Location Of Amino Acid Substitution Research Articles

SARS-CoV-2 Point Mutation and Deletion Spectra and Their Association with Different Disease Outcomes.

ABSTRACTMutant spectra of RNA viruses are important to understand viral pathogenesis and response to selective pressures. There is a need to characterize the complexity of mutant spectra in coronaviruses sampled from infected patients. In particular, the possible relationship between SARS-CoV-2 mutant spectrum complexity and disease associations has not been established. In the present study, we report an ultradeep sequencing (UDS) analysis of the mutant spectrum of amplicons from the nsp12 (polymerase)- and spike (S)-coding regions of 30 nasopharyngeal isolates (diagnostic samples) of SARS-CoV-2 of the first COVID-19 pandemic wave (Madrid, Spain, April 2020) classified according to the severity of ensuing COVID-19. Low-frequency mutations and deletions, counted relative to the consensus sequence of the corresponding isolate, were overwhelmingly abundant. We show that the average number of different point mutations, mutations per haplotype, and several diversity indices was significantly higher in SARS-CoV-2 isolated from patients who developed mild disease than in those associated with moderate or severe disease (exitus). No such bias was observed with RNA deletions. Location of amino acid substitutions in the three-dimensional structures of nsp12 (polymerase) and S suggest significant structural or functional effects. Thus, patients who develop mild symptoms may be a richer source of genetic variants of SARS-CoV-2 than patients with moderate or severe COVID-19.IMPORTANCE The study shows that mutant spectra of SARS-CoV-2 from diagnostic samples differ in point mutation abundance and complexity and that significantly larger values were observed in virus from patients who developed mild COVID-19 symptoms. Mutant spectrum complexity is not a uniform trait among isolates. The nature and location of low-frequency amino acid substitutions present in mutant spectra anticipate great potential for phenotypic diversification of SARS-CoV-2.

A sequence embedding method for enzyme optimal condition analysis

BackgroundAn enzyme activity is influenced by the external environment. It is important to have an enzyme remain high activity in a specific condition. A usual way is to first determine the optimal condition of an enzyme by either the gradient test or by tertiary structure, and then to use protein engineering to mutate a wild type enzyme for a higher activity in an expected condition.ResultsIn this paper, we investigate the optimal condition of an enzyme by directly analyzing the sequence. We propose an embedding method to represent the amino acids and the structural information as vectors in the latent space. These vectors contain information about the correlations between amino acids and sites in the aligned amino acid sequences, as well as the correlation with the optimal condition. We crawled and processed the amino acid sequences in the glycoside hydrolase GH11 family, and got 125 amino acid sequences with optimal pH condition. We used probabilistic approximation method to implement the embedding learning method on these samples. Based on these embedding vectors, we design a computational score to determine which one has a better optimal condition for two given amino acid sequences and achieves the accuracy 80% on the test proteins in the same family. We also give the mutation suggestion such that it has a higher activity in an expected environment, which is consistent with the previously professional wet experiments and analysis.ConclusionA new computational method is proposed for the sequence based on the enzyme optimal condition analysis. Compared with the traditional process that involves a lot of wet experiments and requires multiple mutations, this method can give recommendations on the direction and location of amino acid substitution with reference significance for an expected condition in an efficient and effective way.

Architecture and self-assembly of the SARS-CoV-2 nucleocapsid protein.

The COVID‐2019 pandemic is the most severe acute public health threat of the twenty‐first century. To properly address this crisis with both robust testing and novel treatments, we require a deep understanding of the life cycle of the causative agent, the SARS‐CoV‐2 coronavirus. Here, we examine the architecture and self‐assembly properties of the SARS‐CoV‐2 nucleocapsid protein, which packages viral RNA into new virions. We determined a 1.4 Å resolution crystal structure of this protein's N2b domain, revealing a compact, intertwined dimer similar to that of related coronaviruses including SARS‐CoV. While the N2b domain forms a dimer in solution, addition of the C‐terminal spacer B/N3 domain mediates formation of a homotetramer. Using hydrogen‐deuterium exchange mass spectrometry, we find evidence that at least part of this putatively disordered domain is structured, potentially forming an α‐helix that self‐associates and cooperates with the N2b domain to mediate tetramer formation. Finally, we map the locations of amino acid substitutions in the N protein from over 38,000 SARS‐CoV‐2 genome sequences. We find that these substitutions are strongly clustered in the protein's N2a linker domain, and that substitutions within the N1b and N2b domains cluster away from their functional RNA binding and dimerization interfaces. Overall, this work reveals the architecture and self‐assembly properties of a key protein in the SARS‐CoV‐2 life cycle, with implications for both drug design and antibody‐based testing.

Identification of the amino acids associated with the novel ribonuclease activity of cusativin via protein engineering and LC‐MS

Ribonucleases (RNases) are widely used in detecting the location of chemical modification in RNA, an approach commonly referred to as, “RNA modification mapping” [1]. The commonly employed RNases include RNase T1 (guanosine‐specific), U2 (purine‐specific) and A (pyrimidine specific). Recently, uridine‐specific (MC1) [2], cytidine‐specific (cusativin) [3] and a non‐specific RNase U2 [4] were identified as potential tools for RNA modification mapping. Of these, RNase cusativin exhibits novel cleavage behavior where it does not cleave the bond between two consecutive cytidine nucleotides (CpC) [3]. This unique feature of cusativin has encouraged us to probe the nucleobase recognition characteristics of the enzyme binding site, which is composed of B1 and B2 sites. We employed amino acid substitutions (through protein engineering), and liquid chromatography coupled with mass spectrometry (LC‐MS) to analyze the contributions of specific amino acids in the enzyme binding site. The mutant proteins generated by specific amino acid substitutions were investigated for alteration of secondary structure, kinetic changes in enzyme activity, and substrate specificity. While single amino acid substitutions revealed minimal effects on the secondary structure, multiple substitutions resulted in significant changes as revealed by circular dichroism studies. However, the mutant proteins, irrespective of the number of substitutions, exhibited remarkable changes in their kinetic behavior with the tested RNA substrate. The LC‐MS analysis of enzymatic digestion products of RNA indicated altered nucleobase specificity depending on the location of amino acid substitutions. While mutation at one location (V99L) behaved more or less like wild type protein, mutation at different position (N97S) but within same B2 binding site pocket revealed generation of digestion products indicative of CpC bond cleavage. However, the N97S mutant also exhibited digestion products that suggest cleavage of RNA at 3′‐end of guanosine. Another mutant protein with amino acid substitutions in both B1 (R69T) and B2 (V99L and T100R) sites also exhibited significant cleavage of CpC bond. But this behavior is also accompanied with more or less equal affinity towards C and G for RNA cleavage. The implications of this preliminary experimental data on the role of specific amino acids in nucleobase recognition of RNA substrate will be discussed.Support or Funding InformationFinancial support of this work was provided by the National Institutes of Health (GM58843), and the University of Cincinnati.

The Spatial Location of Single Amino Acid Substitutions in Proteins of Cold-Adapted Influenza B Viruses and Their Impact upon Cold Adaptation

This review summarizes the locations of unique amino acid substitutions in proteins of cold-adapted influenza B viruses, master donor viruses of live attenuated influenza vaccines: B/Ann Arbor/1/66ca, B/USSR/60/69, B/Leningrad/14/17/55, B/Victoria/2/63/87, and B/Vienna/1/99ca. The analysis is based on data on the viral sequences and information on the functional organization of internal proteins of the influenza B virus. A description is given of the main features of the structure of internal proteins from influenza B virus and a recent understanding of the functioning of their domains. We describe 35 substitutions in proteins of the ribonucleoprotein complex [PB1 (3), PB2 (10), PA (11), NP (11)] of cold-adapted influenza B viruses. In total, there are 44 amino acid positions that are considered to be associated with cold adaptation. Protein domains involved in protein–protein interactions are the most susceptible to changes. In the polymerase complex, the largest number of replacements were found in C-terminal domains of PA and PB2 proteins, forming the external parts of the polymerase complex structure and providing interaction among subunits, with cellular factors, and with the nucleoprotein (NP). Substitutions in the NP are located in the areas responsible for its oligomerization (flexible N-terminal fragment), as well as for interaction with the polymerase complex (the outer surface of the NP head and body domains). All the cold-adapted viruses had changes in the seventh segment of the genome, leading to amino acid substitutions in the matrix protein or BM2 proton channel. An analysis of the location of amino acid substitutions allows it to be theorized that an important role is played by the internal gene constellation in attenuation of cold-adapted viruses.

Low frequency of amino acid alterations following therapeutic immunization with HIV-1 Gag p24-like peptides

In chronic HIV-1 infection, the efficacy of a cellular immune response may decline if the virus evolves into variants not recognized by host immune response. The aim of this study was to explore HIV-1 immune escape mutations imposed by therapeutic immunization by investigating sequence variations that might contribute to relapse of viremia in an immunized, HIV-1-infected cohort. We have previously immunized HIV-1-infected individuals on antiretroviral therapy (ART) with a mixture of four short peptides (Vacc-4x) corresponding to p24. Long postimmunization periods without ART allowed longitudinal sequence studies of regions corresponding to Vacc-4x. Regions of gag p24 including the locations of the Vacc-4x peptides, were sequenced before start of ART, and after postimmunization ART stop (n = 27). Rates and locations of amino acid substitutions were then related to peptide-specific T-cell responses and known epitopes presented by Vacc-4x. The overall rate of amino acid substitutions was low during 35 months (median) of postimmunization viremia, with similar rates of substitution within the regions corresponding to Vacc-4x peptides and other p24 regions despite durable Vacc-4x-specific T-cell responses. Postimmunization amino acid substitutions within Vacc-4x regions were detected in only six patients, and only two of them had measurable T-cell responses against the relevant peptide. The results suggested low prevalence of evolutionary selection of p24 despite new and long-lasting Vacc-4x-specific T-cell responses. The conserved Vacc-4x sequences might therefore be particularly suited for therapeutic immunization. Generally, studies of longitudinal sequence variations after immunization might be valuable when assessing immune escape in HIV vaccine trials.

Identification of Non-Permissive HLA Allele Mismatch Combinations and Amino Acid Substitution Responsible for Acute Graft-Versus-Host Disease in Unrelated Allogeneic Bone Marrow Transplantation.

Background: In the allogenic hematopoietic stem cell transplantation from unrelated donors (UR-HSCT), an effect of HLA locus mismatch in allele level on clinical outcome has been clarified. However, the effect of each HLA allele mismatch combinations is little known, and its molecular mechanism to induce acute graft versus host disease (aGVHD) remained to be elucidated.Methods: Consecutive 4866 patients transplanted with T cell replete marrow from a serologically HLA-A, -B and -DR antigen-matched donor through Japan Marrow Donor Program were registered in this cohort study. All HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 alleles were retrospectively typed in 2171 pairs, and partly in the other pairs. The impact of the HLA allele mismatch combinations in HLA six loci and amino acid substitution positions in HLA-C and HLA-DPB1 locus on aGVHD and survival was analyzed using a multivariable Cox regression model.Results: Significant high-risk HLA allele mismatch combinations compared with match for severe aGVHD were identified; four in HLA-A allele (donor A*0206- patient A*0201 (n=108) hazard ratio (HR): 1.77, A*0206-A*0207 (n=20) : 3.24, A*2601-A*2603 (n=32): 1.96, A*2602-A*2601 (n=24): 2.18), six in HLA-B (B*1507-B*1501 (n=14): 2.95, B*4002-B*4003 (n=14): 2.44, B*4002-B*4006 (n=85): 1.69, B*4003-B*4006 (n=7): 3.85, B*4006-B*4002 (n=60): 1.62, B*4403-B*4402 (n=4) : 5.78), 11 in HLA-C, six in HLA-DRB1, zero in HLA-DQB1 and two in HLA-DPB1. Amino acid substitutions of position 80 of HLA-C at which donor had Asp80 and patient Lys80 (Asp80-Lys80) and Ser77-Asp77 were first elucidated as significant risk factors for severe aGVHD. These two amino acid substitutions were completely linked, and HR for severe aGVHD was 1.49 (1.01–2.21). As position 80 is ligand for NK cell receptor KIR2DL as a result, further analysis was performed in the KIR2DL ligand match in the GVH vector population. Notably, particular amino acid substitution at positions 95, 156 and 163 of HLA-C was a significant risk factor for severe aGVHD. HR of Leu95-Ile95, Arg156-Leu156, Leu156-Trp156, Trp156-Leu156 and Thr163-Leu163 substitutions were 1.74 (95%CI: 1.06–2.86), 2.10 (1.16–3.81), 5.22 (1.65–16.4), 4.64 (1.04–20.7) and 1.82 (1.11–2.99), respectively. The amplitude of hydropathy scales were 0.7, 8.3, 4.7, 4.7 and 4.7, respectively. Amino acid substitutions of any other positions of HLA-C were not significant risk factors. When analyzing the location of amino acid substitution in HLA-C, residues located in the T-cell receptor contact have marginal impact on severe aGVHD (HR: 1.45 trend P=0.096), and no other locations were significant. In HLA-DPB1 mismatch combinations, there was no obvious tendency to associate the positons of amino acid substitutions with severe aGVHD and grades 2–4 aGVHD.Conclusion: These findings provide evidences to elucidate the mechanism of aGVHD on the base of HLA molecule. Furthermore, the identification of non-permissive and possible permissive mismatch would be beneficial for the selection of suitable donor and international donor exchange for UR-HSCT.

Identification of residues on CD40 and its ligand which are critical for the receptor-ligand interaction.

Interactions between gp39 (CD40L, TRAP, T-BAM) on activated T cells and CD40 on antigen-presenting cells play an important role in regulating antibody production by B cells, cytokine production by monocytes, and other immune responses which require T cell "help". Using structure-based sequence alignments, a molecular model of gp39, site-directed mutagenesis, and receptor-ligand binding assays, we have identified CD40 and gp39 surface residues which are important for receptor-ligand binding. Binding studies with CD40 or gp39 proteins containing single and double amino acid substitutions showed that CD40 residues Y82, D84, and N86 are involved in gp39 binding, while gp39 residues K143 and Y145 are important for CD40 binding. Analysis of the location of amino acid substitutions in the naturally occurring gp39 mutants expressed by the X-linked hyper-IgM (X-HIM) patients studied to date indicated the E129/G substitution found in the S128/R-E129/G double mutant affects a solvent-accessible residue which might participate in CD40/gp39 binding. Binding studies with E129/G and E129/A gp39 point mutants showed that this residue does not contribute directly to CD40/gp39 binding but that its substitution with a glycine disrupts the gp39 structure. Comparison of the gp39 and CD40 residues involved in receptor-ligand contacts with those previously identified as playing an important role in TNF-beta/TNFR binding suggests that some of the identified residues from contacts similar to those found in the TNF-beta/TNFR while others are unique to the CD40-gp39 interaction.

Locations of amino acid substitutions in bacteriophage T4 tsL56 DNA polymerase predict an N-terminal exonuclease domain.

The amino acid substitutions responsible for the temperature-sensitive (ts) and mutator phenotypes of the classical bacteriophage T4 DNA polymerase mutant tsL56 were determined. tsL56 DNA polymerase has two mutations in the 5' end of the DNA polymerase gene (g43) that produce two amino acid substitutions: codon 89, alanine to threonine, and codon 363, aspartate to asparagine. Both mutations are required for the strong ts and mutator phenotypes. The increased error rate of the tsL56 DNA polymerase is due to a reduction in 3'----5' exonuclease activity relative to polymerase activity (N. Muzyczka, R. L. Poland, and M. J. Bessman, J. Biol. Chem. 247:7116-7122, 1972). Thus, the locations of the tsL56 mutations suggest that the 3'----5' exonuclease domain resides in the N-terminal region. Several other ts DNA polymerase mutant strains isolated with tsL56 also have mutator or antimutator phenotypes. The nucleotide changes in these important mutant strains were also determined. This mutant collection, combined with collections of g43 amber mutants and mutants selected on the basis of a strong mutator phenotype (L. J. Reha-Krantz, J. Mol. Biol. 202:711-724, 1988), contains nearly 70 different DNA polymerase mutations. The numerous T4 DNA polymerase mutations are valuable for DNA polymerase structure-function and fidelity studies.

Amino acid substitutions that reduce the affinity of penicillin-binding protein 3 of Escherichia coli for cephalexin.

The location of amino acid substitutions that allow an enzyme to discriminate between the binding of its normal substrate and a substrate analogue may be used to identify regions of the polypeptide that fold to form the substrate binding site. We have isolated a large number of cephalexin-resistant mutants of Escherichia coli in which the resistance is due to the production of altered forms of penicillin-binding protein 3 that have reduced affinity for the antibiotic. Using three mutagens, and a variety of selection procedures, we obtained only five classes of mutants which could be distinguished by their patterns of cross-resistance to other beta-lactam antibiotics. The three classes of mutants that showed the highest levels of resistance to cephalexin were cross-resistant to several other cephalosporins but not to penicillins or to the monobactam, aztreonam. The penicillin-binding protein 3 gene from 46 independent mutants was cloned and sequenced. Each member of the five classes of cephalexin-resistant mutants had the same amino acid substitution in penicillin-binding protein 3. The mutants that showed the highest levels of resistance to cephalexin had alterations of either Thr-308 to Pro, Val-344 to Gly, or Asn-361 to Ser. The Thr-308 to Pro substitution had occurred within the beta-lactam-binding site since the adjacent residue (Ser-307) has been shown to be acylated by benzylpenicillin. The Asn-361 to Ser change occurred in a region that showed substantial similarity to regions in both penicillin-binding protein 1A and 1B and may also define a residue that is located within the beta-lactam-binding site in the three-dimensional structure of the enzyme.