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The FoldX web server: an online force field
Abstract:FoldX is an empirical force field that was developed for the rapid evaluation of the effect of mutations on the stability, folding and dynamics of proteins and nucleic acids. The core functionality of FoldX, namely the calculation of the free energy of a macromolecule based on its high-resolution 3D structure, is now publicly available through a web server at . The current release allows the calculation of the stability of a protein, calculation of the positions of the protons and the prediction of water bridges, prediction of metal binding sites and the analysis of the free energy of complex formation. Alanine scanning, the systematic truncation of side chains to alanine, is also included. In addition, some reporting functions have been added, and it is now possible to print both the atomic interaction networks that constitute the protein, print the structural and energetic details of the interactions per atom or per residue, as well as generate a general quality report of the pdb structure. This core functionality will be further extended as more FoldX applications are developed.
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Side-by-side comparison of Notch- and C83 binding to γ-secretase in a complete membrane model at physiological temperature
RSC advances
2020
摘要
γ-Secretase cleaves the C99 fragment of the amyloid precursor protein, leading to formation of aggregated β-amyloid peptide central to Alzheimer's disease, and Notch, essential for cell regulation. Recent cryogenic electron microscopy (cryo-EM) structures indicate major changes upon substrate binding, a β-sheet recognition motif, and a possible helix unwinding to expose peptide bonds towards nucleophilic attack. Here we report side-by-side comparison of the 303 K dynamics of the two proteins in realistic membranes using molecular dynamics simulations. Our ensembles agree with the cryo-EM data (full-protein Cα-RMSD = 1.62–2.19 Å) but reveal distinct presenilin helix conformation states and thermal β-strand to coil transitions of C83 and Notch100. We identify distinct 303 K hydrogen bond dynamics and water accessibility of the catalytic sites. The RKRR motif (1758–1761) contributes significantly to Notch binding and serves as a “membrane anchor” that prevents Notch displacement. Water that transiently hydrogen bonds to G1753 and V1754 probably represents the catalytic nucleophile. At 303 K, Notch and C83 binding induce different conformation states, with Notch mostly present in a closed state with shorter Asp–Asp distance. This may explain the different outcome of Notch and C99 cleavage, as the latter is more imprecise with many products. Our identified conformation states may aid efforts to develop conformation-selective drugs that target C99 and Notch cleavage differently, e.g. Notch-sparing γ-secretase modulators.
De novo variants in CAMK2A and CAMK2B cause neurodevelopmental disorders
Annals of clinical and translational neurology
2018
摘要
α (CAMK2A) and β (CAMK2B) isoforms of Calcium/calmodulin‐dependent protein kinase II (CaMKII) play a pivotal role in neuronal plasticity and in learning and memory processes in the brain. Here, we explore the possible involvement of α‐ and β‐CaMKII variants in neurodevelopmental disorders.
Autonomous zinc-finger nuclease pairs for targeted chromosomal deletion
Nucleic acids research
2010
摘要
Zinc-finger nucleases (ZFNs) have been successfully used for rational genome engineering in a variety of cell types and organisms. ZFNs consist of a non-specific FokI endonuclease domain and a specific zinc-finger DNA-binding domain. Because the catalytic domain must dimerize to become active, two ZFN subunits are typically assembled at the cleavage site. The generation of obligate heterodimeric ZFNs was shown to significantly reduce ZFN-associated cytotoxicity in single-site genome editing strategies. To further expand the application range of ZFNs, we employed a combination of in silico protein modeling, in vitro cleavage assays, and in vivo recombination assays to identify autonomous ZFN pairs that lack cross-reactivity between each other. In the context of ZFNs designed to recognize two adjacent sites in the human HOXB13 locus, we demonstrate that two autonomous ZFN pairs can be directed simultaneously to two different sites to induce a chromosomal deletion in ∼10% of alleles. Notably, the autonomous ZFN pair induced a targeted chromosomal deletion with the same efficacy as previously published obligate heterodimeric ZFNs but with significantly less toxicity. These results demonstrate that autonomous ZFNs will prove useful in targeted genome engineering approaches wherever an application requires the expression of two distinct ZFN pairs.
Interaction of ZIKV NS5 and STAT2 Explored by Molecular Modeling, Docking, and Simulations Studies
IWBBIO
2019
摘要
ZIKV NS5 has been associated with inhibition of type I IFN during the host antiviral response. The protein-protein interaction may promote the proteasomal degradation of STAT2, although the entire mechanism is still unknown. In this study, a three-dimensional model of the full STAT2 protein (C-score = \(-0.62\)) was validated. Likewise, the top scored docked complex NS5-STAT2 is presented among several other models; the top model shows a total stabilizing energy for the complex of \(-77.942\) kcal mol\(^{-1}\) and a Gibbs binding free energy of \(-4.30\) kcal mol\(^{-1}\). The analysis of the complex has revealed that the interaction is limited to three domains known as N-terminal from STAT2 and Mtase-Thumb from NS5; both located in the ordered regions of these proteins. Key residues involved in the interaction interface that showed the highest frequency among the models are stabilized by electrostatic interactions, hydrophobic interactions, salt bridges, and ionic interactions. Therefore, our findings support the experimental preliminaries observations reported in the literature and present additional structural characterization that will help in the drug design efforts against ZIKV NS5.
Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases
Nature
2008
摘要
Xeroderma pigmentosum is a monogenic disease characterized by hypersensitivity to ultraviolet light. The cells of xeroderma pigmentosum patients are defective in nucleotide excision repair, limiting their capacity to eliminate ultraviolet-induced DNA damage, and resulting in a strong predisposition to develop skin cancers. The use of rare cutting DNA endonucleases—such as homing endonucleases, also known as meganucleases—constitutes one possible strategy for repairing DNA lesions. Homing endonucleases have emerged as highly specific molecular scalpels that recognize and cleave DNA sites, promoting efficient homologous gene targeting through double-strand-break-induced homologous recombination. Here we describe two engineered heterodimeric derivatives of the homing endonuclease I-CreI, produced by a semi-rational approach. These two molecules—Amel3–Amel4 and Ini3–Ini4—cleave DNA from the human XPC gene (xeroderma pigmentosum group C), in vitro and in vivo. Crystal structures of the I-CreI variants complexed with intact and cleaved XPC target DNA suggest that the mechanism of DNA recognition and cleavage by the engineered homing endonucleases is similar to that of the wild-type I-CreI. Furthermore, these derivatives induced high levels of specific gene targeting in mammalian cells while displaying no obvious genotoxicity. Thus, homing endonucleases can be designed to recognize and cleave the DNA sequences of specific genes, opening up new possibilities for genome engineering and gene therapy in xeroderma pigmentosum patients whose illness can be treated ex vivo.
Current challenges in genome annotation through structural biology and bioinformatics.
Current opinion in structural biology
2012
摘要
With the huge volume in genomic sequences being generated from high-throughout sequencing projects the requirement for providing accurate and detailed annotations of gene products has never been greater. It is proving to be a huge challenge for computational biologists to use as much information as possible from experimental data to provide annotations for genome data of unknown function. A central component to this process is to use experimentally determined structures, which provide a means to detect homology that is not discernable from just the sequence and permit the consequences of genomic variation to be realized at the molecular level. In particular, structures also form the basis of many bioinformatics methods for improving the detailed functional annotations of enzymes in combination with similarities in sequence and chemistry.
DNA-binding specificity prediction with FoldX.
Methods in enzymology
2011
摘要
With the advent of Synthetic Biology, a field between basic science and applied engineering, new computational tools are needed to help scientists reach their goal, their design, optimizing resources. In this chapter, we present a simple and powerful method to either know the DNA specificity of a wild-type protein or design new specificities by using the protein design algorithm FoldX. The only basic requirement is having a good resolution structure of the complex. Protein-DNA interaction design may aid the development of new parts designed to be orthogonal, decoupled, and precise in its target. Further, it could help to fine-tune the systems in terms of specificity, discrimination, and binding constants. In the age of newly developed devices and invented systems, computer-aided engineering promises to be an invaluable tool.
An Evolutionary Trade-Off between Protein Turnover Rate and Protein Aggregation Favors a Higher Aggregation Propensity in Fast Degrading Proteins
PLoS Comput. Biol.
2011
摘要
We previously showed the existence of selective pressure against protein aggregation by the enrichment of aggregation-opposing ‘gatekeeper’ residues at strategic places along the sequence of proteins. Here we analyzed the relationship between protein lifetime and protein aggregation by combining experimentally determined turnover rates, expression data, structural data and chaperone interaction data on a set of more than 500 proteins. We find that selective pressure on protein sequences against aggregation is not homogeneous but that short-living proteins on average have a higher aggregation propensity and fewer chaperone interactions than long-living proteins. We also find that short-living proteins are more often associated to deposition diseases. These findings suggest that the efficient degradation of high-turnover proteins is sufficient to preclude aggregation, but also that factors that inhibit proteasomal activity, such as physiological ageing, will primarily affect the aggregation of short-living proteins.
Mutations in Extracellular Matrix Genes NID1 and LAMC1 Cause Autosomal Dominant Dandy–Walker Malformation and Occipital Cephaloceles
Human mutation
2013
摘要
We performed whole‐exome sequencing of a family with autosomal dominant Dandy–Walker malformation and occipital cephaloceles and detected a mutation in the extracellular matrix (ECM) protein‐encoding gene NID1. In a second family, protein interaction network analysis identified a mutation in LAMC1, which encodes a NID1‐binding partner. Structural modeling of the NID1–LAMC1 complex demonstrated that each mutation disrupts the interaction. These findings implicate the ECM in the pathogenesis of Dandy–Walker spectrum disorders.
Sailfish enables alignment-free isoform quantification from RNA-seq reads using lightweight algorithms
Nature Biotechnology
2013
摘要
We introduce Sailfish, a computational method for quantifying the abundance of previously annotated RNA isoforms from RNA-seq data. Because Sailfish entirely avoids mapping reads, a time-consuming step in all current methods, it provides quantification estimates much faster than do existing approaches (typically 20 times faster) without loss of accuracy. By facilitating frequent reanalysis of data and reducing the need to optimize parameters, Sailfish exemplifies the potential of lightweight algorithms for efficiently processing sequencing reads.
Mutational Locally Enhanced Sampling (MULES) for quantitative prediction of the effects of mutations at protein–protein interfaces
2012
摘要
We have successfully developed and validated with experiment a new computational method for quantitatively predicting the effects of mutations at a protein–protein interface. From over 500 ns of explicitly solvated molecular dynamics, Mutational Locally Enhanced Sampling (MULES) shows significantly improved accuracy over post-processing methods for a prototypical set of mutations, with a maximum mean unsigned error to experiment of 0.5 kcal mol−1 and comparable or better precision. The technique in principle allows the effect of any mutation to be calculated, whether natural or non-natural. The versatility, quantitative accuracy, high precision and speed of MULES compared to existing computational prediction techniques enhance its potential for modelling changes to the interface in a systematic way, thereby aiding peptide and protein interaction design.
PupaSuite: finding functional single nucleotide polymorphisms for large-scale genotyping purposes
Nucleic Acids Res.
2006
摘要
We have developed a web tool, PupaSuite, for the selection of single nucleotide polymorphisms (SNPs) with potential phenotypic effect, specifically oriented to help in the design of large-scale genotyping projects. PupaSuite uses a collection of data on SNPs from heterogeneous sources and a large number of pre-calculated predictions to offer a flexible and intuitive interface for selecting an optimal set of SNPs. It improves the functionality of PupaSNP and PupasView programs and implements new facilities such as the analysis of user's data to derive haplotypes with functional information. A new estimator of putative effect of polymorphisms has been included that uses evolutionary information. Also SNPeffect database predictions have been included. The PupaSuite web interface is accessible through and through .
Modifications to glucose-6-phosphate dehydrogenase for industrial applications: predictions and tests
Journal of Cheminformatics
2013
摘要
We have been combining computational methods with experimental tests to gently modify glucose-6-phosphate dehydrogenase (G6PDH) from Leuconostoc mesenteroides for two specific goals. Firstly, it should be immobilized on an inorganic substrate as part of a longer-term project involving microreactor methods. Secondly, the natural enzyme is found as a homodimer, but we would like to have active monomers which would be better suited to the use of small pores in an aerogel based-system.
Our approach has involved consideration of available conservation information, structural properties and simple methods for estimating free energy changes. The approach relies completely on the crystal structure deposited in the protein data bank [1].
Covalent binding to a fixed surface involves introducing side-chains with appropriate reactive groups such as the thiols in cysteines, but there is some choice as to groups which can be incorporated into the inorganic surface. Here, the only considerations have been surface accessibility, distance from the active site and evolutionary conservation. One would rather substitute a naturally variable residue than a conserved site.
The second aim is more ambitious and speculative. The enzyme is naturally homo-dimeric, but a monomer might be easier to incorporate in the pores of the substrate. The approach here has been to identify sites at the monomer-monomer interface. From this small set of residues, a crude, but fast free energy estimator [2] was used to check substitutions which should destabilize the dimer while not destabilizing the monomer. First results from analytical chromatography and light scattering suggest a substantial monomer population with activity reduced by perhaps two orders of magnitude.
CAOS—Episodic Cerebellar Ataxia, Areflexia, Optic Atrophy, and Sensorineural Hearing Loss
Journal of child neurology
2015
摘要
We describe the molecular basis of a distinctive syndrome characterized by infantile stress-induced episodic weakness, ataxia, and sensorineural hearing loss, with permanent areflexia and optic nerve pallor. Whole exome sequencing identified a deleterious heterozygous c.2452 G>A, p.(E818K) variant in the ATP1A3 gene and structural analysis predicted its protein-destabilizing effect. This variant has not been reported in context with rapid-onset dystonia parkinsonism and alternating hemiplegia of childhood, the 2 main diseases associated with ATP1A3. The clinical presentation in the family described here differs categorically from these diseases in age of onset, clinical course, cerebellar over extrapyramidal movement disorder predominance, and peripheral nervous system involvement. While this paper was in review, a highly resembling phenotype was reported in additional patients carrying the same c.2452 G>A variant. Our findings substantiate this variant as the cause of a unique inherited autosomal dominant neurologic syndrome that constitutes a third allelic disease of the ATP1A3 gene.
Increasing Affinity of Interferon-γ Receptor 1 to Interferon-γ by Computer-Aided Design
BioMed research international
2013
摘要
We describe a computer-based protocol to design protein mutations increasing binding affinity between ligand and its receptor. The method was applied to mutate interferon-γ receptor 1 (IFN-γ-Rx) to increase its affinity to natural ligand IFN-γ, protein important for innate immunity. We analyzed all four available crystal structures of the IFN-γ-Rx/IFN-γ complex to identify 40 receptor residues forming the interface with IFN-γ. For these 40 residues, we performed computational mutation analysis by substituting each of the interface receptor residues by the remaining standard amino acids. The corresponding changes of the free energy were calculated by a protocol consisting of FoldX and molecular dynamics calculations. Based on the computed changes of the free energy and on sequence conservation criteria obtained by the analysis of 32 receptor sequences from 19 different species, we selected 14 receptor variants predicted to increase the receptor affinity to IFN-γ. These variants were expressed as recombinant proteins in Escherichia coli, and their affinities to IFN-γ were determined experimentally by surface plasmon resonance (SPR). The SPR measurements showed that the simple computational protocol succeeded in finding two receptor variants with affinity to IFN-γ increased about fivefold compared to the wild-type receptor.
Molecular modeling and molecular dynamic simulation of the effects of variants in the TGFBR2 kinase domain as a paradigm for interpretation of variants obtained by next generation sequencing
PloS one
2017
摘要
Variants in the TGFBR2 kinase domain cause several human diseases and can increase propensity for cancer. The widespread application of next generation sequencing within the setting of Individualized Medicine (IM) is increasing the rate at which TGFBR2 kinase domain variants are being identified. However, their clinical relevance is often uncertain. Consequently, we sought to evaluate the use of molecular modeling and molecular dynamics (MD) simulations for assessing the potential impact of variants within this domain. We documented the structural differences revealed by these models across 57 variants using independent MD simulations for each. Our simulations revealed various mechanisms by which variants may lead to functional alteration; some are revealed energetically, while others structurally or dynamically. We found that the ATP binding site and activation loop dynamics may be affected by variants at positions throughout the structure. This prediction cannot be made from the linear sequence alone. We present our structure-based analyses alongside those obtained using several commonly used genomics-based predictive algorithms. We believe the further mechanistic information revealed by molecular modeling will be useful in guiding the examination of clinically observed variants throughout the exome, as well as those likely to be discovered in the near future by clinical tests leveraging next-generation sequencing through IM efforts.
Identification of functional SNPs in VEGF gene and in silico analysis of damaging SNPs based on data procured from dbSNP database
2016
摘要
VEGF (vascular endothelial growth factor) gene is known to cause angiogenesis and participate in many diseases like prostate cancer and diabetic retinopathy. Several single nucleotide polymorphisms (SNPs) have been described in the VEGF gene, some of which have been reported to be associated with differential expression of VEGF in vitro. In the present study, polymorphism of VEGF gene was studied in relation to damaging mutations. Of total SNPs in VEGF gene, 38 were nonsynonymous SNPs (nsSNPs), 47 were synonymous SNPs, 66 were in non-coding regions, which comprised of 13 SNPs in 5'UTR region and 53 SNPs were in 3'UTR region. The rest were in the intronic region. SIFT (Sorting Intolerant from Tolerant) analysis showed that L163P, H200W, P312T, R288W and G365V mutations were damaging, whereas PolyPhen predicted P163L and H200W nsSNPs as damaging. Among the predicted nsSNPs, rs4645843, rs1800620 were identified as deleterious and damaging by the SIFT and PolyPhen programs. Protein structural analysis with these amino acid variants was performed by using I-Mutant, Swiss PDB viewer, ANOLEA (Atomic Non-Local Environment Assessment), MUSTER (MUlti-Sources ThreadER) and NOMAD-Ref servers to check their molecular dynamics and energy minimization calculations. This in silico analysis suggested that mutations in VEGF, such as, G365V, P312T and R288W, could directly or indirectly destabilize the amino acid interactions and hydrogen bond networks, thus explaining the functional deviations of protein to some extent. Thus screening for VEGF G365V, P312T and R288W variants in the population-based study may be useful to check disease susceptibility.
AUTO-MUTE: web-based tools for predicting stability changes in proteins due to single amino acid replacements.
Protein engineering, design & selection : PEDS
2010
摘要
Utilizing cutting-edge supervised classification and regression algorithms, three web-based tools have been developed for predicting stability changes upon single residue substitutions in proteins with known native structures. Trained models classify independent mutant test sets with accuracies ranging from 87 to 94%. Attributes representing each mutant protein are based on a computational mutagenesis methodology relying on a four-body statistical potential, illustrating a novel integration of both energy-based and machine learning approaches. The servers are written in PHP and hosted on a Linux platform, and they can be freely accessed online along with detailed data sets, documentation and performance results at http://proteins.gmu.edu/automute.
Tertiary structural motif sequence statistics enable facile prediction and design of peptides that bind anti-apoptotic Bfl-1 and Mcl-1
2018
摘要
Understanding the relationship between protein sequence and structure well enough to rationally design novel proteins or protein complexes is a longstanding goal in protein science. The Protein Data Bank (PDB) is a key resource for defining sequence-structure relationships that has supported the development of critical resources such as rotamer libraries and backbone torsional statistics that quantify the probabilities of protein sequences adopting different structures. Here, we show that well-defined, non-contiguous structural motifs (TERMs) in the PDB can also provide rich information useful for protein-peptide interaction prediction and design. Specifically, we show that it is possible to rapidly predict the binding energies of peptides to Bcl-2 family proteins as accurately as can be done with widely used structure-based tools, without explicit atomistic modeling. One benefit of a TERM-based approach is that prediction performance is less sensitive to the details of the input structure than are methods that evaluate energies using precise atomic coordinates. We show that protein design using TERM energies (dTERMen) can generate highly novel and diverse peptides to target anti-apoptotic proteins Bfl-1 and Mcl-1. 15 of 17 peptides designed using dTERMen bound tightly to their intended targets, and these peptides have just 15 - 38% sequence identity to any known native Bcl-2 family protein ligand. High-resolution structures of four designed peptides bound to their targets provided opportunities to analyze strengths and limitations of this approach. Dramatic success designing peptides using dTERMen, which comprised going from input structure to experimental validation of high-affinity binders in approximately one month, provides strong motivation for further developing TERM-based approaches to design.
Tertiary structural motif sequence statistics enable facile prediction and design of peptides that bind anti-apoptotic Bfl-1 and Mcl-1
bioRxiv
2018
摘要
Understanding the relationship between protein sequence and structure well enough to design new proteins with desired functions is a longstanding goal in protein science. Here, we show that recurring tertiary structural motifs (TERMs) in the PDB provide rich information for protein-peptide interaction prediction and design. TERM statistics can be used to predict peptide binding energies for Bcl-2 family proteins as accurately as widely used structure-based tools. Furthermore, design using TERM energies (dTERMen) rapidly and reliably generates high-affinity peptide binders of anti-apoptotic proteins Bfl-1 and Mcl-1 with just 15%-38% sequence identity to any known native Bcl-2 family protein ligand. High-resolution structures of four designed peptides bound to their targets provide opportunities to analyze the strengths and limitations of the computational design method. Our results support dTERMen as a powerful approach that can complement existing tools for protein engineering.