Engineering of weak helper interactions for high-efficiency FRET probes.

Fluorescence resonance energy transfer (FRET)-based detection of protein interactions is limited by the very narrow range of FRET-permitting distances. We show two different strategies for the rational design of weak helper interactions that co-recruit donor and acceptor fluorophores for a more robust detection of bimolecular FRET: (i) in silico design of electrostatically driven encounter complexes and (ii) fusion of tunable domain-peptide interaction modules based on WW or SH3 domains. We tested each strategy for optimization of FRET between (m)Citrine and mCherry, which do not natively interact. Both approaches yielded comparable and large increases in FRET efficiencies with little or no background. Helper-interaction modules can be fused to any pair of fluorescent proteins and could, we found, enhance FRET between mTFP1 and mCherry as well as between mTurquoise2 and mCitrine. We applied enhanced helper-interaction FRET (hiFRET) probes to study the binding between full-length H-Ras and Raf1 as well as the drug-induced interaction between Raf1 and B-Raf.

Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases.

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.

BriX: a database of protein building blocks for structural analysis, modeling and design.

High-resolution structures of proteins remain the most valuable source for understanding their function in the cell and provide leads for drug design. Since the availability of sufficient protein structures to tackle complex problems such as modeling backbone moves or docking remains a problem, alternative approaches using small, recurrent protein fragments have been employed. Here we present two databases that provide a vast resource for implementing such fragment-based strategies. The BriX database contains fragments from over 7000 non-homologous proteins from the Astral collection, segmented in lengths from 4 to 14 residues and clustered according to structural similarity, summing up to a content of 2 million fragments per length. To overcome the lack of loops classified in BriX, we constructed the Loop BriX database of non-regular structure elements, clustered according to end-to-end distance between the regular residues flanking the loop. Both databases are available online (http://brix.crg.es) and can be accessed through a user-friendly web-interface. For high-throughput queries a web-based API is provided, as well as full database downloads. In addition, two exciting applications are provided as online services: (i) user-submitted structures can be covered on the fly with BriX classes, representing putative structural variation throughout the protein and (ii) gaps or low-confidence regions in these structures can be bridged with matching fragments.

Molecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus.

Homing endonucleases recognize long target DNA sequences generating an accurate double-strand break that promotes gene targeting through homologous recombination. We have modified the homodimeric I-CreI endonuclease through protein engineering to target a specific DNA sequence within the human RAG1 gene. Mutations in RAG1 produce severe combined immunodeficiency (SCID), a monogenic disease leading to defective immune response in the individuals, leaving them vulnerable to infectious diseases. The structures of two engineered heterodimeric variants and one single-chain variant of I-CreI, in complex with a 24-bp oligonucleotide of the human RAG1 gene sequence, show how the DNA binding is achieved through interactions in the major groove. In addition, the introduction of the G19S mutation in the neighborhood of the catalytic site lowers the reaction energy barrier for DNA cleavage without compromising DNA recognition. Gene-targeting experiments in human cell lines show that the designed single-chain molecule preserves its in vivo activity with higher specificity, further enhanced by the G19S mutation. This is the first time that an engineered meganuclease variant targets the human RAG1 locus by stimulating homologous recombination in human cell lines up to 265 bp away from the cleavage site. Our analysis illustrates the key features for à la carte procedure in protein-DNA recognition design, opening new possibilities for SCID patients whose illness can be treated ex vivo.

A graphical interface for the FoldX forcefield.

SUMMARY: A graphical user interface for the FoldX protein design program has been developed as a plugin for the YASARA molecular graphics suite. The most prominent FoldX commands such as free energy difference upon mutagenesis and interaction energy calculations can now be run entirely via a windowed menu system and the results are immediately shown on screen. AVAILABILITY AND IMPLEMENTATION: The plugin is written in Python and is freely available for download at http://foldxyasara.switchlab.org/ and supported on Linux, MacOSX and MS Windows.

Using protein design algorithms to understand the molecular basis of disease caused by protein-DNA interactions: the Pax6 example.

Quite often a single or a combination of protein mutations is linked to specific diseases. However, distinguishing from sequence information which mutations have real effects in the protein's function is not trivial. Protein design tools are commonly used to explain mutations that affect protein stability, or protein-protein interaction, but not for mutations that could affect protein-DNA binding. Here, we used the protein design algorithm FoldX to model all known missense mutations in the paired box domain of Pax6, a highly conserved transcription factor involved in eye development and in several diseases such as aniridia. The validity of FoldX to deal with protein-DNA interactions was demonstrated by showing that high levels of accuracy can be achieved for mutations affecting these interactions. Also we showed that protein-design algorithms can accurately reproduce experimental DNA-binding logos. We conclude that 88% of the Pax6 mutations can be linked to changes in intrinsic stability (77%) and/or to its capabilities to bind DNA (30%). Our study emphasizes the importance of structure-based analysis to understand the molecular basis of diseases and shows that protein-DNA interactions can be analyzed to the same level of accuracy as protein stability, or protein-protein interactions.

PepX: a structural database of non-redundant protein-peptide complexes.

Although protein-peptide interactions are estimated to constitute up to 40% of all protein interactions, relatively little information is available for the structural details of these interactions. Peptide-mediated interactions are a prime target for drug design because they are predominantly present in signaling and regulatory networks. A reliable data set of nonredundant protein-peptide complexes is indispensable as a basis for modeling and design, but current data sets for protein-peptide interactions are often biased towards specific types of interactions or are limited to interactions with small ligands. In PepX (http://pepx.switchlab.org), we have designed an unbiased and exhaustive data set of all protein-peptide complexes available in the Protein Data Bank with peptide lengths up to 35 residues. In addition, these complexes have been clustered based on their binding interfaces rather than sequence homology, providing a set of structurally diverse protein-peptide interactions. The final data set contains 505 unique protein-peptide interface clusters from 1431 complexes. Thorough annotation of each complex with both biological and structural information facilitates searching for and browsing through individual complexes and clusters. Moreover, we provide an additional source of data for peptide design by annotating peptides with naturally occurring backbone variations using fragment clusters from the BriX database.

Directed evolution of prenylated FMN-dependent Fdc supports efficient in vivo isobutene production.

Isobutene is a high value gaseous alkene used as fuel additive and a chemical building block. As an alternative to fossil fuel derived isobutene, we here develop a modified mevalonate pathway for the production of isobutene from glucose in vivo. The final step in the pathway consists of the decarboxylation of 3-methylcrotonic acid, catalysed by an evolved ferulic acid decarboxylase (Fdc) enzyme. Fdc belongs to the prFMN-dependent UbiD enzyme family that catalyses reversible decarboxylation of (hetero)aromatic acids or acrylic acids with extended conjugation. Following a screen of an Fdc library for inherent 3-methylcrotonic acid decarboxylase activity, directed evolution yields variants with up to an 80-fold increase in activity. Crystal structures of the evolved variants reveal that changes in the substrate binding pocket are responsible for increased selectivity. Solution and computational studies suggest that isobutene cycloelimination is rate limiting and strictly dependent on presence of the 3-methyl group.

Computer design of obligate heterodimer meganucleases allows efficient cutting of custom DNA sequences.

Meganucleases cut long (>12 bp) unique sequences in genomes and can be used to induce targeted genome engineering by homologous recombination in the vicinity of their cleavage site. However, the use of natural meganucleases is limited by the repertoire of their target sequences, and considerable efforts have been made to engineer redesigned meganucleases cleaving chosen targets. Homodimeric meganucleases such as I-CreI have provided a scaffold, but can only be modified to recognize new quasi-palindromic DNA sequences, limiting their general applicability. Other groups have used dimer-interface redesign and peptide linkage to control heterodimerization between related meganucleases such as I-DmoI and I-CreI, but until now there has been no application of this aimed specifically at the scaffolds from existing combinatorial libraries of I-CreI. Here, we show that engineering meganucleases to form obligate heterodimers results in functional endonucleases that cut non-palindromic sequences. The protein design algorithm (FoldX v2.7) was used to design specific heterodimer interfaces between two meganuclease monomers, which were themselves engineered to recognize different DNA sequences. The new monomers favour functional heterodimer formation and prevent homodimer site recognition. This design massively increases the potential repertoire of DNA sequences that can be specifically targeted by designed I-CreI meganucleases and opens the way to safer targeted genome engineering.

How protein stability and new functions trade off.

Numerous studies have noted that the evolution of new enzymatic specificities is accompanied by loss of the protein's thermodynamic stability (DeltaDeltaG), thus suggesting a tradeoff between the acquisition of new enzymatic functions and stability. However, since most mutations are destabilizing (DeltaDeltaG>0), one should ask how destabilizing mutations that confer new or altered enzymatic functions relative to all other mutations are. We applied DeltaDeltaG computations by FoldX to analyze the effects of 548 mutations that arose from the directed evolution of 22 different enzymes. The stability effects, location, and type of function-altering mutations were compared to DeltaDeltaG changes arising from all possible point mutations in the same enzymes. We found that mutations that modulate enzymatic functions are mostly destabilizing (average DeltaDeltaG = +0.9 kcal/mol), and are almost as destabilizing as the "average" mutation in these enzymes (+1.3 kcal/mol). Although their stability effects are not as dramatic as in key catalytic residues, mutations that modify the substrate binding pockets, and thus mediate new enzymatic specificities, place a larger stability burden than surface mutations that underline neutral, non-adaptive evolutionary changes. How are the destabilizing effects of functional mutations balanced to enable adaptation? Our analysis also indicated that many mutations that appear in directed evolution variants with no obvious role in the new function exert stabilizing effects that may compensate for the destabilizing effects of the crucial function-altering mutations. Thus, the evolution of new enzymatic activities, both in nature and in the laboratory, is dependent on the compensatory, stabilizing effect of apparently "silent" mutations in regions of the protein that are irrelevant to its function.

Genome-wide prediction of SH2 domain targets using structural information and the FoldX algorithm.

Current experiments likely cover only a fraction of all protein-protein interactions. Here, we developed a method to predict SH2-mediated protein-protein interactions using the structure of SH2-phosphopeptide complexes and the FoldX algorithm. We show that our approach performs similarly to experimentally derived consensus sequences and substitution matrices at predicting known in vitro and in vivo targets of SH2 domains. We use our method to provide a set of high-confidence interactions for human SH2 domains with known structure filtered on secondary structure and phosphorylation state. We validated the predictions using literature-derived SH2 interactions and a probabilistic score obtained from a naive Bayes integration of information on coexpression, conservation of the interaction in other species, shared interaction partners, and functions. We show how our predictions lead to a new hypothesis for the role of SH2 domains in signaling.

Reconstruction of protein backbones from the BriX collection of canonical protein fragments.

As modeling of changes in backbone conformation still lacks a computationally efficient solution, we developed a discretisation of the conformational states accessible to the protein backbone similar to the successful rotamer approach in side chains. The BriX fragment database, consisting of fragments from 4 to 14 residues long, was realized through identification of recurrent backbone fragments from a non-redundant set of high-resolution protein structures. BriX contains an alphabet of more than 1,000 frequently observed conformations per peptide length for 6 different variation levels. Analysis of the performance of BriX revealed an average structural coverage of protein structures of more than 99% within a root mean square distance (RMSD) of 1 Angstrom. Globally, we are able to reconstruct protein structures with an average accuracy of 0.48 Angstrom RMSD. As expected, regular structures are well covered, but, interestingly, many loop regions that appear irregular at first glance are also found to form a recurrent structural motif, albeit with lower frequency of occurrence than regular secondary structures. Larger loop regions could be completely reconstructed from smaller recurrent elements, between 4 and 8 residues long. Finally, we observed that a significant amount of short sequences tend to display strong structural ambiguity between alpha helix and extended conformations. When the sequence length increases, this so-called sequence plasticity is no longer observed, illustrating the context dependency of polypeptide structures.

The stability effects of protein mutations appear to be universally distributed.

How the thermodynamic stability effects of protein mutations (DeltaDeltaG) are distributed is a fundamental property related to the architecture, tolerance to mutations (mutational robustness), and evolutionary history of proteins. The stability effects of mutations also dictate the rate and dynamics of protein evolution, with deleterious mutations being the main inhibitory factor. Using the FoldX algorithm that attempts to computationally predict DeltaDeltaG effects of mutations, we deduced the overall distributions of stability effects for all possible mutations in 21 different globular, single domain proteins. We found that these distributions are strikingly similar despite a range of sizes and folds, and largely follow a bi-Gaussian function: The surface residues exhibit a narrow distribution with a mildly destabilizing mean DeltaDeltaG ( approximately 0.6 kcal/mol), whereas the core residues exhibit a wider distribution with a stronger destabilizing mean ( approximately 1.4 kcal/mol). Since smaller proteins have a higher fraction of surface residues, the relative weight of these single distributions correlates with size. We also found that proteins evolved in the laboratory follow an essentially identical distribution, whereas de novo designed folds show markedly less destabilizing distributions (i.e. they seem more robust to the effects of mutations). This bi-Gaussian model provides an analytical description of the predicted distributions of mutational stability effects. It comprises a novel tool for analyzing proteins and protein models, for simulating the effect of mutations under evolutionary processes, and a quantitative description of mutational robustness.

Quantifying information transfer by protein domains: analysis of the Fyn SH2 domain structure.

BACKGROUND: Efficient communication between distant sites within a protein is essential for cooperative biological response. Although often associated with large allosteric movements, more subtle changes in protein dynamics can also induce long-range correlations. However, an appropriate formalism that directly relates protein structural dynamics to information exchange between functional sites is still lacking. RESULTS: Here we introduce a method to analyze protein dynamics within the framework of information theory and show that signal transduction within proteins can be considered as a particular instance of communication over a noisy channel. In particular, we analyze the conformational correlations between protein residues and apply the concept of mutual information to quantify information exchange. Mapping out changes of mutual information on the protein structure then allows visualizing how distal communication is achieved. We illustrate the approach by analyzing information transfer by the SH2 domain of Fyn tyrosine kinase, obtained from Monte Carlo dynamics simulations. Our analysis reveals that the Fyn SH2 domain forms a noisy communication channel that couples residues located in the phosphopeptide and specificity binding sites and a number of residues at the other side of the domain near the linkers that connect the SH2 domain to the SH3 and kinase domains. We find that for this particular domain, communication is affected by a series of contiguous residues that connect distal sites by crossing the core of the SH2 domain. CONCLUSION: As a result, our method provides a means to directly map the exchange of biological information on the structure of protein domains, making it clear how binding triggers conformational changes in the protein structure. As such it provides a structural road, next to the existing attempts at sequence level, to predict long-range interactions within protein structures.

Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets.

The last decade has seen the emergence of a universal method for precise and efficient genome engineering. This method relies on the use of sequence-specific endonucleases such as homing endonucleases. The structures of several of these proteins are known, allowing for site-directed mutagenesis of residues essential for DNA binding. Here, we show that a semi-rational approach can be used to derive hundreds of novel proteins from I-CreI, a homing endonuclease from the LAGLIDADG family. These novel endonucleases display a wide range of cleavage patterns in yeast and mammalian cells that in most cases are highly specific and distinct from I-CreI. Second, rules for protein/DNA interaction can be inferred from statistical analysis. Third, novel endonucleases can be combined to create heterodimeric protein species, thereby greatly enhancing the number of potential targets. These results describe a straightforward approach for engineering novel endonucleases with tailored specificities, while preserving the activity and specificity of natural homing endonucleases, and thereby deliver new tools for genome engineering.

Rational design of a CD4 mimic that inhibits HIV-1 entry and exposes cryptic neutralization epitopes.

The conserved surfaces of the human immunodeficiency virus (HIV)-1 envelope involved in receptor binding represent potential targets for the development of entry inhibitors and neutralizing antibodies. Using structural information on a CD4-gp120-17b antibody complex, we have designed a 27-amino acid CD4 mimic, CD4M33, that presents optimal interactions with gp120 and binds to viral particles and diverse HIV-1 envelopes with CD4-like affinity. This mini-CD4 inhibits infection of both immortalized and primary cells by HIV-1, including primary patient isolates that are generally resistant to inhibition by soluble CD4. Furthermore, CD4M33 possesses functional properties of CD4, including the ability to unmask conserved neutralization epitopes of gp120 that are cryptic on the unbound glycoprotein. CD4M33 is a prototype of inhibitors of HIV-1 entry and, in complex with envelope proteins, a potential component of vaccine formulations, or a molecular target in phage display technology to develop broad-spectrum neutralizing antibodies.

The FoldX web server: an online force field.

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 http://foldx.embl.de/. 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.

SNPeffect: a database mapping molecular phenotypic effects of human non-synonymous coding SNPs.

Single nucleotide polymorphisms (SNPs) are an increasingly important tool for genetic and biomedical research. However, the accumulated sequence information on allelic variation is not matched by an understanding of the effect of SNPs on the functional attributes or 'molecular phenotype' of a protein. Towards this aim we developed SNPeffect, an online resource of human non-synonymous coding SNPs (nsSNPs) mapping phenotypic effects of allelic variation in human genes. SNPeffect contains 31 659 nsSNPs from 12 480 human proteins. The current release of SNPeffect incorporates data on protein stability, integrity of functional sites, protein phosphorylation and glycosylation, subcellular localization, protein turnover rates, protein aggregation, amyloidosis and chaperone interaction. The SNP entries are accessible through both a search and browse interface and are linked to most major biological databases. The data can be displayed as detailed descriptions of individual SNPs or as an overview of all SNPs for a given protein. SNPeffect will be regularly updated and can be accessed at http://snpeffect.vib.be/.

Scorpion-toxin mimics of CD4 in complex with human immunodeficiency virus gp120 crystal structures, molecular mimicry, and neutralization breadth.

The binding surface on CD4 for the HIV-1 gp120 envelope glycoprotein has been transplanted previously onto a scorpion-toxin scaffold. Here, we use X-ray crystallography to characterize atomic-level details of gp120 with this transplant, CD4M33. Despite known envelope flexibility, the conformation of gp120 induced by CD4M33 was so similar to that induced by CD4 that localized measures were required to distinguish ligand-induced differences from lattice variation. To investigate relationships between structure, function, and mimicry, an F23 analog of CD4M33 was devised. Structural and thermodynamic analyses showed F23 to be a better molecular mimic of CD4 than CD4M33. F23 also showed increased neutralization breadth, against diverse isolates of HIV-1, HIV-2, and SIVcpz. Our results lend insight into the stability of the CD4 bound conformation of gp120, define measures that quantify molecular mimicry as a function of evolutionary distance, and suggest how such evaluations might be useful in developing mimetic antagonists with increased neutralization breadth.

Design of miniproteins by the transfer of active sites onto small-size scaffolds.

Natural miniproteins (e.g., animal toxins, protease inhibitors, defensins) can express specific and powerful biological activities by using a stable and minimal (<80 amino acids) structural motif. Artificial activities have been designed on these miniscaffolds by transferring previously identified protein active sites into regions structurally compatible with the site and permissive for sequence mutations. These newly designed miniproteins, presenting a specific and high activity within a small size and well-defined three-dimensional structure, represent novel tools in biology, biotechnology, and medical sciences, and are also useful intermediates to develop new therapeutic agents. The different steps used to design and characterize new bioactive miniproteins are here described in detail. Two successful examples are here reported. The first one is a metal-binding miniprotein (MBP, 37 residues), which possesses a metal specificity resembling that of natural carbonic anhydrase; the second is a CD4 mimic (CD4M33, 27 residues), which is a powerful inhibitor of HIV-1 entry but also a fully functional substitute of the human receptor CD4 and, hence, a potential component of an AIDS vaccine.