Single-site Amino Acid Substitutions Research Articles

A mutational atlas for Parkin proteostasis

Proteostasis can be disturbed by mutations affecting folding and stability of the encoded protein. An example is the ubiquitin ligase Parkin, where gene variants result in autosomal recessive Parkinsonism. To uncover the pathological mechanism and provide comprehensive genotype-phenotype information, variant abundance by massively parallel sequencing (VAMP-seq) is leveraged to quantify the abundance of Parkin variants in cultured human cells. The resulting mutational map, covering 9219 out of the 9300 possible single-site amino acid substitutions and nonsense Parkin variants, shows that most low abundance variants are proteasome targets and are located within the structured domains of the protein. Half of the known disease-linked variants are found at low abundance. Systematic mapping of degradation signals (degrons) reveals an exposed degron region proximal to the so-called “activation element”. This work provides examples of how missense variants may cause degradation either via destabilization of the native protein, or by introducing local signals for degradation.

Vasor: Accurate prediction of variant effects for amino acid substitutions in multidrug resistance protein 3.

The phosphatidylcholine floppase multidrug resistance protein 3 (MDR3) is an essential hepatobiliary transport protein. MDR3 dysfunction is associated with various liver diseases, ranging from severe progressive familial intrahepatic cholestasis to transient forms of intrahepatic cholestasis of pregnancy and familial gallstone disease. Single amino acid substitutions are often found as causative of dysfunction, but identifying the substitution effect in in vitro studies is time and cost intensive. We developed variant assessor of MDR3 (Vasor), a machine learning‐based model to classify novel MDR3 missense variants into the categories benign or pathogenic. Vasor was trained on the largest data set to date that is specific for benign and pathogenic variants of MDR3 and uses general predictors, namely Evolutionary Models of Variant Effects (EVE), EVmutation, PolyPhen‐2, I‐Mutant2.0, MUpro, MAESTRO, and PON‐P2 along with other variant properties, such as half‐sphere exposure and posttranslational modification site, as input. Vasor consistently outperformed the integrated general predictors and the external prediction tool MutPred2, leading to the current best prediction performance for MDR3 single‐site missense variants (on an external test set: F1‐score, 0.90; Matthew's correlation coefficient, 0.80). Furthermore, Vasor predictions cover the entire sequence space of MDR3. Vasor is accessible as a webserver at https://cpclab.uni‐duesseldorf.de/mdr3_predictor/ for users to rapidly obtain prediction results and a visualization of the substitution site within the MDR3 structure. The MDR3‐specific prediction tool Vasor can provide reliable predictions of single‐site amino acid substitutions, giving users a fast way to initially assess whether a variant is benign or pathogenic.

Interactions of a class IIb bacteriocin with a model lipid bilayer, investigated through molecular dynamics simulations

The emergence of antibiotic resistant microorganisms poses an alarming threat to global health. Antimicrobial peptides (AMPs) are considered a possible effective alternative to conventional antibiotic therapies. An understanding of the mechanism of action of AMPs is needed in order to better control and optimize their bactericidal activity. Plantaricin EF is a heterodimeric AMP, consisting of two peptides Plantaricin E (PlnE) and Plantaricin F (PlnF). We studied the behavior of these peptides on the surface of a model lipid bilayer. We identified the residues that facilitate peptide–peptide interactions. We also identified residues that mediate interactions of the dimer with the membrane. PlnE interacts with the membrane through amino acids at both its termini, while only the N terminus of PlnF approaches the membrane. By comparing the activity of single-site mutants of the two-peptide bacteriocin and the simulations of the bacteriocin on the surface of a model lipid bilayer, structure activity relationships are proposed. These studies allow us to generate hypotheses that relate biophysical interactions observed in simulations with the experimentally measured activity. We find that single-site amino acid substitutions result in markedly stronger antimicrobial activity when they strengthen the interactions between the two peptides, while, concomitantly, they weaken peptide–membrane association. This effect is more pronounced in the case of the PlnE mutant (G20A), which interacts the strongest with PlnF and the weakest with the membrane while displaying the highest activity.

Protein cold adaptation strategy via a unique seven-amino acid domain in the icefish ( Chionodraco hamatus ) PEPT1 transporter

Adaptation of organisms to extreme environments requires proteins to work at thermodynamically unfavorable conditions. To adapt to subzero temperatures, proteins increase the flexibility of parts of, or even the whole, 3D structure to compensate for the lower thermal kinetic energy available at low temperatures. This may be achieved through single-site amino acid substitutions in regions of the protein that undergo large movements during the catalytic cycle, such as in enzymes or transporter proteins. Other strategies of cold adaptation involving changes in the primary amino acid sequence have not been documented yet. In Antarctic icefish (Chionodraco hamatus) peptide transporter 1 (PEPT1), the first transporter cloned from a vertebrate living at subzero temperatures, we came upon a unique principle of cold adaptation. A de novo domain composed of one to six repeats of seven amino acids (VDMSRKS), placed as an extra stretch in the cytosolic COOH-terminal region, contributed per se to cold adaptation. VDMSRKS was in a protein region uninvolved in transport activity and, notably, when transferred to the COOH terminus of a warm-adapted (rabbit) PEPT1, it conferred cold adaptation to the receiving protein. Overall, we provide a paradigm for protein cold adaptation that relies on insertion of a unique domain that confers greater affinity and maximal transport rates at low temperatures. Due to its ability to transfer a thermal trait, the VDMSRKS domain represents a useful tool for future cell biology or biotechnological applications.

Three-Dimensional Structure and Determinants of Stability of the Iron–Sulfur Cluster Scaffold Protein IscU from Escherichia coli

The highly conserved protein, IscU, serves as the scaffold for iron-sulfur cluster (ISC) assembly in the ISC system common to bacteria and eukaryotic mitochondria. The apo-form of IscU from Escherichia coli has been shown to populate two slowly interconverting conformational states: one structured (S) and one dynamically disordered (D). Furthermore, single-site amino acid substitutions have been shown to shift the equilibrium between the metamorphic states. Here, we report three-dimensional structural models derived from NMR spectroscopy for the S-state of wild-type (WT) apo-IscU, determined under conditions where the protein was 80% in the S-state and 20% in the D-state, and for the S-state of apo-IscU(D39A), determined under conditions where the protein was ~95% in the S-state. We have used these structures in interpreting the effects of single site amino acid substitutions that alter %S = (100 × [S])/([S] + [D]). These include different residues at the same site, %S: D39V > D39L > D39A > D39G ≈ WT, and alanine substitutions at different sites, %S: N90A > S107A ≈ E111A > WT. Hydrophobic residues at residue 39 appear to stabilize the S-state by decreasing the flexibility of the loops that contain the conserved cysteine residues. The alanine substitutions at positions 90, 107, and 111, on the other hand, stabilize the protein without affecting the loop dynamics. In general, the stability of the S-state correlates with the compactness and thermal stability of the variant.

Protein stability: a single recorded mutation aids in predicting the effects of other mutations in the same amino acid site

Motivation: Accurate prediction of protein stability is important for understanding the molecular underpinnings of diseases and for the design of new proteins. We introduce a novel approach for the prediction of changes in protein stability that arise from a single-site amino acid substitution; the approach uses available data on mutations occurring in the same position and in other positions. Our algorithm, named Pro-Maya (Protein Mutant stAbilitY Analyzer), combines a collaborative filtering baseline model, Random Forests regression and a diverse set of features. Pro-Maya predicts the stability free energy difference of mutant versus wild type, denoted as ΔΔG.Results: We evaluated our algorithm extensively using cross-validation on two previously utilized datasets of single amino acid mutations and a (third) validation set. The results indicate that using known ΔΔG values of mutations at the query position improves the accuracy of ΔΔG predictions for other mutations in that position. The accuracy of our predictions in such cases significantly surpasses that of similar methods, achieving, e.g. a Pearson's correlation coefficient of 0.79 and a root mean square error of 0.96 on the validation set. Because Pro-Maya uses a diverse set of features, including predictions using two other methods, it also performs slightly better than other methods in the absence of additional experimental data on the query positions.Availability: Pro-Maya is freely available via web server at http://bental.tau.ac.il/ProMaya.Contact: nirb@tauex.tau.ac.il; wolf@cs.tau.ac.ilSupplementary Information: Supplementary data are available at Bioinformatics online.

Machine learning integration for predicting the effect of single amino acid substitutions on protein stability

BackgroundComputational prediction of protein stability change due to single-site amino acid substitutions is of interest in protein design and analysis. We consider the following four ways to improve the performance of the currently available predictors: (1) We include additional sequence- and structure-based features, namely, the amino acid substitution likelihoods, the equilibrium fluctuations of the alpha- and beta-carbon atoms, and the packing density. (2) By implementing different machine learning integration approaches, we combine information from different features or representations. (3) We compare classification vs. regression methods to predict the sign vs. the output of stability change. (4) We allow a reject option for doubtful cases where the risk of misclassification is high.ResultsWe investigate three different approaches: early, intermediate and late integration, which respectively combine features, kernels over feature subsets, and decisions. We perform simulations on two data sets: (1) S1615 is used in previous studies, (2) S2783 is the updated version (as of July 2, 2009) extracted also from ProTherm. For S1615 data set, our highest accuracy using both sequence and structure information is 0.842 on cross-validation and 0.904 on testing using early integration. Newly added features, namely, local compositional packing and the mobility extent of the mutated residues, improve accuracy significantly with intermediate integration. For S2783 data set, we also train regression methods to estimate not only the sign but also the amount of stability change and apply risk-based classification to reject when the learner has low confidence and the loss of misclassification is high. The highest accuracy is 0.835 on cross-validation and 0.832 on testing using only sequence information. The percentage of false positives can be decreased to less than 0.005 by rejecting 10 per cent using late integration.ConclusionWe find that in both early and late integration, combining inputs or decisions is useful in increasing accuracy. Intermediate integration allows assessing the contributions of individual features by looking at the assigned weights. Overall accuracy of regression is not better than that of classification but it has less false positives, especially when combined with the reject option. The server for stability prediction for three integration approaches and the data sets are available at .

The Multiphasic Nature of Nonphotochemical Quenching: Implications for Assessment of Photosynthetic Electron Transport Based on Chlorophyll Fluorescence

Defining a quantitative relationship between chlorophyll a fluorescence yield and Photosystem II (PS II) function is important to photosynthesis research. Prior work [Peterson and Havir (2003) Photosynth Res 75: 57-70] indicated an apparent effect of psbS genotype on the in vivo rate constant for photochemistry in PS II (k(P0)). The nuclear psbS gene encodes a 22-kDa pigment-binding antenna protein (PS II-S) essential for photoprotective nonphotochemical quenching (NPQ) in PS II. Ten Arabidopsis thaliana lines were chosen for study, encompassing effects on PS II-S expression level and/or structure due to single-site amino acid substitution. Short-term (i.e. seconds) irradiance-dependent changes in steady state fluorescence yields F(o) and F(m)(open and closed centers, respectively) were evaluated for compliance with the reversible radical pair (RRP) model of PS II. All lines (including normal Nicotiana tabacum and Zea mays) deviated from the RRP scheme in the same way indicating that psbS genotype per se does not alter interactions between the antenna and reaction center and thereby affect k(P0). Rather, observed departures from RRP model behavior are consistent with overestimation of F(m) due to perturbing effects of the saturating multiple turnover flash employed in its measurement. Reversal of direct quenching of singlet states by plastoquinone during the flash could occur but by itself cannot account for the anomalous covariation in F(o) and F(m). Reduction of the PS II acceptor side apparently either amplifies the rate constant for fluorescence or suppresses that of xanthophyll-dependent thermal deactivation (q(E)). A procedure was devised that considers F(o) when correcting maximal fluorescence values for measurement bias. A high degree of consistency in assessment of PS II quantum yield based on corrected fluorescence parameters and simultaneous CO(2) exchange measurements was noted under both steady state and transient conditions (360 mul CO(2)l(-1), 1% O(2)).

How to make foods safer--genetically modified foods.

It is the responsibility of companies developing genetically modified foods, and of regulatory authorities that approve their marketing, to ensure that they are at least as safe as the traditional foods they are intended to replace in the diet. This requires that any novel material introduced into the food material should not be allergenic. If the novel gene has come from an allergenic source, e.g. nuts, it is necessary to demonstrate using immunological procedures applied to the IgE fractions of pooled sera from individuals with confirmed allergies that the novel protein is non-allergenic. When the novel gene is from a non-allergenic source then it is necessary to demonstrate lack of significant amino acid sequence homology to known allergens together with sensitivity to food manufacturing and digestive processes. Consumer confidence in genetically modified foods would be significantly improved if hypoallergenic varieties of crops and food products that are currently allergenic could be developed. Techniques such as antisense technology and single site amino acid substitution have been shown to have such potential.

Mutational analysis of the IgE-binding epitopes of P34/Gly m Bd 30K

Background: Peanuts and soybeans are 2 foods that have been shown to be responsible for many atopic disorders. Because of their nutritional benefit, soybean proteins are now being used increasingly in a number of food products. Previous studies have documented multiple allergens in soybean extracts, including glycinin, β-conglycinin, and the P34/Gly m Bd 30K protein. Objective: Our overall goal was to identify soybean-specific allergens to begin to understand molecular and immunochemical characteristics of legume proteins. The specific aim of the current investigation was to identify the essential amino acid residues necessary for IgE binding in the 5 distinct immunodominant epitopes of P34/Gly m Bd 30K. Methods: Serum IgE from 6 clinically sensitive soybean-allergic individuals was used to identify P34/Gly m Bd 30K in the native and single amino acid substituted peptides with use of the SPOTS peptide synthesis technique to determine critical amino acids required for IgE binding. Results: The intensity of IgE binding and epitope recognition by serum IgE from the individuals varied substantially. With use of serum from 6 clinically soybean-sensitive individuals, 2 of the 5 immunodominant epitopes could be mutagenized to non-IgE binding peptides. Conclusions: Single-site amino acid substitution of the 5 immunodominant epitopes of Gly m Bd 30K with alanine revealed that IgE binding could be reduced or eliminated in epitopes 6 and 16 in the serum obtained from 6 soybean-sensitive patients. (J Allergy Clin Immunol 2000;105:378-84.)

Specific encapsidation of nodavirus RNAs is mediated through the C terminus of capsid precursor protein alpha.

Flock house virus (FHV) is a small icosahedral insect virus with a bipartite, messenger-sense RNA genome. Its T=3 icosahedral capsid is initially assembled from 180 subunits of a single type of coat protein, capsid precursor protein alpha (407 amino acids). Following assembly, the precursor particles undergo a maturation step in which the alpha subunits autocatalytically cleave between Asn363 and Ala364. This cleavage generates mature coat proteins beta (363 residues) and gamma (44 residues) and is required for acquisition of virion infectivity. The X-ray structure of mature FHV shows that gamma peptides located at the fivefold axes of the virion form a pentameric helical bundle, and it has been suggested that this bundle plays a role in release of viral RNA during FHV uncoating. To provide experimental support for this hypothesis, we generated mutant coat proteins that carried deletions in the gamma region of precursor protein alpha. Surprisingly, we found that these mutations interfered with specific recognition and packaging of viral RNA during assembly. The resulting particles contained large amounts of cellular RNAs and varying amounts of the viral RNAs. Single-site amino acid substitution mutants showed that three phenylalanines located at positions 402, 405, and 407 of coat precursor protein alpha were critically important for specific recognition of the FHV genome. Thus, in addition to its hypothesized role in uncoating and RNA delivery, the C-terminal region of coat protein alpha plays a significant role in recognition of FHV RNA during assembly. A possible link between these two functions is discussed.

Host mutations (miaA and rpsL) reduce tetracycline resistance mediated by Tet(O) and Tet(M).

The effects of mutations in host genes on tetracycline resistance mediated by the Tet(O) and Tet(M) ribosomal protection proteins, which originated in Campylobacter spp. and Streptococcus spp., respectively, were investigated by using mutants of Salmonella typhimurium and Escherichia coli. The miaA, miaB, and miaAB double mutants of S. typhimurium specify enzymes for tRNA modification at the adenosine at position 37, adjacent to the anticodon in tRNA. In S. typhimurium, this involves biosynthesis of N6-(4-hydroxyisopentenyl)-2-methylthio-adenosine (ms2io6A). The miaA mutation reduced the level of tetracycline resistance mediated by both Tet(O) and Tet(M), but the latter showed a greater effect, which was ascribed to the isopentenyl (i6) group or to a combination of the methylthioadenosine (ms2) and i6 groups but not to the ms2 group alone (specified by miaB). In addition, mutations in E. coli rpsL genes, generating both streptomycin-resistant and streptomycin-dependent strains, were also shown to reduce the level of tetracycline resistance mediated by Tet(O) and Tet(M). The single-site amino acid substitutions present in the rpsL mutations were pleiotropic in their effects on tetracycline MICs. These mutants affect translational accuracy and kinetics and suggest that Tet(O) and Tet(M) binding to the ribosome may be reduced or slowed in the E. coli rpsL mutants in which the S12 protein is altered. Data from both the miaA and rpsL mutant studies indicate a possible link between stability of the aminoacyl-tRNA in the ribosomal acceptor site and tetracycline resistance mediated by the ribosomal protection proteins.

Substrate recognition by ceramide-activated protein kinase. Evidence that kinase activity is proline-directed.

Signal transduction for tumor necrosis factor-alpha and interleukin-1 involves sphingomyelin hydrolysis to ceramide and stimulation of a ceramide-activated serine/threonine protein kinase (Mathias, S., Younes, A., Kan, C., Orlow, I., Joseph, C., and Kolesnick, R. (1993) Science 259, 519-522). Kinase activity is detected by phosphorylation of a 19-amino acid peptide derived from the sequence surrounding Thr669 of the epidermal growth factor receptor. Thr669 is contained within a -Pro-Leu-Thr-Pro- motif, which conforms to a known recognition sequence for the proline-directed class of serine/threonine protein kinases. The present studies used peptides with single-site amino acid substitutions within this sequence to assess substrate recognition by ceramide-activated protein kinase. Substitution of alanine for the C-terminal but not the N-terminal proline reduced kinase activity by 80%. Similarly, substitution of basic residues for the leucine residue reduced kinase activity by 90%. Substitution of acidic residues for leucine, or its removal, also markedly reduced kinase activity. Surprisingly, addition of a leucine residue between threonine and the C-terminal proline enhanced kinase activity 3-4 fold. The Vmax(app) of the enzyme toward the control peptide containing -Pro-Leu-Thr-Pro- (200 +/- 11 pmol of peptide phosphorylated/min/mg of membrane protein) was enhanced 2.3-fold by ceramide. However, ceramide had no effect on the Km (2.0 +/- 0.4 mM). Membranes containing ceramide-activated protein kinase showed minimal activity toward peptides derived from substrates for casein kinase II, S6 kinase, protein kinase C, and cAMP-dependent protein kinase, but possessed substantial activity toward a calmodulin kinase substrate. However, activities toward these substrates were not enhanced by ceramide. These results suggest that ceramide-activated protein kinase may be a member of the proline-directed class of protein kinases and display specificity for -Leu-Thr-Pro- as a minimal substrate recognition motif.