NPTX1 mutations trigger endoplasmic reticulum stress and cause autosomal dominant cerebellar ataxia.

With more than 40 causative genes identified so far, autosomal dominant cerebellar ataxias exhibit a remarkable genetic heterogeneity. Yet, half the patients are lacking a molecular diagnosis. In a large family with nine sampled affected members, we performed exome sequencing combined with whole-genome linkage analysis. We identified a missense variant in NPTX1, NM_002522.3:c.1165G>A: p.G389R, segregating with the phenotype. Further investigations with whole-exome sequencing and an amplicon-based panel identified four additional unrelated families segregating the same variant, for whom a common founder effect could be excluded. A second missense variant, NM_002522.3:c.980A>G: p.E327G, was identified in a fifth familial case. The NPTX1-associated phenotype consists of a late-onset, slowly progressive, cerebellar ataxia, with downbeat nystagmus, cognitive impairment reminiscent of cerebellar cognitive affective syndrome, myoclonic tremor and mild cerebellar vermian atrophy on brain imaging. NPTX1 encodes the neuronal pentraxin 1, a secreted protein with various cellular and synaptic functions. Both variants affect conserved amino acid residues and are extremely rare or absent from public databases. In COS7 cells, overexpression of both neuronal pentraxin 1 variants altered endoplasmic reticulum morphology and induced ATF6-mediated endoplasmic reticulum stress, associated with cytotoxicity. In addition, the p.E327G variant abolished neuronal pentraxin 1 secretion, as well as its capacity to form a high molecular weight complex with the wild-type protein. Co-immunoprecipitation experiments coupled with mass spectrometry analysis demonstrated abnormal interactions of this variant with the cytoskeleton. In agreement with these observations, in silico modelling of the neuronal pentraxin 1 complex evidenced a destabilizing effect for the p.E327G substitution, located at the interface between monomers. On the contrary, the p.G389 residue, located at the protein surface, had no predictable effect on the complex stability. Our results establish NPTX1 as a new causative gene in autosomal dominant cerebellar ataxias. We suggest that variants in NPTX1 can lead to cerebellar ataxia due to endoplasmic reticulum stress, mediated by ATF6, and associated to a destabilization of NP1 polymers in a dominant-negative manner for one of the variants.

Inhibition of aquaporin-1 prevents myocardial remodeling by blocking the transmembrane transport of hydrogen peroxide.

Pathological remodeling of the myocardium has long been known to involve oxidant signaling, but strategies using systemic antioxidants have generally failed to prevent it. We sought to identify key regulators of oxidant-mediated cardiac hypertrophy amenable to targeted pharmacological therapy. Specific isoforms of the aquaporin water channels have been implicated in oxidant sensing, but their role in heart muscle is unknown. RNA sequencing from human cardiac myocytes revealed that the archetypal AQP1 is a major isoform. AQP1 expression correlates with the severity of hypertrophic remodeling in patients with aortic stenosis. The AQP1 channel was detected at the plasma membrane of human and mouse cardiac myocytes from hypertrophic hearts, where it colocalized with NADPH oxidase-2 and caveolin-3. We show that hydrogen peroxide (H2O2), produced extracellularly, is necessary for the hypertrophic response of isolated cardiac myocytes and that AQP1 facilitates the transmembrane transport of H2O2 through its water pore, resulting in activation of oxidant-sensitive kinases in cardiac myocytes. Structural analysis of the amino acid residues lining the water pore of AQP1 supports its permeation by H2O2 Deletion of Aqp1 or selective blockade of the AQP1 intrasubunit pore inhibited H2O2 transport in mouse and human cells and rescued the myocyte hypertrophy in human induced pluripotent stem cell-derived engineered heart muscle. Treatment of mice with a clinically approved AQP1 inhibitor, Bacopaside, attenuated cardiac hypertrophy. We conclude that cardiac hypertrophy is mediated by the transmembrane transport of H2O2 by the water channel AQP1 and that inhibitors of AQP1 represent new possibilities for treating hypertrophic cardiomyopathies.

Rational antibiotic design: in silico structural comparison of the functional cavities of penicillin-binding proteins and ß-lactamases.

The class of ß-lactam antibiotics has proven highly efficient in targeting bacterial penicillin-binding proteins (PBP) leading to the blocking of the bacterial cell wall synthesis. However, the benefit of these drugs is limited because of bacterial resistance mechanisms; the most widespread resistance involves ß-lactamase enzymes (ßLACT) that inactivate ß-lactam-based molecules. We focused on PBPs and ßLACTs from enterobacteria, and performed a detailed in silico study of PBPs whose inactivation is lethal for the bacteria and of ßLACTs that have a PBP-type catalytic mechanism. The comparison of the sequences and structures of PBPs and ßLACTs shows an almost perfect conservation of the catalytic site, and a high spatial resemblance of the whole functional cavity despite a very low overall sequence identity. Some notable differences in the functional cavity were observed in the vicinity of the catalytic site: four tyrosines are well conserved in the PBPs, whereas the residues occurring at equivalent positions in the ßLACT families present other physicochemical properties. These tyrosines are thus good candidates to be targeted in designing new antibiotic molecules with increased affinity and specificity for PBPs, with the goal of overcoming drug resistance. Our analysis also identified residues that have similar characteristics in most ßLACT families and different properties in PBPs; these are interesting targets for new ligands that specifically inhibit ßLACT proteins. The in silico approach presented here can be extended to other protein systems in view of guiding and improving rational drug design.

The house dust mite allergen Der p 5 binds lipid ligands and stimulates airway epithelial cells through a TLR2-dependent pathway.

BACKGROUND: Protein crystallographic studies suggest that the house dust mite (HDM) allergen Der p 5 potentially interacts with hydrophobic ligands. Der p 5, in association with its ligand(s), might therefore trigger innate immune signalling pathways in the airway epithelium and influence the initiation of the HDM-allergic response. OBJECTIVE: We investigated the lipid binding propensities of recombinant (r)Der p 5 and characterized the signalling pathways triggered by the allergen in airway epithelial cells. METHODS: rDer p 5 was produced in Pichia pastoris and characterized by mass spectrometry, multi-angle light scattering and circular dichroism. Its interactions with hydrophobic ligands were investigated in fluorescence-based lipid binding assays and in-silico docking simulations. Innate immune signalling pathways triggered by rDer p 5 were investigated in airway epithelial cell activation assays in vitro. RESULTS: Biophysical analysis showed that rDer p 5 was monomeric and adopted a similar α-helix-rich fold at both physiological and acidic pH. Spectrofluorimetry experiments showed that rDer p 5 is able to selectively bind lipid ligands, but only under mild acidic pH conditions. Computer-based docking simulations identified potential binding sites for these ligands. This allergen, with putatively associated lipid(s), triggered the production of IL-8 in respiratory epithelial cells through a TLR2-, NF-kB- and MAPK-dependent signalling pathway. CONCLUSIONS AND CLINICAL RELEVANCE: Despite the fact that Der p 5 represents a HDM allergen of intermediate prevalence, our findings regarding its lipid binding and activation of TLR2 indicate that it could participate in the initiation of the HDM-allergic state.

Cross-Reactivity between Major IgE Epitopes of Family 5 Allergens from Dermatophagoides pteronyssinus and Blomia tropicalis.

BACKGROUND: The aim of this work was to understand the molecular features that trigger the cross-reactivity observed between Der p 5 from Dermatophagoides pteronyssinus, Blo t 5 from Blomia tropicalis, and Der f 5 from D. farinae. METHODS: We collected serum from 60 house dust mite (HDM)-allergic patients residing in the Dellys area of Boumerdès province in northern Algeria. The presence of specific IgE to Der p 5, Der f 5, and Blo t 5 was analyzed. We performed in silico analysis of the structure of the different allergens in order to identify epitopes that can elicit the cross-reactivity of the sera. Synthetic peptides corresponding to the linear epitope sequence of Der p 5, Der f 5, and Blo t 5 were used to evaluate its implication in the cross-reactivity between the allergens. We also modified the sequence of the conformational epitope of Der p 5 by site-directed mutagenesis to mimic Blo t 5. RESULTS: Several sera of patients allergic to HDM contained specific IgE antibodies to Der p 5 and Blo t 5. We demonstrated that the linear epitope of Der p 5 and Blo t 5 is not involved in the cross-reactivity of the sera. Furthermore, mutations introduced in the sequence of Der p 5 to mimic Blo t 5 could not modulate the cross-reactivity between them. CONCLUSIONS: The major linear IgE epitopes of Der p 5 and Blo t 5 are involved in species-specific recognition. Our results may be useful for the development of a hypoallergenic vaccine against HDM group 5 allergens.

Immunodominant IgE Epitopes of Der p 5 Allergen.

BACKGROUND: Der p 5 is an important allergen of Dermatophagoides pteronyssinus that plays a key role in allergic airway diseases. Its three dimensional structure (PDB 3MQ1) consists of three anti-parallel α-helices arranged in a helical bundle. Aggregation of Der p5 can modulate its allergenicity. This study aimed to identify the key residues of IgE binding epitopes of Der p 5. METHODS: IgE binding epitopes of Der p 5 were characterized as follow. An in silico prediction of the epitope was performed with the help of SEPPA program. We also made a mapping of the epitope by using an overlapping library of peptides that encompass the sequence of mature Der p 5. Finally, an alanine scanning mutagenesis allowed us to define the key residues of the allergen involved in its interaction with IgE. The integrity of the structure of the different protein's mutants was assessed by far UV circular dichroism. RESULTS: The presented data indicate that the major epitope sequence of Der p 5 is 90DRLMQRKDLDIFEQYNLEM108. Residues L98, D99, I100, F101, E102 and Y104 appear to be important for IgE binding. CONCLUSION: This study highlighted the residues of Der p 5 essential for IgE binding. The identification of the major residues epitope of Der p 5 allergen may participate in the selection and engineering of new hypoallergens used in immunotherapy.

The Hidden Face of Rubisco.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) fixes atmospheric CO2 into organic compounds and is composed of eight copies each of a large subunit (RbcL) and a small subunit (RbcS). Recent reports have revealed unusual RbcS, which are expressed in particular tissues and confer higher catalytic rate, lesser affinity for CO2, and a more acidic profile of the activity versus pH. The resulting Rubisco was proposed to be adapted to a high CO2 environment and recycle CO2 generated by the metabolism. These RbcS belong to a cluster named T (for trichome), phylogenetically distant from cluster M, which gathers well-characterized RbcS expressed in mesophyll or bundle-sheath tissues. Cluster T is largely represented in different plant phyla, including pteridophytes and bryophytes, indicating an ancient origin.

Single Mutations in the Transmembrane Domains of Maize Plasma Membrane Aquaporins Affect the Activity of Monomers within a Heterotetramer.

Aquaporins are channels facilitating the diffusion of water and/or small uncharged solutes across biological membranes. They assemble as homotetramers but some of them also form heterotetramers, especially in plants. In Zea mays, aquaporins belonging to the plasma membrane intrinsic protein (PIP) subfamily are clustered into two groups, PIP1 and PIP2, which exhibit different water-channel activities when expressed in Xenopus oocytes. When PIP1 and PIP2 isoforms are co-expressed, they physically interact to modulate their subcellular localization and channel activity. Here, we demonstrated by affinity chromatography purification that, when co-expressed in Xenopus oocytes, the maize PIP1;2 and PIP2;5 isoforms assemble as homo- and heterodimers within heterotetramers. We built the 3D structure of such heterotetramers by comparative modeling on the basis of the spinach SoPIP2;1 X-ray structure and identified amino acid residues in the transmembrane domains which putatively interact at the interfaces between monomers. Their roles in the water-channel activity, subcellular localization, protein abundance, and physical interaction were investigated by mutagenesis. We highlighted single-residue substitutions that either inactivated PIP2;5 or activated PIP1;2 without affecting their interaction. Interestingly, the Phe220Ala mutation in the transmembrane domain 5 of PIP1;2 activated its water-channel activity and, at the same time, inactivated PIP2;5 within a heterotetramer. Altogether, these data contribute to a better understanding of the interaction mechanisms between PIP isoforms and the role of heterotetramerization on their water-channel activity.

Stability strengths and weaknesses in protein structures detected by statistical potentials: Application to bovine seminal ribonuclease.

We present an in silico method to estimate the contribution of each residue in a protein to its overall stability using three database-derived statistical potentials that are based on inter-residue distances, backbone torsion angles and solvent accessibility, respectively. Residues that contribute very unfavorably to the folding free energy are defined as stability weaknesses, whereas residues that show a highly stabilizing contribution are called stability strengths. Strengths and/or weaknesses on residues that are in spatial contact are clustered into 3-dimensional (3D) stability patches. The identification and analysis of strength- and weakness-containing regions in a protein may reveal structural or functional characteristics, and/or interesting spots to introduce mutations. To illustrate the power of our method, we apply it to bovine seminal ribonuclease. This enzyme catalyzes the degradation of RNA strands, and has the peculiarity of undergoing 3D domain swapping in physiological conditions. The weaknesses and strengths were compared among the monomeric, dimeric and swapped dimeric forms. We identified weaknesses among the catalytic residues and a mixture of weaknesses and strengths among the substrate-binding residues in the three forms. In the regions involved in 3D swapping, we observed an accumulation of weaknesses in the monomer, which disappear in the dimer and especially in the swapped dimer. Moreover, monomeric homologous proteins were found to exhibit less weaknesses in these regions, whereas mutants known to favor unswapped dimerization appear stabilized in this form. Our method has several perspectives for functional annotation, rational prediction of targeted mutations, and mapping of stability changes upon conformational rearrangements.

BeAtMuSiC: Prediction of changes in protein-protein binding affinity on mutations.

The ability of proteins to establish highly selective interactions with a variety of (macro)molecular partners is a crucial prerequisite to the realization of their biological functions. The availability of computational tools to evaluate the impact of mutations on protein-protein binding can therefore be valuable in a wide range of industrial and biomedical applications, and help rationalize the consequences of non-synonymous single-nucleotide polymorphisms. BeAtMuSiC (http://babylone.ulb.ac.be/beatmusic) is a coarse-grained predictor of the changes in binding free energy induced by point mutations. It relies on a set of statistical potentials derived from known protein structures, and combines the effect of the mutation on the strength of the interactions at the interface, and on the overall stability of the complex. The BeAtMuSiC server requires as input the structure of the protein-protein complex, and gives the possibility to assess rapidly all possible mutations in a protein chain or at the interface, with predictive performances that are in line with the best current methodologies.

A conserved cysteine residue is involved in disulfide bond formation between plant plasma membrane aquaporin monomers.

AQPs (aquaporins) are conserved in all kingdoms of life and facilitate the rapid diffusion of water and/or other small solutes across cell membranes. Among the different plant AQPs, PIPs (plasma membrane intrinsic proteins), which fall into two phylogenetic groups, PIP1 and PIP2, play key roles in plant water transport processes. PIPs form tetramers in which each monomer acts as a functional channel. The intermolecular interactions that stabilize PIP oligomer complexes and are responsible for the resistance of PIP dimers to denaturating conditions are not well characterized. In the present study, we identified a highly conserved cysteine residue in loop A of PIP1 and PIP2 proteins and demonstrated by mutagenesis that it is involved in the formation of a disulfide bond between two monomers. Although this cysteine seems not to be involved in regulation of trafficking to the plasma membrane, activity, substrate selectivity or oxidative gating of ZmPIP1s (Zm is Zea mays), ZmPIP2s and hetero-oligomers, it increases oligomer stability under denaturating conditions. In addition, when PIP1 and PIP2 are co-expressed, the loop A cysteine of ZmPIP1;2, but not that of ZmPIP2;5, is involved in the mercury sensitivity of the channels.

Modelling and bioinformatics analysis of the dimeric structure of house dust mite allergens from families 5 and 21: Der f 5 could dimerize as Der p 5.

Allergy represents an increasing thread to public health in both developed and emerging countries and the dust mites Dermatophagoides pteronyssinus (Der p), Blomia tropicalis (Blo t), Dermatophagoides farinae (Der f), Lepidoglyphus destructor (Lep d) and Suidasia medanensis (Sui m) strongly contribute to this problem. Their allergens are classified in several families among which families 5 and 21 which are the subject of this work. Indeed, their biological function as well as the mechanism or epitopes by which they are contributing to the allergic response remain unknown and their tridimensional structures have not been resolved experimentally except for Blo t 5 and Der p 5. Blo t 5 is a monomeric three helical bundle, whereas Der p 5 shows a three helical bundle with a kinked N-terminal helix that assembles in an entangled dimeric structure with a large hydrophobic cavity. This cavity could be involved in the binding of hydrophobic ligands, which in turn could be responsible for the shift of the immune response from tolerance to allergic inflammation. We used molecular modelling approaches to bring out if other house dust mite allergens of families 5 and 21 (Der f 5, Sui m 5, Lep d 5, Der p 21 and Der f 21) could dimerize and form a large cavity in the same way as Der p 5. Monomeric models were first performed with MODELLER using the experimental structures of Der p 5 and Blo t 5 as templates. The ClusPro server processed the selected monomers in order to assess their capacity to form dimeric structures with a positive result for Der p 5 and Der f 5 only. The other allergens (Blo t 5, Sui m 5, Lep d 5, Der p 21 and Der f 21) did not present such a propensity. Moreover, we identified mutations that should destabilize and/or prevent the formation of the Der p 5 dimeric structure. The production of these mutated proteins could help us to understand the role of the dimerization process in the allergic response induced by Der p 5, and if Der p 5 and Der f 5 behave similarly.

Flanking domain stability modulates the aggregation kinetics of a polyglutamine disease protein.

Spinocerebellar Ataxia Type 3 (SCA3) is one of nine polyglutamine (polyQ) diseases that are all characterized by progressive neuronal dysfunction and the presence of neuronal inclusions containing aggregated polyQ protein, suggesting that protein misfolding is a key part of this disease. Ataxin-3, the causative protein of SCA3, contains a globular, structured N-terminal domain (the Josephin domain) and a flexible polyQ-containing C-terminal tail, the repeat-length of which modulates pathogenicity. It has been suggested that the fibrillogenesis pathway of ataxin-3 begins with a non-polyQ-dependent step mediated by Josephin domain interactions, followed by a polyQ-dependent step. To test the involvement of the Josephin domain in ataxin-3 fibrillogenesis, we have created both pathogenic and nonpathogenic length ataxin-3 variants with a stabilized Josephin domain, and have both stabilized and destabilized the isolated Josephin domain. We show that changing the thermodynamic stability of the Josephin domain modulates ataxin-3 fibrillogenesis. These data support the hypothesis that the first stage of ataxin-3 fibrillogenesis is caused by interactions involving the non-polyQ containing Josephin domain and that the thermodynamic stability of this domain is linked to the aggregation propensity of ataxin-3.

PoPMuSiC 2.1: a web server for the estimation of protein stability changes upon mutation and sequence optimality.

BACKGROUND: The rational design of modified proteins with controlled stability is of extreme importance in a whole range of applications, notably in the biotechnological and environmental areas, where proteins are used for their catalytic or other functional activities. Future breakthroughs in medical research may also be expected from an improved understanding of the effect of naturally occurring disease-causing mutations on the molecular level. RESULTS: PoPMuSiC-2.1 is a web server that predicts the thermodynamic stability changes caused by single site mutations in proteins, using a linear combination of statistical potentials whose coefficients depend on the solvent accessibility of the mutated residue. PoPMuSiC presents good prediction performances (correlation coefficient of 0.8 between predicted and measured stability changes, in cross validation, after exclusion of 10% outliers). It is moreover very fast, allowing the prediction of the stability changes resulting from all possible mutations in a medium size protein in less than a minute. This unique functionality is user-friendly implemented in PoPMuSiC and is particularly easy to exploit. Another new functionality of our server concerns the estimation of the optimality of each amino acid in the sequence, with respect to the stability of the structure. It may be used to detect structural weaknesses, i.e. clusters of non-optimal residues, which represent particularly interesting sites for introducing targeted mutations. This sequence optimality data is also expected to have significant implications in the prediction and the analysis of particular structural or functional protein regions. To illustrate the interest of this new functionality, we apply it to a dataset of known catalytic sites, and show that a much larger than average concentration of structural weaknesses is detected, quantifying how these sites have been optimized for function rather than stability. CONCLUSION: The freely available PoPMuSiC-2.1 web server is highly useful for identifying very rapidly a list of possibly relevant mutations with the desired stability properties, on which subsequent experimental studies can be focused. It can also be used to detect sequence regions corresponding to structural weaknesses, which could be functionally important or structurally delicate regions, with obvious applications in rational protein design.

Evidence that interaction between conserved residues in transmembrane helices 2, 3, and 7 are crucial for human VPAC1 receptor activation.

The VPAC(1) receptor belongs to family B of G protein-coupled receptors (GPCR-B) and is activated upon binding of the vasoactive intestinal peptide (VIP). Despite the recent determination of the structure of the N terminus of several members of this receptor family, little is known about the structure of the transmembrane (TM) region and about the molecular mechanisms leading to activation. In the present study, we designed a new structural model of the TM domain and combined it with experimental mutagenesis experiments to investigate the interaction network that governs ligand binding and receptor activation. Our results suggest that this network involves the cluster of residues Arg(188) in TM2, Gln(380) in TM7, and Asn(229) in TM3. This cluster is expected to be altered upon VIP binding, because Arg(188) has been shown previously to interact with Asp(3) of VIP. Several point mutations at positions 188, 229, and 380 were experimentally characterized and were shown to severely affect VIP binding and/or VIP-mediated cAMP production. Double mutants built from reciprocal residue exchanges exhibit strong cooperative or anticooperative effects, thereby indicating the spatial proximity of residues Arg(188), Gln(380), and Asn(229). Because these residues are highly conserved in the GPCR-B family, they can moreover be expected to have a general role in mediating function.

The first peptides: the evolutionary transition between prebiotic amino acids and early proteins.

The issues we attempt to tackle here are what the first peptides did look like when they emerged on the primitive earth, and what simple catalytic activities they fulfilled. We conjecture that the early functional peptides were short (3-8 amino acids long), were made of those amino acids, Gly, Ala, Val and Asp, that are abundantly produced in many prebiotic synthesis experiments and observed in meteorites, and that the neutralization of Asp's negative charge is achieved by metal ions. We further assume that some traces of these prebiotic peptides still exist, in the form of active sites in present-day proteins. Searching these proteins for prebiotic peptide candidates led us to identify three main classes of motifs, bound mainly to Mg(2+) ions: D(F/Y)DGD corresponding to the active site in RNA polymerases, DGD(G/A)D present in some kinds of mutases, and DAKVGDGD in dihydroxyacetone kinase. All three motifs contain a DGD submotif, which is suggested to be the common ancestor of all active peptides. Moreover, all three manipulate phosphate groups, which was probably a very important biological function in the very first stages of life. The statistical significance of our results is supported by the frequency of these motifs in today's proteins, which is three times higher than expected by chance, with a P-value of 3 x 10(-2). The implications of our findings in the context of the appearance of life and the possibility of an experimental validation are discussed.

Fast and accurate predictions of protein stability changes upon mutations using statistical potentials and neural networks: PoPMuSiC-2.0.

MOTIVATION: The rational design of proteins with modified properties, through amino acid substitutions, is of crucial importance in a large variety of applications. Given the huge number of possible substitutions, every protein engineering project would benefit strongly from the guidance of in silico methods able to predict rapidly, and with reasonable accuracy, the stability changes resulting from all possible mutations in a protein. RESULTS: We exploit newly developed statistical potentials, based on a formalism that highlights the coupling between four protein sequence and structure descriptors, and take into account the amino acid volume variation upon mutation. The stability change is expressed as a linear combination of these energy functions, whose proportionality coefficients vary with the solvent accessibility of the mutated residue and are identified with the help of a neural network. A correlation coefficient of R = 0.63 and a root mean square error of sigma(c) = 1.15 kcal/mol between measured and predicted stability changes are obtained upon cross-validation. These scores reach R = 0.79, and sigma(c) = 0.86 kcal/mol after exclusion of 10% outliers. The predictive power of our method is shown to be significantly higher than that of other programs described in the literature. AVAILABILITY: http://babylone.ulb.ac.be/popmusic

SODa: an Mn/Fe superoxide dismutase prediction and design server.

BACKGROUND: Superoxide dismutases (SODs) are ubiquitous metalloenzymes that play an important role in the defense of aerobic organisms against oxidative stress, by converting reactive oxygen species into nontoxic molecules. We focus here on the SOD family that uses Fe or Mn as cofactor. RESULTS: The SODa webtool http://babylone.ulb.ac.be/soda predicts if a target sequence corresponds to an Fe/Mn SOD. If so, it predicts the metal ion specificity (Fe, Mn or cambialistic) and the oligomerization mode (dimer or tetramer) of the target. In addition, SODa proposes a list of residue substitutions likely to improve the predicted preferences for the metal cofactor and oligomerization mode. The method is based on residue fingerprints, consisting of residues conserved in SOD sequences or typical of SOD subgroups, and of interaction fingerprints, containing residue pairs that are in contact in SOD structures. CONCLUSION: SODa is shown to outperform and to be more discriminative than traditional techniques based on pairwise sequence alignments. Moreover, the fact that it proposes selected mutations makes it a valuable tool for rational protein design.

Mn/Fe superoxide dismutase interaction fingerprints and prediction of oligomerization and metal cofactor from sequence.

Fe- and Mn-containing superoxide dismutase (sod) enzymes are closely related and similar in both amino acid sequence and structure, but differ in their mode of oligomerization and in their specificity for the Fe or Mn cofactor. The goal of the present work is to identify and analyze the sequence and structure characteristics that ensure the cofactor specificities and the oligomerization modes. For that purpose, 374 sod sequences and 17 sod crystal structures were collected and aligned. These alignments were searched for residues and inter-residue interactions that are conserved within the whole sod family, or alternatively, that are specific to a given sod subfamily sharing common characteristics. This led us to define key residues and inter-residue interaction fingerprints in each subfamily. The comparison of these fingerprints allows, on a rational basis, the design of mutants likely to modulate the activity and/or specificity of the target sod, in good agreement with the available experimental results on known mutants. The key residues and interaction fingerprints are furthermore used to predict if a novel sequence corresponds to a sod enzyme, and if so, what type of sod it is. The predictions of this fingerprint method reach much higher scores and present much more discriminative power than the commonly used method that uses pairwise sequence comparisons.

Enhancing the stability and solubility of TEV protease using in silico design.

The ability to rationally increase the stability and solubility of recombinant proteins has long been a goal of biotechnology and has significant implications for biomedical research. Poorly soluble enzymes, for example, result in the need for larger reaction volumes, longer incubation times, and more restricted reaction conditions, all of which increase the cost and have a negative impact on the feasibility of the process. Rational design is achieved here by means of the PoPMuSiC program, which performs in silico predictions of stability changes upon single-site mutations. We have used this program to increase the stability of the tobacco etch virus (TEV) protein. TEV is a 27-kDa nuclear inclusion protease with stringent specificity that is commonly used for the removal of solubility tags during protein purification protocols. However, while recombinant TEV can be produced in large quantities, a limitation is its relatively poor solubility (generally approximately 1 mg/mL), which means that large volumes and often long incubation times are required for efficient cleavage. Following PoPMuSiC analysis of TEV, five variants predicted to be more stable than the wild type were selected for experimental analysis of their stability, solubility, and activity. Of these, two were found to enhance the solubility of TEV without compromising its functional activity. In addition, a fully active double mutant was found to remain soluble at concentrations in excess of 40 mg/mL. This modified TEV appears thus as an interesting candidate to be used in recombinant protein technology.