perturbing the primary conformations of the switch regions. Moreover, PUM binding reduced the distances between key residues in the β and β’ subunits that extend to accommodate the DNA. Conclusion: This study’s findings present structural insights that would contribute to the structure-based design of potent and selective PUM inhibitors.

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A few key residues determine the high redox potential shift in azurin mutants

Organic & Biomolecular Chemistry ◽

10.1039/c5ob01819f ◽

2015 ◽

Vol 13 (45) ◽

pp. 11003-11013 ◽

Cited By ~ 20

Author(s):

Laura Zanetti-Polzi ◽

Carlo A. Bortolotti ◽

Isabella Daidone ◽

Massimiliano Aschi ◽

Andrea Amadei ◽

...

Keyword(s):

Redox Potential ◽

Potential Shift ◽

Key Residues ◽

High Redox Potential

The changes in the redox potential of Azurin upon mutation stem from the effects of a few key residues, including non-mutated ones, rather than being the result of a generalized rearrangement.

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Bioinformatics-based prediction of conformational epitopes for Enterovirus A71 and Coxsackievirus A16

Scientific Reports ◽

10.1038/s41598-021-84891-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Liping Wang ◽

Miao Zhu ◽

Yulu Fang ◽

Hao Rong ◽

Liuying Gao ◽

...

Keyword(s):

Foot And Mouth Disease ◽

Distribution Patterns ◽

Vital Role ◽

Coxsackievirus A16 ◽

Essential Information ◽

Site Distribution ◽

The North ◽

Conformational Epitopes ◽

Enterovirus A71 ◽

Antigenic Evolution

AbstractEnterovirus A71 (EV-A71), Coxsackievirus A16 (CV-A16) and CV-A10 are the major causative agents of hand, foot and mouth disease (HFMD). The conformational epitopes play a vital role in monitoring the antigenic evolution, predicting dominant strains and preparing vaccines. In this study, we employed a Bioinformatics-based algorithm to predict the conformational epitopes of EV-A71 and CV-A16 and compared with that of CV-A10. Prediction results revealed that the distribution patterns of conformational epitopes of EV-A71 and CV-A16 were similar to that of CV-A10 and their epitopes likewise consisted of three sites: site 1 (on the “north rim” of the canyon around the fivefold vertex), site 2 (on the “puff”) and site 3 (one part was in the “knob” and the other was near the threefold vertex). The reported epitopes highly overlapped with our predicted epitopes indicating the predicted results were reliable. These data suggested that three-site distribution pattern may be the basic distribution role of epitopes on the enteroviruses capsids. Our prediction results of EV-A71 and CV-A16 can provide essential information for monitoring the antigenic evolution of enterovirus.

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Structure activity relationships and the binding mode of quinolinone-pyrimidine hybrids as reversal agents of multidrug resistance mediated by P-gp

Scientific Reports ◽

10.1038/s41598-021-96226-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jerónimo Laiolo ◽

Priscila Ailin Lanza ◽

Oscar Parravicini ◽

Cecilia Barbieri ◽

Daniel Insuasty ◽

...

Keyword(s):

Multidrug Resistance ◽

Molecular Complexes ◽

Binding Mode ◽

Reversal Agents ◽

Structure Activity Relationships ◽

Structure Activity ◽

Doxorubicin Toxicity ◽

Key Residues ◽

Nanomolar Range ◽

Experimental Structure

AbstractP-gp-associated multidrug resistance is a major impediment to the success of chemotherapy. With the aim of finding non-toxic and effective P-gp inhibitors, we investigated a panel of quinolin-2-one-pyrimidine hybrids. Among the active compounds, two of them significantly increased intracellular doxorubicin and rhodamine 123 accumulation by inhibiting the efflux mediated by P-gp and restored doxorubicin toxicity at nanomolar range. Structure–activity relationships showed that the number of methoxy groups, an optimal length of the molecule in its extended conformation, and at least one flexible methylene group bridging the quinolinone to the moiety bearing the pyrimidine favored the inhibitory potency of P-gp. The best compounds showed a similar binding pattern and interactions to those of doxorubicin and tariquidar, as revealed by MD and hybrid QM/MM simulations performed with the recent experimental structure of P-gp co-crystallized with paclitaxel. Analysis of the molecular interactions stabilizing the different molecular complexes determined by MD and QTAIM showed that binding to key residues from TMH 4–7 and 12 is required for inhibition.

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C. elegans germ granules require both assembly and localized regulators for mRNA repression

Nature Communications ◽

10.1038/s41467-021-21278-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Scott Takeo Aoki ◽

Tina R. Lynch ◽

Sarah L. Crittenden ◽

Craig A. Bingman ◽

Marvin Wickens ◽

...

Keyword(s):

Liquid Droplet ◽

Scaffolding Protein ◽

P Granules ◽

C Elegans ◽

Cytoplasmic Rna ◽

And Function ◽

Rnp Granules ◽

Key Residues ◽

Reporter Mrna

AbstractCytoplasmic RNA–protein (RNP) granules have diverse biophysical properties, from liquid to solid, and play enigmatic roles in RNA metabolism. Nematode P granules are paradigmatic liquid droplet granules and central to germ cell development. Here we analyze a key P granule scaffolding protein, PGL-1, to investigate the functional relationship between P granule assembly and function. Using a protein–RNA tethering assay, we find that reporter mRNA expression is repressed when recruited to PGL-1. We determine the crystal structure of the PGL-1 N-terminal region to 1.5 Å, discover its dimerization, and identify key residues at the dimer interface. Mutations of those interface residues prevent P granule assembly in vivo, de-repress PGL-1 tethered mRNA, and reduce fertility. Therefore, PGL-1 dimerization lies at the heart of both P granule assembly and function. Finally, we identify the P granule-associated Argonaute WAGO-1 as crucial for repression of PGL-1 tethered mRNA. We conclude that P granule function requires both assembly and localized regulators.

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HPF1 remodels the active site of PARP1 to enable the serine ADP-ribosylation of histones

Nature Communications ◽

10.1038/s41467-021-21302-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Fa-Hui Sun ◽

Peng Zhao ◽

Nan Zhang ◽

Lu-Lu Kong ◽

Catherine C. L. Wong ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Active Site ◽

Negative Charge ◽

Structural Data ◽

Dna Breaks ◽

Adp Ribosylation ◽

Local Conformation ◽

Key Residues ◽

Structural Insights

AbstractUpon binding to DNA breaks, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other factors to initiate DNA repair. Serine is the major residue for ADP-ribosylation upon DNA damage, which strictly depends on HPF1. Here, we report the crystal structures of human HPF1/PARP1-CAT ΔHD complex at 1.98 Å resolution, and mouse and human HPF1 at 1.71 Å and 1.57 Å resolution, respectively. Our structures and mutagenesis data confirm that the structural insights obtained in a recent HPF1/PARP2 study by Suskiewicz et al. apply to PARP1. Moreover, we quantitatively characterize the key residues necessary for HPF1/PARP1 binding. Our data show that through salt-bridging to Glu284/Asp286, Arg239 positions Glu284 to catalyze serine ADP-ribosylation, maintains the local conformation of HPF1 to limit PARP1 automodification, and facilitates HPF1/PARP1 binding by neutralizing the negative charge of Glu284. These findings, along with the high-resolution structural data, may facilitate drug discovery targeting PARP1.

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Molecular epidemiology of coxsackievirus A16 circulating in children in Beijing, China from 2010 to 2019

World Journal of Pediatrics ◽

10.1007/s12519-021-00451-y ◽

2021 ◽

Author(s):

Ya-Fang Hu ◽

Li-Ping Jia ◽

Fang-Yuan Yu ◽

Li-Ying Liu ◽

Qin-Wei Song ◽

...

Keyword(s):

Molecular Epidemiology ◽

Evolutionary Rate ◽

Foot And Mouth Disease ◽

Recent Common Ancestor ◽

Rt Pcr ◽

Coxsackievirus A16 ◽

Most Recent Common Ancestor ◽

Etiological Agents ◽

High Level ◽

And Control

Abstract Background Coxsackievirus A16 (CVA16) is one of the major etiological agents of hand, foot and mouth disease (HFMD). This study aimed to investigate the molecular epidemiology and evolutionary characteristics of CVA16. Methods Throat swabs were collected from children with HFMD and suspected HFMD during 2010–2019. Enteroviruses (EVs) were detected and typed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and RT-PCR. The genotype, evolutionary rate, the most recent common ancestor, population dynamics and selection pressure of CVA16 were analyzed based on viral protein gene (VP1) by bioinformatics software. Results A total of 4709 throat swabs were screened. EVs were detected in 3180 samples and 814 were CVA16 positive. More than 81% of CVA16-positive children were under 5 years old. The prevalence of CVA16 showed obvious periodic fluctuations with a high level during 2010–2012 followed by an apparent decline during 2013–2017. However, the activities of CVA16 increased gradually during 2018–2019. All the Beijing CVA16 strains belonged to sub-genotype B1, and B1b was the dominant strain. One B1c strain was detected in Beijing for the first time in 2016. The estimated mean evolutionary rate of VP1 gene was 4.49 × 10–3 substitution/site/year. Methionine gradually fixed at site-23 of VP1 since 2012. Two sites were detected under episodic positive selection, one of which (site-223) located in neutralizing linear epitope PEP71. Conclusions The dominant strains of CVA16 belonged to clade B1b and evolved in a fast evolutionary rate during 2010–2019 in Beijing. To provide more favorable data for HFMD prevention and control, it is necessary to keep attention on molecular epidemiological and evolutionary characteristics of CVA16.

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