Capsid Assembly is Regulated by Amino Acid Residues Asparagine 47 and 48 in The VP2 Protein of Porcine Parvovirus

Abstract Background: Porcine parvovirus (PPV) is a major cause of reproductive failure in swine, and has caused huge losses throughout the world. Viral protein 2 (VP2) of PPV is a major structural protein that can self-assemble into virus-like particles (VLP) with hemagglutination (HA) activity. In order to identify the essential residues involved in the mechanism of capsid assembly and to further understand the function of HA, we analyzed a series of deletion mutants and site-directed mutations within the N-terminal of VP2 in the Escherichia coli (E. coli) system. Results: Our results showed that deletion of first 47 amino acids from the N-terminal of VP2 protein did not affect capsid assembly, and further truncation to residue 48 Asparagine (Asn, N) caused detrimental effects. Site-directed mutagenesis experiments demonstrated that residue 47Asn reduced the assembly efficiency of PPV VLP, while residue 48Asn destroyed the stability, hemagglutination, and self-assembly characteristics of the PPV VP2 protein. These findings indicated that the residues 47Asn and 48Asn are important amino acid sites to capsid assembly and HA activity. Results from Native PAGE inferred that macromolecular polymers were critical intermediates of the VP2 protein during the capsid assembly process. Site-directed mutation at 48Asn did not affect the association of monomers to form into oligomers, but destroyed the ability of oligomers to assemble into macromolecular particles, influencing both capsid assembly and HA activity. Conclusions: These results demonstrated that PPV capsid assembly is a complex process that is regulated by amino acids 47Asn and 48Asn, which are located at the N-terminal of VP2 and closely related to the association of macromolecular particles. Our findings provide valuable information on the mechanisms of PPV capsid assembly and the possibility of chimeric VLP vaccine development by replacing as much as 47 amino acids at the N-terminal of VP2 protein.

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Capsid assembly is regulated by amino acid residues asparagine 47 and 48 in the VP2 protein of porcine parvovirus

Veterinary Microbiology ◽

10.1016/j.vetmic.2020.108974 ◽

2020 ◽

pp. 108974

Author(s):

Jucai Wang ◽

Yunchao Liu ◽

Yumei Chen ◽

Teng Zhang ◽

Aiping Wang ◽

...

Keyword(s):

Amino Acid ◽

Porcine Parvovirus ◽

Capsid Assembly ◽

Amino Acid Residues ◽

Vp2 Protein

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Site-directed mutagenesis at aspartate and glutamate residues of xylanase from Bacillus pumilus

Biochemical Journal ◽

10.1042/bj2880117 ◽

1992 ◽

Vol 288 (1) ◽

pp. 117-121 ◽

Cited By ~ 48

Author(s):

E P Ko ◽

H Akatsuka ◽

H Moriyama ◽

A Shinmyo ◽

Y Hata ◽

...

Keyword(s):

Amino Acids ◽

Reaction Mechanism ◽

Amino Acid ◽

Bacillus Pumilus ◽

Specific Activity ◽

Sequence Similarity ◽

Site Directed Mutagenesis ◽

Dimensional Structure ◽

Amino Acid Sequence Similarity ◽

Directed Mutagenesis

To elucidate the reaction mechanism of xylanase, the identification of amino acids essential for its catalysis is of importance. Studies have indicated the possibility that the reaction mechanism of xylanase is similar to that of hen's egg lysozyme, which involves acidic amino acid residues. On the basis of this assumption, together with the three-dimensional structure of Bacillus pumilus xylanase and its amino acid sequence similarity to other xylanases of different origins, three acidic amino acids, namely Asp-21, Glu-93 and Glu-182, were selected for site-directed mutagenesis. The Asp residue was altered to either Ser or Glu, and the Glu residues to Ser or Asp. The purified mutant xylanases D21E, D21S, E93D, E93S, E182D and E182S showed single protein bands of about 26 kDa on SDS/PAGE. C.d. spectra of these mutant enzymes show no effect on the secondary structure of xylanase, except that of D21E, which shows a little variation. Furthermore, mutations of Glu-93 and Glu-182 resulted in a drastic decrease in the specific activity of xylanase as compared with mutation of Asp-21. On the basis of these results we propose that Glu-93 and Glu-182 are the best candidates for the essential catalytic residues of xylanase.

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Whole Genome Sequence Analysis and Homology Modelling of a 3C Like Peptidase and a Non-Structural Protein 3 of the SARS-CoV-2 Shows Protein Ligand Interaction with an Aza-Peptide and a Noncovalent Lead Inhibitor with Possible Antiviral Properties

10.26434/chemrxiv.11846943.v7 ◽

2020 ◽

Author(s):

Arun Shanker ◽

Divya Bhanu ◽

Anjani Alluri

Keyword(s):

Amino Acids ◽

Binding Sites ◽

Tertiary Structure ◽

Vaccine Development ◽

Homology Modelling ◽

Whole Genome Sequence ◽

Whole Genome ◽

Structural Protein ◽

Ligand Interaction ◽

Percent Identity

The family of viruses belonging to Coronaviridae mainly consist of virulent pathogens that have a zoonotic property, Severe Acute Respiratory Syndrome (SARS-CoV) and Middle East Respiratory Syndrome (MERS-CoV) of this family have emerged before and now the SARS-CoV-2 has emerged in China. Characterization of spike glycoproteins, polyproteins and other viral proteins from viruses are important for vaccine development. Homology modelling of these proteins with known templates offers the opportunity to discover ligand binding sites and explore the possible antiviral properties of these protein ligand complexes. Any information emerging from these protein models can be used for vaccine development. In this study we did a complete bioinformatic analysis, sequence alignment, comparison of multiple sequences and homology modelling of the <a>SARS-CoV-2 </a>whole genome sequences, the spike protein and the polyproteins for homology with known proteins, we also analysed receptor binding sites in these models for possible binding with ligands that exhibit antiviral properties. Our results showed that the tertiary structure of the polyprotein isolate SARS-CoV-2_HKU-SZ-001_2020 had 98.94 percent identity with SARS-Coronavirus NSP12 bound to NSP7 and NSP8 co-factors. <a>Our results indicate that a part of the viral genome </a><a>(residues 3268 -3573 in Frame 2 with 306 amino acids) of the SARS-CoV-2 virus isolate Wuhan-Hu-1 (Genbank Accession Number MN908947.3) </a>when modelled with template 2a5i of the PDB database had 96 percent identity with a 3C like peptidase of SARS-CoV which has ability to bind with Aza-Peptide Epoxide (APE) which is known for irreversible inhibition of SARS-CoV main peptidase. The part of the genome (residues 1568-1882 in Frame 2 with 315 amino acids) when modelled with template 3e9s of the PDB database had 82 percent identity with a papain-like protease/deubiquitinase which when complexed with ligand GRL0617 acts as inhibitor which can block SARS-CoV replication. The regions studied was conserved in more than 90 genomes of SARS-CoV-2. It is possible that these viral inhibiters can be used for vaccine development for the SARS-CoV-2.

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Identification of amino acids essential for DNA binding and dimerization in p67SRF: implications for a novel DNA-binding motif

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.123-132.1993 ◽

1993 ◽

Vol 13 (1) ◽

pp. 123-132

Author(s):

A D Sharrocks ◽

H Gille ◽

P E Shaw

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Dna Binding ◽

Transcriptional Activation ◽

Serum Response Factor ◽

Alpha Helix ◽

Site Directed Mutagenesis ◽

Binding Motif ◽

Amino Acid Peptide ◽

Serum Response

The serum response factor (p67SRF) binds to a palindromic sequence in the c-fos serum response element (SRE). A second protein, p62TCF binds in conjunction with p67SRF to form a ternary complex, and it is through this complex that growth factor-induced transcriptional activation of c-fos is thought to take place. A 90-amino-acid peptide, coreSRF, is capable for dimerizing, binding DNA, and recruiting p62TCF. By using extensive site-directed mutagenesis we have investigated the role of individual coreSRF amino acids in DNA binding. Mutant phenotypes were defined by gel retardation and cross-linking analyses. Our results have identified residues essential for either DNA binding or dimerization. Three essential basic amino acids whose conservative mutation severely reduced DNA binding were identified. Evidence which is consistent with these residues being on the face of a DNA binding alpha-helix is presented. A phenylalanine residue and a hexameric hydrophobic box are identified as essential for dimerization. The amino acid phasing is consistent with the dimerization interface being presented as a continuous region on a beta-strand. A putative second alpha-helix acts as a linker between these two regions. This study indicates that p67SRF is a member of a protein family which, in common with many DNA binding proteins, utilize an alpha-helix for DNA binding. However, this alpha-helix is contained within a novel domain structure.

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Alteration of amino acids in VP2 of very virulent infectious bursal disease virus results in tissue culture adaptation and attenuation in chickens

Journal of General Virology ◽

10.1099/0022-1317-83-1-121 ◽

2002 ◽

Vol 83 (1) ◽

pp. 121-129 ◽

Cited By ~ 95

Author(s):

A. A. W. M. van Loon ◽

N. de Haas ◽

I. Zeyda ◽

E. Mundt

Keyword(s):

Amino Acids ◽

Tissue Culture ◽

Amino Acid ◽

Disease Virus ◽

Infectious Bursal Disease Virus ◽

Site Directed Mutagenesis ◽

Infectious Bursal Disease ◽

Single Amino Acid ◽

Culture Adaptation ◽

Bursal Disease

Reverse genetics technology offers the possibility to study the influence of particular amino acids of infectious bursal disease virus (IBDV) on adaptation to tissue culture. Genomic segments A and B of the very virulent (vv) IBDV field strain UK661 were completely cloned and sequenced, and the strain was rescued from full-length cDNA copies of both segments (UK661rev). Using site-directed mutagenesis, alteration of a single amino acid in the segment A-encoded VP2 (A284T) resulted in a limited capacity of UK661 to replicate in tissue culture. Additional alteration of a second amino acid (Q253H) increased replication efficiency in tissue culture. The second mutant (UK661-Q253H-A284T) was used to infect chickens and results were compared with UK661 and UK661rev. Whereas UK661 and UK661rev induced 100% morbidity and 50–80% mortality, UK661-Q253H-A284T proved to be strikingly attenuated, producing neither morbidity nor mortality. Moreover, UK661-Q253H-A284T-infected animals were protected from challenge infection. Thus, alteration of two specific amino acids in the VP2 region of IBDV resulted in tissue culture adaptation and attenuation in chickens of vvIBDV. The data demonstrate that VP2 plays a decisive role in pathogenicity of IBDV.

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Probing the Molecular Determinant for the Catalytic Efficiency of l-Arabinose Isomerase from Bacillus licheniformis

Applied and Environmental Microbiology ◽

10.1128/aem.02254-09 ◽

2010 ◽

Vol 76 (5) ◽

pp. 1653-1660 ◽

Cited By ~ 23

Author(s):

Ponnandy Prabhu ◽

Marimuthu Jeya ◽

Jung-Kul Lee

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Bacillus Licheniformis ◽

Catalytic Efficiency ◽

Homology Model ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Dynamics Simulation ◽

High Turnover ◽

Arabinose Isomerase

ABSTRACT Bacillus licheniformis l-arabinose isomerase (l-AI) is distinguished from other l-AIs by its high degree of substrate specificity for l-arabinose and its high turnover rate. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis to alter individual screened residues, was used to study the molecular determinants for the catalytic efficiency of B. licheniformis l-AI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type B. licheniformis l-AI was identified as an important residue affecting the catalytic efficiency of B. licheniformis l-AI. Further insights into the function of residue Y333 were obtained by replacing it with other aromatic, nonpolar hydrophobic amino acids or polar amino acids. Replacing Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward l-arabinose. In contrast, the activities of mutants containing a hydrophobic amino acid (Ala, Val, or Leu) at position 333 decreased as the size of the hydrophobic side chain of the amino acid decreased. However, mutants containing hydrophilic and charged amino acids, such as Asp, Glu, and Lys, showed almost no activity with l-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in the catalytic efficiency of B. licheniformis l-AI.

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Intestinal peptidases form functional complexes with the neutral amino acid transporter B0AT1

Biochemical Journal ◽

10.1042/bj20120307 ◽

2012 ◽

Vol 446 (1) ◽

pp. 135-148 ◽

Cited By ~ 40

Author(s):

Stephen J. Fairweather ◽

Angelika Bröer ◽

Megan L. O'Mara ◽

Stefan Bröer

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Brush Border ◽

Brush Border Membrane ◽

Amino Acid Transporter ◽

The Body ◽

Site Directed Mutagenesis ◽

Substrate Affinity ◽

Xenopus Laevis Oocytes ◽

Acid Transporter

The brush-border membrane of the small intestine and kidney proximal tubule are the major sites for the absorption and re-absorption of nutrients in the body respectively. Transport of amino acids is mediated through the action of numerous secondary active transporters. In the mouse, neutral amino acids are transported by B0AT1 [broad neutral (0) amino acid transporter 1; SLC6A19 (solute carrier family 6 member 19)] in the intestine and by B0AT1 and B0AT3 (SLC6A18) in the kidney. Immunoprecipitation and Blue native electrophoresis of intestinal brush-border membrane proteins revealed that B0AT1 forms complexes with two peptidases, APN (aminopeptidase N/CD13) and ACE2 (angiotensin-converting enzyme 2). Physiological characterization of B0AT1 expressed together with these peptidases in Xenopus laevis oocytes revealed that APN increased the substrate affinity of the transporter up to 2.5-fold and also increased its surface expression (Vmax). Peptide competition experiments, in silico modelling and site-directed mutagenesis of APN suggest that the catalytic site of the peptidase is involved in the observed changes of B0AT1 apparent substrate affinity, possibly by increasing the local substrate concentration. These results provide evidence for the existence of B0AT1-containing digestive complexes in the brush-border membrane, interacting differentially with various peptidases, and responding to the dynamic needs of nutrient absorption in the intestine and kidney.

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Genomic diversity of SARS-CoV-2 can be accelerated by a mutation in the nsp14 gene

10.1101/2020.12.23.424231 ◽

2020 ◽

Author(s):

Kosuke Takada ◽

Mahoko Takahashi Ueda ◽

Tokiko Watanabe ◽

So Nakagawa

Keyword(s):

Amino Acid ◽

Substitution Rate ◽

Nucleotide Substitution ◽

Acid Sites ◽

Genomic Diversity ◽

Nucleotide Substitution Rate ◽

Structural Protein ◽

Wild Type ◽

Gamma Delta ◽

Evolutionarily Conserved

AbstractNucleotide substitution rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is relatively low compared to the other RNA viruses because coronaviruses including SARS-CoV-2 encode non-structural protein 14 (nsp14) that is an error-correcting exonuclease protein. In this study, to understand genome evolution of SARS-CoV-2 in the current pandemic, we examined mutations of SARS-CoV-2 nsp14 which could inhibit its error-correcting function. First, to obtain functionally important sites of nsp14, we examined 62 representative coronaviruses belonging to alpha, beta, gamma, delta, and unclassified coronaviruses. As a result, 99 out of 527 amino acid sites of nsp14 were evolutionarily conserved. We then examined nsp14 sequences obtained from 28,082 SARS-CoV-2 genomes and identified 6 amino acid changes in nsp14 mutants that were not detected in the 62 representative coronaviruses. We examined genome substitution rates of these mutants and found that an nsp14 mutant with a proline to leucine change at position 203 (P203L) showed a higher substitution rate (35.9 substitutions/year) than SARS-CoV-2 possessing wild-type nsp14 (19.8 substitutions/year). We confirmed that the substitution rate of the P203L is significantly higher than those of other variants containing mutations in structural proteins. Although the number of SARS-CoV-2 variants containing P203L mutation of nsp14 is limited (26), these mutants appeared at least 10 times independently in the current pandemic. These results indicated that the molecular function of nsp14 is important for survival of various coronaviruses including SARS-CoV-2 and that some mutations such as P203L of nsp14 inhibiting its error-correcting function are removed rapidly due to their deleterious effects.

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Senescence and entrenchment in evolution of amino acid sites

Nature Communications ◽

10.1038/s41467-020-18366-z ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 1

Author(s):

A. V. Stolyarova ◽

E. Nabieva ◽

V. V. Ptushenko ◽

A. V. Favorov ◽

A. V. Popova ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Adaptive Evolution ◽

Protein Evolution ◽

Environmental Changes ◽

Acid Sites ◽

Analytical Modelling ◽

Site Change ◽

A Site ◽

Positively Selected Sites

Abstract Amino acid propensities at a site change in the course of protein evolution. This may happen for two reasons. Changes may be triggered by substitutions at epistatically interacting sites elsewhere in the genome. Alternatively, they may arise due to environmental changes that are external to the genome. Here, we design a framework for distinguishing between these alternatives. Using analytical modelling and simulations, we show that they cause opposite dynamics of the fitness of the allele currently occupying the site: it tends to increase with the time since its origin due to epistasis (“entrenchment”), but to decrease due to random environmental fluctuations (“senescence”). By analysing the genomes of vertebrates and insects, we show that the amino acids originating at negatively selected sites experience strong entrenchment. By contrast, the amino acids originating at positively selected sites experience senescence. We propose that senescence of the current allele is a cause of adaptive evolution.

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Directed evolution and structural analysis of N-carbamoyl-D-amino acid amidohydrolase provide insights into recombinant protein solubility in Escherichia coli

Biochemical Journal ◽

10.1042/bj20061457 ◽

2007 ◽

Vol 402 (3) ◽

pp. 429-437 ◽

Cited By ~ 18

Author(s):

Shimin Jiang ◽

Chunhong Li ◽

Weiwen Zhang ◽

Yuanheng Cai ◽

Yunliu Yang ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Recombinant Protein ◽

Protein Solubility ◽

Acid Sites ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Triple Mutant ◽

E Coli ◽

Acid Amidohydrolase

One of the greatest bottlenecks in producing recombinant proteins in Escherichia coli is that over-expressed target proteins are mostly present in an insoluble form without any biological activity. DCase (N-carbamoyl-D-amino acid amidohydrolase) is an important enzyme involved in semi-synthesis of β-lactam antibiotics in industry. In the present study, in order to determine the amino acid sites responsible for solubility of DCase, error-prone PCR and DNA shuffling techniques were applied to randomly mutate its coding sequence, followed by an efficient screening based on structural complementation. Several mutants of DCase with reduced aggregation were isolated. Solubility tests of these and several other mutants generated by site-directed mutagenesis indicated that three amino acid residues of DCase (Ala18, Tyr30 and Lys34) are involved in its protein solubility. In silico structural modelling analyses suggest further that hydrophilicity and/or negative charge at these three residues may be responsible for the increased solubility of DCase proteins in E. coli. Based on this information, multiple engineering designated mutants were constructed by site-directed mutagenesis, among them a triple mutant A18T/Y30N/K34E (named DCase-M3) could be overexpressed in E. coli and up to 80% of it was soluble. DCase-M3 was purified to homogeneity and a comparative analysis with wild-type DCase demonstrated that DCase-M3 enzyme was similar to the native DCase in terms of its kinetic and thermodynamic properties. The present study provides new insights into recombinant protein solubility in E. coli.

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