Neighboring Residues Research Articles

Neighborhood Preference of Amino Acids in Protein Structures and its Applications in Protein Structure Assessment

Amino acids form protein 3D structures in unique manners such that the folded structure is stable and functional under physiological conditions. Non-specific and non-covalent interactions between amino acids exhibit neighborhood preferences. Based on structural information from the protein data bank, a statistical energy function was derived to quantify amino acid neighborhood preferences. The neighborhood of one amino acid is defined by its contacting residues, and the energy function is determined by the neighboring residue types and relative positions. The neighborhood preference of amino acids was exploited to facilitate structural quality assessment, which was implemented in the neighborhood preference program NEPRE. The source codes are available via https://github.com/LiuLab-CSRC/NePre.

Reporter Assays for Ebola Virus Nucleoprotein Oligomerization, Virion-Like Particle Budding, and Minigenome Activity Reveal the Importance of Nucleoprotein Amino Acid Position 111.

For highly pathogenic viruses, reporter assays that can be rapidly performed are critically needed to identify potentially functional mutations for further study under maximal containment (e.g., biosafety level 4 [BSL-4]). The Ebola virus nucleoprotein (NP) plays multiple essential roles during the viral life cycle, yet few tools exist to study the protein under BSL-2 or equivalent containment. Therefore, we adapted reporter assays to measure NP oligomerization and virion-like particle (VLP) production in live cells and further measured transcription and replication using established minigenome assays. As a proof-of-concept, we examined the NP-R111C substitution, which emerged during the 2013–2016 Western African Ebola virus disease epidemic and rose to high frequency. NP-R111C slightly increased NP oligomerization and VLP budding but slightly decreased transcription and replication. By contrast, a synthetic charge-reversal mutant, NP-R111E, greatly increased oligomerization but abrogated transcription and replication. These results are intriguing in light of recent structures of NP oligomers, which reveal that the neighboring residue, K110, forms a salt bridge with E349 on adjacent NP molecules. By developing and utilizing multiple reporter assays, we find that the NP-111 position mediates a complex interplay between NP’s roles in protein structure, virion budding, and transcription and replication.

Pivotal role of the redox-active tyrosine in driving the water splitting catalyzed by photosystem II.

Photosynthetic water oxidation is catalyzed by the Mn4Ca cluster in photosystem II (PSII). The nearby redox-active tyrosine (YZ) serves as a direct electron acceptor of the Mn4Ca cluster and it forms a low-barrier H-bond (LBHB) with a neighboring histidine residue (D1-His190). Experimental evidence indicates that YZ oxidation triggers changes in the hydrogen bonding network that precede proton abstraction from the Mn4Ca cluster. In order to characterize such changes, we compare ab initio molecular dynamics simulations of different states of the catalytic cycle of PSII with dynamics of isolated tyrosine models (namely, p-cresol) in different oxidation states. The systematic comparison of the H-bond networks in different simulated systems suggests that the YZ oxidation leads to a water hydration pattern which is more similar to that of the neutral p-cresol rather than that of the p-cresol anion. Our simulations also reveal the twofold nature of the interactions between YZ and the Mn4Ca cluster. Firstly, the YZ oxidation triggers rapid structural changes of the H-bond pattern in the proximity of the cluster which have been observed to propagate on the ps time scale on the Ca2+ hydration shell up to other water molecules in the proximity of the cluster. Secondly, it is clear that YZ interacts with the Mn4Ca cluster also through Coulombic interactions mediated by CP43-Arg357 through the remaining positive charge of the pair. Our results are able to identify, for the first time, the structural rearrangements guided by the oxidation of YZ necessary for the evolution of the water splitting reaction in PSII. Based on these findings, we propose a mechanism of structural changes which is functional towards the progression of the catalytic cycle in PSII.

A Method to Calculate the Relative Binding Free Energy Differences of α-Helical Stapled Peptides.

Hydrocarbon-stapled peptides are a class of bioactive α-helical ligands developed to target protein-protein interactions. Peptide stapling has benefited from the development of several chemical reactions to modulate their membrane permeability and binding affinity. However, in most current programs, choosing the best stapling positions is usually a trial-and-error process. Here, we develop a protocol to obtain optimal stapling positions computationally. Our method is based on molecular dynamics simulations and free energy calculations with nonequilibrium approaches; here, we predict the binding poses, hot-spot residues, and binding affinity differences of a set of perfluoroarene stapled α-helical peptides of the BIM BH3 peptide to the BCLXL receptor. The prediction of the hot-spot residues within the target peptide through computational alanine scanning anticipates not only the key residues for the receptor-peptide complex formation but also which positions should be avoided when applying the stapling groups. The staple moieties introduce local conformational changes not only in the replaced positions but also on their neighbor residues of the template peptide further affecting their binding behavior. Our approach is successful at rank-ordering the binding affinities of these stapled peptides with respect to the BIM BH3 peptide.

Do sequence neighbours of intrinsically disordered regions promote structural flexibility in intrinsically disordered proteins?

Intrinsically disordered proteins (IDPs) are crucial players in various cellular activities. Several experimental and computational analyses have been conducted to study structural pliability and functional potential of IDPs. In spite of active research in past few decades, what induces structural disorder in IDPs and how is still elusive. Many studies testify that sequential and spatial neighbours often play important roles in determining structural and functional behaviour of proteins. Considering this fact, we assessed sequence neighbours of intrinsically disordered regions (IDRs) to understand if they have any role to play in inducing structural flexibility in IDPs. Our analysis includes 97% eukaryotic IDPs and 3% from bacteria and viruses. Physicochemical and structural parameters including amino acid propensity, hydrophobicity, secondary structure propensity, relative solvent accessibility, B-factor and atomic packing density are used to characterise the neighbouring residues of IDRs (NRIs). We show that NRIs exhibit a unique nature, which makes them stand out from both ordered and disordered residues. They show correlative occurrences of residue pairs like Ser-Thr and Gln-Asn, indicating their tendency to avoid strong biases of order or disorder promoting amino acids. We also find differential preferences of amino acids between N- and C-terminal neighbours, which might indicate a plausible directional effect on the dynamics of adjacent IDRs. We designed an efficient prediction tool using Random Forest to distinguish the NRIs from the ordered residues. Our findings will contribute to understand the behaviour of IDPs, and may provide potential lead in deciphering the role of IDRs in protein folding and assembly.

Energetic Self-Folding Mechanism in α-Helices.

A novel energetic route driving the folding of a polyalanine peptide from an extended conformation to its α-helix native conformation is described, supported by a new method to compute mean potential energy surfaces accurately in terms of the dihedral angles of the peptide chain from extensive molecular dynamics simulations. The energetic self-folding (ESF) route arises specifically from the balance between the intrinsic propensity of alanine residues toward the αR conformation and two, opposite, effects: the destabilizing interaction with neighbor residues and the stabilizing formation of native hydrogen bonds, with the latter being dominant for large peptide lengths. The ESF mechanism provides simple but robust support to the nucleation-elongation or zipper models and offers a quantitative energetic funnel picture of the folding process. The mechanism is validated by the reasonable agreement between the computed folding energies and the experimental values.

Functional importance of the oligomer formation of the cyanobacterial H+ pump Gloeobacter rhodopsin

Many microbial rhodopsins self-oligomerize, but the functional consequences of oligomerization have not been well clarified. We examined the effects of oligomerization of a H+ pump, Gloeobacter rhodopsin (GR), by using nanodisc containing trimeric and monomeric GR. The monomerization did not appear to affect the unphotolyzed GR. However, we found a significant impact on the photoreaction: The monomeric GR showed faint M intermediate formation and negligible H+ transfer reactions. These changes reflected the elevated pKa of the Asp121 residue, whose deprotonation is a prerequisite for the functional photoreaction. Here, we focused on His87, which is a neighboring residue of Asp121 and conserved among eubacterial H+ pumps but replaced by Met in an archaeal H+ pump. We found that the H87M mutation removes the “monomerization effects”: Even in the monomeric state, H87M contained the deprotonated Asp121 and showed both M formation and distinct H+ transfer reactions. Thus, for wild-type GR, monomerization probably strengthens the Asp121-His87 interaction and thereby elevates the pKa of Asp121 residue. This strong interaction might occur due to the loosened protein structure and/or the disruption of the interprotomer interaction of His87. Thus, the trimeric assembly of GR enables light-induced H+ transfer reactions through adjusting the positions of key residues.

Dynamics of human defensin 5 (HD5) self-assembly in solution: Molecular simulations/insights

Human α -defensin 5 (HD5) is a 32-residue cysteine-rich host-defense peptide that exhibits broad-spectrum antimicrobial activity and plays an essential role in innate immunity in the human gut and other organ systems. Although its antimicrobial mechanism of action remains unclear, the high salt concentration seems to attenuate the antimicrobial function of HD5 via an unknown mechanism. In this work, we employ Molecular Dynamics (MD) simulations to analyse the oligomerization behaviour of HD5 when exposed to different salt concentration. We demonstrate that the presence of salt, such as sodium chloride (NaCl), promotes HD5 to form higher-order oligomers (up to heptamers) in our simulations. In addition, we also analyse the electrostatic interactions between the two Glu residues (E14 and E21) and their neighbouring residues. Our data confirm that the E14 residue is essential for the structural integrity, whereas the E21 residue contributes to the dimerization of HD5, suggesting that these Glu residues are important for the antimicrobial function of this peptide.

Abstract 1655: A structural approach for prioritizing variants in personalized Oncology

Abstract In personalized oncology, treatment strategies take individualized genomic aberrations into consideration and relies heavily on how the cancer genomes are interpreted. With immense efforts put into cohort studies, knowledge of pathogenicity at the gene level had been accumulated. On the other hand, the increasing amount of personalized clinical trials and the accompanying need of interpreting pathogenecity at the sub-gene level emphasize the importance of resolving heterogeneous significance within genes. There had been some initiatives where people identified mutation hotspots within genes, looking at either highly recurrent single residues or mutation clusters. The hotspot clusters can be inferred from protein sequences as well as from protein structures, where residues distant in sequence may be brought together through protein folding. Nonetheless, these information are not well utilized in routine clinical settings yet. In this study, we aim to build a consensus list of prevalent mutational events for molecular stratification of patients. This is achieved by collecting sequence and structural mutation hotspots from different algorithms, and further combine structural information to identify their neighboring residues in protein structures. Specifically, around 1400 residue hotspots are collected from CancerHotspots and DominoEffect. An additional of around 250 structural hotspots are identified by three structural algorithms, namely 3DHotSpots, HotMAPS and HotSpot3D. These results are mostly derived from TCGA PanCancer cohorts, with CancerHotspots included more other studies in the analysis. With this consensus information in place, we further set up the mechanism to map from genomic variants to protein sequence and protein structures. This enables us to annotate the variants found in patient genome and see whether they co-localize with hotspot residues, in their structural neighborhood, or in structural informative domains like interaction interfaces. In summary, a higher prevalence of variants might imply higher significance and more well-studied roles than other low prevalent mutant alleles. Transferring this knowledge from the public cohorts helps researchers quickly prioritize variants found in patient genomes. Annotating structural significance of variants further provides us with more confidence in identifying driving events. With these procedures automated, the goal is to help facilitate clinical evaluations to meet the growing number of patients recruited to personalized oncology trials. Citation Format: Siao-Han Wong, Brors Benedikt. A structural approach for prioritizing variants in personalized Oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1655.

Tailoring a Soluble Diiron Monooxygenase for Synthesis of Aromatic N-oxides

The aromatic N-oxides have received increased attention over the last few years due to their potential application in medicine, agriculture and organic chemistry. As a green alternative in their synthesis, the biocatalytic method employing whole cells of Escherichia coli bearing phenol monooxygenase like protein PmlABCDEF (from here on – PML monooxygenase) has been introduced. In this work, site-directed mutagenesis was used to study the contributions of active site neighboring residues I106, A113, G109, F181, F200, F209 to the regiospecificity of N-oxidation. Based on chromogenic indole oxidation screening, a collection of PML mutants with altered catalytic properties was created. Among the tested mutants, the A113G variant acquired the most distinguishable N-oxidations capacity. This new variant of PML was able to produce dioxides (quinoxaline-1,4-dioxide, 2,5-dimethylpyrazine-1,4-dioxide) and specific mono-N-oxides (2,3,5-trimethylpyrazine-1-oxide) that were unachievable using the wild type PML. This mutant also featured reshaped regioselectivity as N-oxidation shifted towards quinazoline-1-oxide compared to quinazoline-3-oxide that is produced by the wild type PML.

Structural Basis of Sarco/Endoplasmic Reticulum Ca2+-ATPase 2b Regulation via Transmembrane Helix Interplay

Sarco/endoplasmic reticulum (ER) Ca2+-ATPase 2b (SERCA2b) is a ubiquitously expressed membrane protein that facilitates Ca2+ uptake from the cytosol to the ER. SERCA2b includes a characteristic 11th transmembrane helix (TM11) followed by a luminal tail, but the structural basis of SERCA regulation by these C-terminal segments remains unclear. Here, we determined the crystal structures of SERCA2b and its C-terminal splicing variant SERCA2a, both in the E1-2Ca2+-adenylyl methylenediphosphonate (AMPPCP) state. Despite discrepancies with the previously reported structural model of SERCA2b, TM11 was found to be located adjacent to TM10 and to interact weakly with a part of the L8/9 loop and theN-terminal end of TM10, thereby inhibiting the SERCA2b catalytic cycle. Accordingly, mutational disruption of the interactions between TM11 and its neighboring residues caused SERCA2b to display SERCA2a-like ATPase activity. We propose that TM11 serves as a key modulator of SERCA2b activity by fine-tuning the intramolecular interactions with other transmembrane regions.

PyFeat: a Python-based effective feature generation tool for DNA, RNA and protein sequences.

Extracting useful feature set which contains significant discriminatory information is a critical step in effectively presenting sequence data to predict structural, functional, interaction and expression of proteins, DNAs and RNAs. Also, being able to filter features with significant information and avoid sparsity in the extracted features require the employment of efficient feature selection techniques. Here we present PyFeat as a practical and easy to use toolkit implemented in Python for extracting various features from proteins, DNAs and RNAs. To build PyFeat we mainly focused on extracting features that capture information about the interaction of neighboring residues to be able to provide more local information. We then employ AdaBoost technique to select features with maximum discriminatory information. In this way, we can significantly reduce the number of extracted features and enable PyFeat to represent the combination of effective features from large neighboring residues. As a result, PyFeat is able to extract features from 13 different techniques and represent context free combination of effective features. The source code for PyFeat standalone toolkit and employed benchmarks with a comprehensive user manual explaining its system and workflow in a step by step manner are publicly available. https://github.com/mrzResearchArena/PyFeat/blob/master/RESULTS.md. Toolkit, source code and manual to use PyFeat: https://github.com/mrzResearchArena/PyFeat/. Supplementary data are available at Bioinformatics online.

Enhanced phosphorylation of T153 in soluble tau is a defining biochemical feature of the A152T tau risk variant

Pathogenic mutations in the tau gene (microtubule associated protein tau, MAPT) are linked to the onset of tauopathy, but the A152T variant is unique in acting as a risk factor for a range of disorders including Alzheimer’s disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and dementia with Lewy bodies (DLB). In order to provide insight into the mechanism by which A152T modulates disease risk, we developed a novel mouse model utilizing somatic brain transgenesis with adeno-associated virus (AAV) to drive tau expression in vivo, and validated the model by confirming the distinct biochemical features of A152T tau in postmortem brain tissue from human carriers. Specifically, TauA152T-AAV mice exhibited increased tau phosphorylation that unlike animals expressing the pathogenic P301L mutation remained localized to the soluble fraction. To investigate the possibility that the A152T variant might alter the phosphorylation state of tau on T152 or the neighboring T153 residue, we generated a novel antibody that revealed significant accumulation of soluble tau species that were hyperphosphorylated on T153 (pT153) in TauA152T-AAV mice, which were absent the soluble fraction of TauP301L-AAV mice. Providing new insight into the role of A152T in modifying risk of tauopathy, as well as validating the TauA152T-AAV model, we demonstrate that the presence of soluble pT153-positive tau species in human postmortem brain tissue differentiates A152T carriers from noncarriers, independent of disease classification. These results implicate both phosphorylation of T153 and an altered solubility profile in the mechanism by which A152T modulates disease risk.

Evolutionary Stability of Salt Bridges Hints Its Contribution to Stability of Proteins

The contribution of newly designed salt bridges to protein stabilization remains controversial even today. In order to solve this problem, we investigated salt bridges from two aspects: spatial distribution and evolutionary characteristics of salt bridges. Firstly, we analyzed spatial distribution of salt bridges in proteins, elucidating the basic requirements of forming salt bridges. Then, from an evolutionary point of view, the evolutionary characteristics of salt bridges as well as their neighboring residues were investigated in our study. The results demonstrate that charged residues appear more frequently than other neutral residues at certain positions of sequence even under evolutionary pressure, which are able to form electrostatic interactions that could increase the evolutionary stability of corresponding amino acid regions, enhancing their importance to stability of proteins. As a corollary, we conjectured that the newly designed salt bridges with more contribution to proteins, not only, are qualified spatial distribution of salt bridges, but also, are needed to further increase the evolutionary stability of corresponding amino acid regions. Based on analysis, the 8 mutations were accordingly constructed in the 1,4-α-glucan branching enzyme (EC 2.4.1.18, GBE) from Geobacillus thermoglucosidans STB02, of which 7 mutations improved thermostability of GBE. The enhanced thermostability of 7 mutations might be a result of additional salt bridges on residue positions that at least one of amino acids positions is conservative, improving their contribution of stabilization to proteins.

The proton and metal binding sites responsible for the pH-dependent green-red bioluminescence color tuning in firefly luciferases

Firefly luciferases produce yellow-green light under physiological and alkaline conditions, however at acidic pH, higher temperatures or in the presence of heavy metals the color changes to red, a property called pH-sensitivity. Despite many decades of studies, the proton and metal binding sites responsible for pH-sensitivity remain enigmatic. Previously we suggested that the salt bridge E311/R337 keeps a closed conformation of the luciferin phenolate binding site. Here we further investigated the effect of this salt bridge and mutations of the neighbor residues H310 and E/N354, on metal and pH-sensitivity of firefly luciferases emitting distinct bioluminescence colors (Cratomorphus distinctus: 548 nm; Macrolampis sp2: 569 nm). The substitutions of H310 and E/N354 modulate metal sensitivity, whereas the carboxylate of E311 may work as the catalytic base essential for green bioluminescence and pH-sensitivity. Modeling studies showed that H310, E311 and E354 side-chains coordinate Zinc, constituting the metal binding site and the pH-sensor. Electrostatic potential and pKa calculations suggest that the external couple H310/E354 is affected by pH, whereas E311/R337 make a stabilized internal pair which retains excited oxyluciferin ejected proton near its phenolate group into a high energy state, promoting yellow-green bioluminescence. Protonation or metal binding weaken these electrostatic gates and their ability to retain the excited oxyluciferin released proton near its phenolate, promoting red light emission.

Salvaging the Thermodynamic Destabilization of Interface Histidine in Transmembrane β-Barrels.

The ability of histidine to participate in a wide range of stabilizing polar interactions preferentially populates this residue in functionally important sites of proteins. Histidine possesses an amphiphilic and electrostatic nature that is essential for amino acids residing at membrane interfaces. However, the frequency of occurrence of histidine at membrane interfaces, particularly transmembrane β-barrels, is lower than those of other aromatic residues. Here, we carry out comprehensive energetic measurements using equilibrium folding of the outer membrane enzyme PagP to address the contribution of a C-terminal interface histidine to barrel stability. We show that placing histidine at the C-terminus universally destabilizes PagP by 4.0–8.0 kcal mol–1 irrespective of the neighboring residue. Spectroscopic and electrophoretic measurements indicate that the altered stability may arise from a loss of barrel compaction. Isoleucine, methionine, and valine salvage this destabilization marginally (in addition to tyrosine, which shows an exceptionally high folding free energy value), when placed at the penultimate position, at the expense of an altered folding pathway. Double-mutant cycle analysis indicates that the coupling energy between the terminal and penultimate residues in PagP-X160H161 increases when the level of intrinsic destabilization by the terminal H161 is high. Our observations that neighboring residues cannot salvage the energetic destabilization of histidine may explain why histidine is less abundant at membrane interfaces.

Geometrical Description of Protein Structural Motifs.

We present a geometrical method that can identify secondary structural motifs in proteins via angular correlations. The method uses crystal structure coordinates to calculate angular and radial signatures of each residue relative to an external reference point as the number of nearest-neighbor residues increases. We apply our approach to the blue copper protein amicyanin using the copper cofactor as the external reference point. We define a signature termed Δβ which describes the change in angular correlation as the span of nearest neighbor residues increases. We find that three turn regions of amicyanin harbor residues with Δβ near zero, while residues in other secondary structures have Δβ greater than zero: for β-strands, Δβ changes gradually residue by residue along the strand. Extension of our analysis to other blue copper proteins demonstrated that the noted structural trends are general. Importantly, our geometrical description of the folded protein accounts for all forces holding the structure together. Through this analysis, we identified some of the turns in amicyanin as symmetrical anchor points.

Gating-induced large aqueous volumetric remodeling and aspartate tolerance in the voltage sensor domain of Shaker K+ channels

Neurons encode electrical signals with critically tuned voltage-gated ion channels and enzymes. Dedicated voltage sensor domains (VSDs) in these membrane proteins activate coordinately with an unresolved structural change. Such change conveys the transmembrane translocation of four positively charged arginine side chains, the voltage-sensing residues (VSRs; R1-R4). Countercharges and lipid phosphohead groups likely stabilize these VSRs within the low-dielectric core of the protein. However, the role of hydration, a sign-independent charge stabilizer, remains unclear. We replaced all VSRs and their neighboring residues with negatively charged aspartates in a voltage-gated potassium channel. The ensuing mild functional effects indicate that hydration is also important in VSR stabilization. The voltage dependency of the VSR aspartate variants approached the expected arithmetic summation of charges at VSR positions, as if negative and positive side chains faced similar pathways. In contrast, aspartates introduced between R2 and R3 did not affect voltage dependence as if the side chains moved outside the electric field or together with it, undergoing a large displacement and volumetric remodeling. Accordingly, VSR performed osmotic work at both internal and external aqueous interfaces. Individual VSR contributions to volumetric works approached arithmetical additivity but were largely dissimilar. While R1 and R4 displaced small volumes, R2 and R3 volumetric works were massive and vectorially opposed, favoring large aqueous remodeling during VSD activation. These diverse volumetric works are, at least for R2 and R3, not compatible with VSR translocation across a unique stationary charge transfer center. Instead, VSRs may follow separated pathways across a fluctuating low-dielectric septum.

Exploring the role of post-translational modifications in regulating α-synuclein interactions by studying the effects of phosphorylation on nanobody binding.

Intracellular deposits of α-synuclein in the form of Lewy bodies are major hallmarks of Parkinson's disease (PD) and a range of related neurodegenerative disorders. Post-translational modifications (PTMs) of α-synuclein are increasingly thought to be major modulators of its structure, function, degradation and toxicity. Among these PTMs, phosphorylation near the C-terminus at S129 has emerged as a dominant pathogenic modification as it is consistently observed to occur within the brain and cerebrospinal fluid (CSF) of post-mortem PD patients, and its level appears to correlate with disease progression. Phosphorylation at the neighboring tyrosine residue Y125 has also been shown to protect against α-synuclein toxicity in a Drosophila model of PD. In the present study we address the potential roles of C-terminal phosphorylation in modulating the interaction of α-synuclein with other protein partners, using a single domain antibody fragment (NbSyn87) that binds to the C-terminal region of α-synuclein with nanomolar affinity. The results reveal that phosphorylation at S129 has negligible effect on the binding affinity of NbSyn87 to α-synuclein while phosphorylation at Y125, only four residues away, decreases the binding affinity by a factor of 400. These findings show that, despite the fact that α-synuclein is intrinsically disordered in solution, selective phosphorylation can modulate significantly its interactions with other molecules and suggest how this particular form of modification could play a key role in regulating the normal and aberrant function of α-synuclein.

Computational Prediction and Analysis for Tyrosine Post-Translational Modifications via Elastic Net.

The tyrosine residue has been identified as suffering three major post-translational modifications (PTMs) including nitration, sulfation, and phosphorylation, which could be involved in different physiological and pathological processes. Multiple tyrosine residues of the whole protein may be modified concurrently, where PTM of a single tyrosine may affect modification of other neighboring tyrosine residues. Hence, it is significant and beneficial to predict nitration, sulfation, and phosphorylation of tyrosine residues in the whole protein sequence. Here, we introduce elastic net to perform feature selection and develop a predictor named TyrPred for predicting nitrotyrosine, sulfotyrosine, and kinase-specific tyrosine phosphorylation sites on the basis of support vector machine. We critically evaluate the performance of TyrPred and compare it with other existing tools. The satisfying results show that using elastic net to mine important features for training can considerably improve the prediction performance. Feature optimization indicates that evolutionary information is significant and contributes to the prediction model. The online tool is established at http://computbiol.ncu.edu.cn/TyrPred . We anticipate that TyrPred can provide useful complements to the existing approaches in this field.