Changes In Amino Acid Residues Research Articles

Multiple Roles for Sialylated Glycans in Determining the Cardiopulmonary Tropism of Adeno-Associated Virus 4

Adeno-associated virus 4 (AAV4) is one of the most divergent serotypes among known AAV isolates. Mucins or O-linked sialoglycans have been identified as the primary attachment receptors for AAV4 in vitro. However, little is known about the role(s) played by sialic acid interactions in determining AAV4 tissue tropism in vivo. In the current study, we first characterized two loss-of-function mutants obtained by screening a randomly mutated AAV4 capsid library. Both mutants harbored several amino acid residue changes localized to the 3-fold icosahedral symmetry axes on the AAV4 capsid and displayed low transduction efficiency in vitro. This defect was attributed to decreased cell surface binding as well as uptake of mutant virions. These results were further corroborated by low transgene expression and recovery of mutant viral genomes in cardiac and lung tissue following intravenous administration in mice. Pharmacokinetic analysis revealed rapid clearance of AAV4 mutants from the blood circulation in conjunction with low hemagglutination potential ex vivo. These results were recapitulated with mice pretreated intravenously with sialidase, directly confirming the role of sialic acids in determining AAV4 tissue tropism. Taken together, our results support the notion that blood-borne AAV4 particles interact sequentially with O-linked sialoglycans expressed abundantly on erythrocytes followed by cardiopulmonary tissues and subsequently for viral cell entry.

Kinesins – How the ATPase Cycle Modulates Function

Kinesins are necessary for a wide range of essential cellular functions, and while these molecular motors share common structural motifs, key amino acid residue changes confer unique mechanochemical properties to each kinesin, which in turn specify cellular function. We have defined the ATPase cycle of three processive kinesin motors using presteady‐state kinetic and thermodynamic methodologies. Our results for conventional Kinesin‐1, Kinesin‐5 Eg5, and Kinesin‐7 CENP‐E show that although each is processive and they share key characteristics, there are distinct mechanistic differences that alter the head‐head communication and microtubule interactions. The results for CENP‐E will be presented in comparison to Kinesin‐1 and Kinesin‐5 Eg5. Supported by NIH GM54141.

The structure of Human Microplasmin in Complex with Textilinin-1, an Aprotinin-like Inhibitor from the Australian Brown Snake

Textilinin-1 is a Kunitz-type serine protease inhibitor from Australian brown snake venom. Its ability to potently and specifically inhibit human plasmin (Ki = 0.44 nM) makes it a potential therapeutic drug as a systemic anti-bleeding agent. The crystal structures of the human microplasmin-textilinin-1 and the trypsin-textilinin-1 complexes have been determined to 2.78 Å and 1.64 Å resolution respectively, and show that textilinin-1 binds to trypsin in a canonical mode but to microplasmin in an atypical mode with the catalytic histidine of microplasmin rotated out of the active site. The space vacated by the histidine side-chain in this complex is partially occupied by a water molecule. In the structure of microplasminogen the χ1 dihedral angle of the side-chain of the catalytic histidine is rotated by 67° from its “active” position in the catalytic triad, as exemplified by its location when microplasmin is bound to streptokinase. However, when textilinin-1 binds to microplasmin the χ1 dihedral angle of this amino acid residue changes by −157° (i.e. in the opposite rotation direction compared to microplasminogen). The unusual mode of interaction between textilinin-1 and plasmin explains textilinin-1′s selectivity for human plasmin over plasma kallikrein. This difference can be exploited in future drug design efforts.

Influence of Halide Binding on the Hydrogen Bonding Network in the Active Site of Salinibacter Sensory Rhodopsin I

In nature, organisms are subjected to a variety of environmental stimuli to which they respond and adapt. They can show avoidance or attractive behaviors away from or toward such stimuli in order to survive in the various environments in which they live. One such stimuli is light, to which, for example, the receptor sensory rhodopsin I (SRI) has been found to respond by regulating both negative and positive phototaxis in, e.g., the archaeon Halobacterium salinarum. Interestingly, to date, all organisms having SRI-like proteins live in highly halophilic environments, suggesting that salt significantly influences the properties of SRIs. Taking advantage of the discovery of the highly stable SRI homologue from Salinibacter ruber (SrSRI), which maintains its color even in the absence of salt, the importance of the chloride ion for the color tuning and for the slow M-decay, which is thought to be essential for the phototaxis function of SRIs, has been reported previously [Suzuki, D., et al. (2009) J. Mol. Biol.392, 48-62]. Here the effects of the anion binding on the structure and structural changes of SRI during its photocycle are investigated by means of Fourier transform infrared (FTIR) spectroscopy and electrochemical experiments. Our results reveal that, among other things, the structural change and proton movement of a characteristic amino acid residue, Asp102 in SrSRI, is suppressed by the binding of an anion in its vicinity, both in the K- and M-intermediate. The presence of this anion also effects the extent of chromophore distrotion, and tentative results indicate an influence on the number and/or properties of internal water molecules. In addition, a photoinduced proton transfer could only be observed in the absence of the bound anion. Possible proton movement pathways, including the residues Asp102 and the putative Cl binding site His131, are discussed. In conclusion, the results show that the anion binding to SRI is not only important for the color tuning, and for controlling the photocycle kinetics, but also induces some structural changes which facilitate the observed properties.

Isolation and mutation trend analysis of influenza A virus subtype H9N2 in Egypt

BackgroundAvian influenza virus H9N2 is a panzootic pathogen that affects poultry causing mild to moderate respiratory distress but has been associated with high morbidity and considerable mortality. Interspecies transmission of H9N2 from avian species to mammalian hosts does occur. The virus possesses human virus-like receptor specificity and it can infect humans producing flu-like illness.MethodsRecently, mild influenza like symptoms were detected in H5N1 vaccinated flocks. Influenza A subtype H9N2 was isolated from the infected flock. The virus evolution was investigated by sequencing the viral genes to screen the possible virus recombination. The viral amino acid sequences from the isolated H9N2 strains were compared to other related sequences from the flu data base that were used to assess the robustness of the mutation trend. Changes in the species-associated amino acid residues or those that enabled virulence to mammals were allocated.ResultsPhylogenetic analyses of haemagglutinin and neuraminidase genes showed that the recently isolated Egyptian strain belonged to the H9N2 sub-lineage that prevails in Israel. The six internal segments of the isolated virus were found to be derived from the same sub-lineage with no new evidence of reassortment. The results demonstrated conserved genetic and biological constitution of H9N2 viruses in the Middle East. The recently isolated H9N2 virus from chicken in Egypt possessed amino acids that could enable the virus to replicate in mammals and caused severe disease in domestic chickens.ConclusionThe study highlights the importance of continuous monitoring of the mutations evolved in avian influenza viruses and its impact on virulence to avian species in addition to its importance in the emergence of new strains with the capacity to be a pandemic candidate.

Aggregation and conformational changes of bovine β-lactoglobulin subjected to dynamic high-pressure microfluidization in relation to antigenicity

Our previous research indicated that dynamic high-pressure microfluidization (DHPM) had a significant effect on the antigenicity of β-lactoglobulin (β-LG). In this study, aggregation and conformational changes subjected to DHPM (0.1–160MPa) were investigated in relation to antigenicity. When DHPM pressure increased from 0.1 to 80MPa, disaggregation of β-LG samples and partial unfolding of the molecule were accompanied by an increase in β-LG antigenicity, which was reflected in the decrease of particle size, increase of free sulfhydryl (SH) contents and β-strands contents, and slight exposure of aromatic amino acid residues. At pressures above 80MPa, the reaggregation of β-LG may contribute to the decrease in antigenicity, which was reflected by an increase in particle size, the formation of aggregates, a decrease of in SH and β-strands contents, and slight changes in aromatic amino acid residues. Aggregation and conformational changes of β-LG under DHPM was related to its antigenicity.

A new crystal lattice structure of Helicobacter pylori neutrophil-activating protein (HP-NAP).

A new crystal lattice structure of Helicobacter pylori neutrophil-activating protein (HP-NAP) has been determined in two forms: the native state (Apo) at 2.20 Å resolution and an iron-loaded form (Fe-load) at 2.50 Å resolution. The highly solvated packing of the dodecameric shell is suitable for crystallographic study of the metal ion-uptake pathway. Like other bacterioferritins, HP-NAP forms a spherical dodecamer with 23 symmetry including two kinds of channels. Iron loading causes a series of conformational changes of amino-acid residues (Trp26, Asp52 and Glu56) at the ferroxidase centre.

Predicting the functional impact of protein mutations: application to cancer genomics

As large-scale re-sequencing of genomes reveals many protein mutations, especially in human cancer tissues, prediction of their likely functional impact becomes important practical goal. Here, we introduce a new functional impact score (FIS) for amino acid residue changes using evolutionary conservation patterns. The information in these patterns is derived from aligned families and sub-families of sequence homologs within and between species using combinatorial entropy formalism. The score performs well on a large set of human protein mutations in separating disease-associated variants (∼19 200), assumed to be strongly functional, from common polymorphisms (∼35 600), assumed to be weakly functional (area under the receiver operating characteristic curve of ∼0.86). In cancer, using recurrence, multiplicity and annotation for ∼10 000 mutations in the COSMIC database, the method does well in assigning higher scores to more likely functional mutations (‘drivers’). To guide experimental prioritization, we report a list of about 1000 top human cancer genes frequently mutated in one or more cancer types ranked by likely functional impact; and, an additional 1000 candidate cancer genes with rare but likely functional mutations. In addition, we estimate that at least 5% of cancer-relevant mutations involve switch of function, rather than simply loss or gain of function.

Enhancement of the hydrolysis activity of β-galactosidase from Geobacillus stearothermophilus by saturation mutagenesis

Thermostable β-galactosidase (BgaB) from Geobacillus stearothermophilus is characterized by its thermoactivity in the hydrolysis of lactose to produce lactose-free milk products. However, BgaB has limited activity toward lactose. We established a method for screening evolved mutants with high hydrolysis activity based on prediction of substrate binding sites. Seven amino acid residues were identified as candidates for substrate binding to galactose. To study the hydrolysis activity of these residues, we constructed mutants by site-saturation mutagenesis of these residue sites, and each variant was screened for its hydrolysis activity. The first round of mutagenesis showed that changes in amino acid residues of Arg109, Tyr272, and Glu351 resulted in altered hydrolysis activity, including greater activity toward ortho-nitrophenyl-β-d-galactopyranoside (oNPG). The mutants R109V and R109L displayed changes in the optimum pH from 7.0 to 6.5, and the mutant R109V/L displayed different substrate affinity and catalytic efficiency (kcat/Km). Mutant R109G showed complete loss of BgaB enzymatic activity, suggesting that Arg109 plays a significant role in maintaining hydrolysis activity. The optimum pH of mutant E351R increased from 7.0 to 7.5 and this mutant showed a prominent increase in catalytic efficiency with oNPG and lactose as substrates.

Comparison of the Antibacterial Properties of Phage Endolysins SAL-1 and LysK

In spite of the high degree of amino acid sequence similarity between the newly discovered phage endolysin SAL-1 and the phage endolysin LysK, SAL-1 has an approximately 2-fold-lower MIC against several Staphylococcus aureus strains and higher bacterial cell-wall-hydrolyzing activity than LysK. The amino acid residue change contributing the most to this enhanced enzymatic activity is a change from glutamic acid to glutamine at the 114th residue.

Exon variability of gene encoding glycerol-3-phosphate dehydrogenase ofIxodes ricinusticks

We have previously found apparent differences in Gpdh allele frequences between borrelia infected and uninfected Ixodes ricinus as revealed by native gel electrophoresis of allozyme polymorphisms. The present study deals with the genetic basis of the observed allozyme polymorphism. Multiple sequence alignment of 36 Gpdh open reading frames identified a total of 40 polymorphic nucleotide sites. Of the 40 polymorphic nucleotide sites, 34 were silent (did not result in amino acid residue change), while six were active causing a change in the amino acid chain. All polymorphic amino acid sites were situated within the N-terminal NAD-binding domain, whereas the C-terminal substrate-binding domain was highly conserved. Analysis of the obtained Gpdh sequences and GPDH allozyme polymorphisms for individual ticks pointed to amino acid changes at positions 61 (glycine-to-glutamic acid), 64 (serine-to-cysteine) and 102 (glycine-to-arginine) as a key for differential mobility of GPDH allozymes in an electric field. Our findings are discussed in the context of the molecular basis of I. ricinus host finding behavior.

Single amino acid residue changes in subsite −1 of levansucrase from Zymomonas mobilis 10232 strongly influence the enzyme activities and products

The -1 subsite of bacterial fructansucrases (FSs) (levansucrases and inulosucrases) plays an important role in the substrate recognition, binding and catalysis. Three residues (for example W47, W118 and R193, Zymomonas mobilis levansucrase numbering) at the -1 subsite are completely conserved among FSs. Site-directed mutational analysis showed that the substitutions of the three strictly conserved amino acid residues, W47N, W47H, W118N, W118H, R193K and R193H, significantly decreased enzyme activities and synthesis rates of levan, while the size of the synthesized oligosaccharides had been influenced. These experimental results, combined with 3D structure modeling, lead to our proposal that a single amino acid residue change in subsite -1 of levansucrase can influence change to the size and polarity of the sucrose binding pocket with a concomitant change to substrate binding and catalysis, and thus having an overall influence on the enzyme activities and products.

Elucidation of Metal‐Ion Accumulation Induced by Hydrogen Bonds on Protein Surfaces by Using Porous Lysozyme Crystals Containing RhIIIIons as the Model Surfaces

Metal-ion accumulation on protein surfaces is a crucial step in the initiation of small-metal clusters and the formation of inorganic materials in nature. This event is expected to control the nucleation, growth, and position of the materials. There remain many unknowns, as to how proteins affect the initial process at the atomic level, although multistep assembly processes of the materials formation by both native and model systems have been clarified at the macroscopic level. Herein the cooperative effects of amino acids and hydrogen bonds promoting metal accumulation reactions are clarified by using porous hen egg white lysozyme (HEWL) crystals containing Rh(III) ions, as model protein surfaces for the reactions. The experimental results reveal noteworthy implications for initiation of metal accumulation, which involve highly cooperative dynamics of amino acids and hydrogen bonds: i) Disruption of hydrogen bonds can induce conformational changes of amino-acid residues to capture Rh(III) ions. ii) Water molecules pre-organized by hydrogen bonds can stabilize Rh(III) coordination as aqua ligands. iii) Water molecules participating in hydrogen bonds with amino-acid residues can be replaced by Rh(III) ions to form polynuclear structures with the residues. iv) Rh(III) aqua complexes are retained on amino-acid residues through stabilizing hydrogen bonds even at low pH (approximately 2). These metal-protein interactions including hydrogen bonds may promote native metal accumulation reactions and also may be useful in the preparation of new inorganic materials that incorporate proteins.

Expanding the Substrate Spectrum of Cytochrome P450 2A6 towards Bulky Substrates*

Molecular breeding and biotransformation of cytochrome P450s (P450) are considered two of the emerging areas today. To further expand the substrate spectrum of P450 2A6, N-terminal modification was done to improve its functional expression and consequently, the sensitivity of mutant library screening. Two random libraries screened yielded no gain-of- function mutant, while site-directed mutagenesis generated a new mutant P450 2A6 (N297Q/I300A) with expanded substrate specificity compared with the wild type enzyme and previously reported mutants. The new mutant was able to catalyze the oxidation of 6-benzyloxyindole (6-OBzl-indole) to form colored indigoid dimers as analyzed with mass spectrometry. Both amino acid residue changes were required for the reaction. The results are interpreted in the context of the recently resolved crystal structures of P450 2A6 mutants. The I300A change expands the active-site cavity in both vertical and horizontal directions of the heme plane to be able to fit in substrates of similar nature. The Asn297 is a functionally conserved residue and the N297Q change may be related to hydrogen bond formation.

Adamantane Resistance in Seasonal Human Influenza A Viruses from Calgary, Alberta (January 2007 to August 2008)

The available antivirals for the treatment and prophylaxis of influenza A infections include the adamantanes (amantadine and rimantadine), which are matrix (M2) protein inhibitors, and the neuraminidase inhibitors (oseltamivir and zanamivir). Resistance to the adamantanes is conferred by mutations at amino acid positions 26, 27, 30, 31 or 34 within the M2 protein of influenza A viruses. A significant increase in adamantane resistance has been reported worldwide since 2003, reflected by a similar increase in Canada. The present study reports on the frequency of adamantane resistance in seasonal influenza A viruses in Calgary, Alberta, for the period between January 2007 and August 2008, as an update to the previous report. Positive influenza A samples (221 original patient specimens and 34 isolates obtained by viral culture) were analyzed for changes in the critical amino acid residues of the M2 gene. The amplification and sequencing of regions that confer adamantane resistance directly from RNA extracts of clinical samples (without previous culture) makes this approach a fast and efficient process for monitoring resistance. The results showed that the frequency of resistance varied from 37.5% to 49.2% in circulating influenza A H3N2 virus strains (n=213) between January 2007 and April 2007. The frequency of resistance increased to 100% in May 2007, after which all H3N2 viruses were resistant until the end of the monitoring period. All resistant H3N2 viruses contained the serine to asparagine substitution at amino acid position 31. Resistance was not observed in the H1N1 viruses tested (n=39) within this monitoring period. The level of adamantane resistance in H3N2 viruses continues to remain high since resistant viruses became the prevalent circulating strains in 2005. Recent reports have indicated that the currently circulating swine-origin influenza A H1N1 subtype viruses are adamantane resistant. It is, thus, important to continue to monitor seasonal influenza A viruses for antiviral resistance markers to ensure optimal prophylaxis and treatment.

IgG2m4, an engineered antibody isotype with reduced Fc function

The Fc region of an antibody mediates effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and plays a key role in the in vivo half-life of an antibody. In designing antibody therapeutics, it is sometimes desirable that the antibody has altered Fc-mediated properties. In the case of a "benign blocker" antibody, it is often desirable to diminish or abolish the ADCC and CDC functions while retaining its PK profile. Here, we report a novel engineered IgG isotype, IgG2m4, with reduced Fc functionality. IgG2m4 is based on the IgG2 isotype with four key amino acid residue changes derived from IgG4 (H268Q, V309L, A330S and P331S). An IgG2m4 antibody has an overall reduction in complement and Fcγ receptor binding in in vitro binding analyses while maintaining the normal in vivo serum half-life in rhesus.

A single nucleotide mutation results in loss of enzymatic activity in the hyaluronate lyase gene of Streptococcus pyogenes

Group A streptococci produce a variety of extracellular proteins, many of which are considered to be virulence factors. One of these is hyaluronate lyase (HylA), an enzyme capable of degrading the extracellular matrix of the host as well as the bacterial capsule. The current study examined three genotypes of hylA (full, truncated and deleted). Only isolates containing a full-length gene produced an enzymatically active hyaluronate lyase; however, truncation of the protein was not the reason for loss of activity. A single nucleotide substitution, resulting in an amino acid change at position 199 of the lyase was present in a highly-conserved region of the protein in isolates not producing active enzyme. In serotypes 4 and 22, those producing active enzymes, this residue was an aspartic acid, in serotypes not showing hyaluronate lyase activity, it was a valine. Site-directed mutagenesis indicated the loss of enzymatic activity of the hyaluronate lyase is in part determined by the mutation resulting in an amino acid residue change. This mutation results in an inactive form of the enzyme and is found in the more virulent serotypes of Streptococcus pyogenes, suggesting that hyaluronate lyase could interfere with the disease process, in essence being an anti-virulence factor.

Physicochemical property changes of amino acid residues that accompany missense mutations in SCN1A affect epilepsy phenotype severity

Background:Several different missense mutations in the voltage-gated sodium channel subunit gene SCN1A have been identified in epileptic patients with benign phenotype and patients with severe phenotype. However, the reason why...

Molecular Characterization of Clinical Isolates of Human Immunodeficiency Virus Resistant to the Protease Inhibitor Darunavir

Darunavir is the most recently approved human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) and is active against many HIV type 1 PR variants resistant to earlier-generation PIs. Darunavir shows a high genetic barrier to resistance development, and virus strains with lower sensitivity to darunavir have a higher number of PI resistance-associated mutations than viruses resistant to other PIs. In this work, we have enzymologically and structurally characterized a number of highly mutated clinically derived PRs with high levels of phenotypic resistance to darunavir. With 18 to 21 amino acid residue changes, the PR variants studied in this work are the most highly mutated HIV PR species ever studied by means of enzyme kinetics and X-ray crystallography. The recombinant proteins showed major defects in substrate binding, while the substrate turnover was less affected. Remarkably, the overall catalytic efficiency of the recombinant PRs (5% that of the wild-type enzyme) is still sufficient to support polyprotein processing and particle maturation in the corresponding viruses. The X-ray structures of drug-resistant PRs complexed with darunavir suggest that the impaired inhibitor binding could be explained by change in the PR-inhibitor hydrogen bond pattern in the P2' binding pocket due to a substantial shift of the aminophenyl moiety of the inhibitor. Recombinant virus phenotypic characterization, enzyme kinetics, and X-ray structural analysis thus help to explain darunavir resistance development in HIV-positive patients.

GATA4 and NKX2.5 gene analysis in Chinese Uygur patients with congenital heart disease

Background Congenital heart disease (CHD) is the most common developmental anomaly in newborns. The germline mutations in GATA4 and NKX2.5 genes have been identified as responsible for CHD. The frequency of GATA4 and NKX2.5 mutations in Chinese Uygur patients with CHD and the correlation between their genotype and CHD phenotype are unknown. Methods We examined the coding region of GATA4 and NKX2.5 genes in 62 Chinese Uygur patients with CHD and 117 Chinese Uygur individuals as the controls by denaturing high performance liquid chromatography (DHPLC) and sequencing. Results Two heterozygous missense mutations of c.1220C>A and c.1273G>A in GATA4 gene, which cause the amino acid residue changes of P407Q and D425N in GATA4, were found in a patient with tetralogy of Fallot and a patient with ventricular septal defect, respectively. The two patients did not have atrioventricular conduct defects or non-cardiac abnormalities. The two mutations are expected to affect the protein function. There were no reported NKX2.5 mutations in the patients. Conclusion Our results provided the primary data on CHD phenotype associated with GATA4 mutation in the Chinese Uygur population.