Accurate profiling of full-length Fv in highly homologous antibody libraries using UMI tagged short reads.

Deep parallel sequencing (NGS) is a viable tool for monitoring scFv and Fab library dynamics in many antibody engineering high-throughput screening efforts. Although very useful, the commonly used Illumina NGS platform cannot handle the entire sequence of scFv or Fab in a single read, usually focusing on specific CDRs or resorting to sequencing VH and VL variable domains separately, thus limiting its utility in comprehensive monitoring of selection dynamics. Here we present a simple and robust method for deep sequencing repertoires of full length scFv, Fab and Fv antibody sequences. This process utilizes standard molecular procedures and unique molecular identifiers (UMI) to pair separately sequenced VH and VL. We show that UMI assisted VH-VL matching allows for a comprehensive and highly accurate mapping of full length Fv clonal dynamics in large highly homologous antibody libraries, as well as identification of rare variants. In addition to its utility in synthetic antibody discovery processes, our method can be instrumental in generating large datasets for machine learning (ML) applications, which in the field of antibody engineering has been hampered by conspicuous paucity of large scale full length Fv data.

The α-helical structure of prodomains promotes translocation of intrinsically disordered neuropeptide hormones into the endoplasmic reticulum.

Different neuropeptide hormones, which are either too small to adopt a stable conformation or are predicted to be intrinsically disordered, are synthesized as larger precursors containing a prodomain in addition to an N-terminal signal peptide. We analyzed the biogenesis of three unstructured neuropeptide hormones and observed that translocation of these precursors into the lumen of the endoplasmic reticulum (ER) is critically dependent on the presence of the prodomain. The hormone domains could be deleted from the precursors without interfering with ER import and secretion, whereas constructs lacking the prodomain remained in the cytosol. Domain-swapping experiments revealed that the activity of the prodomains to promote productive ER import resides in their ability to adopt an α-helical structure. Removal of the prodomain from the precursor did not interfere with co-translational targeting of the nascent chain to the Sec61 translocon but with its subsequent productive translocation into the ER lumen. Our study reveals a novel function of prodomains to enable import of small or intrinsically disordered secretory proteins into the ER based on their ability to adopt an α-helical conformation.

"Redirecting an anti-IL-1ß antibody to bind a new, unrelated and computationally predicted epitope on hIL-17A".

Antibody engineering technology is at the forefront of therapeutic antibody development. The primary goal for engineering a therapeutic antibody is the generation of an antibody with a desired specificity, affinity, function, and developability profile. Mature antibodies are considered antigen specific, which may preclude their use as a starting point for antibody engineering. Here, we explore the plasticity of mature antibodies by engineering novel specificity and function to a pre-selected antibody template. Using a small, focused library, we engineered AAL160, an anti-IL-1ß antibody, to bind the unrelated antigen IL-17A, with the introduction of seven mutations. The final redesigned antibody, 11.003, retains favorable biophysical properties, binds IL-17A with sub-nanomolar affinity, inhibits IL-17A binding to its cognate receptor and is functional in a cell-based assay. The epitope of the engineered antibody can be computationally predicted based on the sequence of the template antibody, as is confirmed by the crystal structure of the 11.003/IL-17A complex. The structures of the 11.003/IL-17A and the AAL160/IL-1ß complexes highlight the contribution of germline residues to the paratopes of both the template and re-designed antibody. This case study suggests that the inherent plasticity of antibodies allows for re-engineering of mature antibodies to new targets, while maintaining desirable developability profiles.

iDBPs: a web server for the identification of DNA binding proteins.

SUMMARY: The iDBPs server uses the three-dimensional (3D) structure of a query protein to predict whether it binds DNA. First, the algorithm predicts the functional region of the protein based on its evolutionary profile; the assumption is that large clusters of conserved residues are good markers of functional regions. Next, various characteristics of the predicted functional region as well as global features of the protein are calculated, such as the average surface electrostatic potential, the dipole moment and cluster-based amino acid conservation patterns. Finally, a random forests classifier is used to predict whether the query protein is likely to bind DNA and to estimate the prediction confidence. We have trained and tested the classifier on various datasets and shown that it outperformed related methods. On a dataset that reflects the fraction of DNA binding proteins (DBPs) in a proteome, the area under the ROC curve was 0.90. The application of the server to an updated version of the N-Func database, which contains proteins of unknown function with solved 3D-structure, suggested new putative DBPs for experimental studies. AVAILABILITY: http://idbps.tau.ac.il/

The ConSurf-DB: pre-calculated evolutionary conservation profiles of protein structures.

ConSurf-DB is a repository for evolutionary conservation analysis of the proteins of known structures in the Protein Data Bank (PDB). Sequence homologues of each of the PDB entries were collected and aligned using standard methods. The evolutionary conservation of each amino acid position in the alignment was calculated using the Rate4Site algorithm, implemented in the ConSurf web server. The algorithm takes into account the phylogenetic relations between the aligned proteins and the stochastic nature of the evolutionary process explicitly. Rate4Site assigns a conservation level for each position in the multiple sequence alignment using an empirical Bayesian inference. Visual inspection of the conservation patterns on the 3D structure often enables the identification of key residues that comprise the functionally important regions of the protein. The repository is updated with the latest PDB entries on a monthly basis and will be rebuilt annually. ConSurf-DB is available online at http://consurfdb.tau.ac.il/

Detection of functionally important regions in "hypothetical proteins" of known structure.

Structural genomics initiatives provide ample structures of "hypothetical proteins" (i.e., proteins of unknown function) at an ever increasing rate. However, without function annotation, this structural goldmine is of little use to biologists who are interested in particular molecular systems. To this end, we used (an improved version of) the PatchFinder algorithm for the detection of functional regions on the protein surface, which could mediate its interactions with, e.g., substrates, ligands, and other proteins. Examination, using a data set of annotated proteins, showed that PatchFinder outperforms similar methods. We collected 757 structures of hypothetical proteins and their predicted functional regions in the N-Func database. Inspection of several of these regions demonstrated that they are useful for function prediction. For example, we suggested an interprotein interface and a putative nucleotide-binding site. A web-server implementation of PatchFinder and the N-Func database are available at http://patchfinder.tau.ac.il/.

Computational Design of Epitope-Specific Functional Antibodies.

The ultimate goal of protein design is to introduce new biological activity. We propose a computational approach for designing functional antibodies by focusing on functional epitopes, integrating large-scale statistical analysis with multiple structural models. Machine learning is used to analyze these models and predict specific residue-residue contacts. We use this approach to design a functional antibody to counter the proinflammatory effect of the cytokine interleukin-17A (IL-17A). X-ray crystallography confirms that the designed antibody binds the targeted epitope and the interaction is mediated by the designed contacts. Cell-based assays confirm that the antibody is functional. Importantly, this approach does not rely on a high-quality 3D model of the designed complex or even a solved structure of the target. As demonstrated here, this approach can be used to design biologically active antibodies, removing some of the main hurdles in antibody design and in drug discovery.

Using a combined computational-experimental approach to predict antibody-specific B cell epitopes.

Antibody epitope mapping is crucial for understanding B cell-mediated immunity and required for characterizing therapeutic antibodies. In contrast to T cell epitope mapping, no computational tools are in widespread use for prediction of B cell epitopes. Here, we show that, utilizing the sequence of an antibody, it is possible to identify discontinuous epitopes on its cognate antigen. The predictions are based on residue-pairing preferences and other interface characteristics. We combined these antibody-specific predictions with results of cross-blocking experiments that identify groups of antibodies with overlapping epitopes to improve the predictions. We validate the high performance of this approach by mapping the epitopes of a set of antibodies against the previously uncharacterized D8 antigen, using complementary techniques to reduce method-specific biases (X-ray crystallography, peptide ELISA, deuterium exchange, and site-directed mutagenesis). These results suggest that antibody-specific computational predictions and simple cross-blocking experiments allow for accurate prediction of residues in conformational B cell epitopes.

Identification of DNA-binding proteins using structural, electrostatic and evolutionary features.

DNA-binding proteins (DBPs) participate in various crucial processes in the life-cycle of the cells, and the identification and characterization of these proteins is of great importance. We present here a random forests classifier for identifying DBPs among proteins with known 3D structures. First, clusters of evolutionarily conserved regions (patches) on the surface of proteins were detected using the PatchFinder algorithm; earlier studies showed that these regions are typically the functionally important regions of proteins. Next, we trained a classifier using features like the electrostatic potential, cluster-based amino acid conservation patterns and the secondary structure content of the patches, as well as features of the whole protein, including its dipole moment. Using 10-fold cross-validation on a dataset of 138 DBPs and 110 proteins that do not bind DNA, the classifier achieved a sensitivity and a specificity of 0.90, which is overall better than the performance of published methods. Furthermore, when we tested five different methods on 11 new DBPs that did not appear in the original dataset, only our method annotated all correctly. The resulting classifier was applied to a collection of 757 proteins of known structure and unknown function. Of these proteins, 218 were predicted to bind DNA, and we anticipate that some of them interact with DNA using new structural motifs. The use of complementary computational tools supports the notion that at least some of them do bind DNA.

Disruption of the Aspergillus fumigatus ECM33 homologue results in rapid conidial germination, antifungal resistance and hypervirulence.

The ECM33/SPS2 family of glycosylphosphatidylinositol-anchored proteins plays an important role in maintaining fungal cell wall integrity and virulence. However, the precise molecular role of these proteins is unknown. In this work, AfuEcm33, the gene encoding the ECM33 homologue in the important pathogenic fungus Aspergillus fumigatus, has been cloned and its function analysed. It is shown that disruption of AfuEcm33 results in rapid conidial germination, increased cell-cell adhesion, resistance to the antifungal agent caspofungin and increased virulence in an immunocompromised mouse model for disseminated aspergillosis. These results suggest that the protein encoded by AfuEcm33 is involved in key aspects of cell wall morphogenesis and plays an important role in A. fumigatus virulence.

In silico identification of functional regions in proteins.

MOTIVATION: In silico prediction of functional regions on protein surfaces, i.e. sites of interaction with DNA, ligands, substrates and other proteins, is of utmost importance in various applications in the emerging fields of proteomics and structural genomics. When a sufficient number of homologs is found, powerful prediction schemes can be based on the observation that evolutionarily conserved regions are often functionally important, typically, only the principal functionally important region of the protein is detected, while secondary functional regions with weaker conservation signals are overlooked. Moreover, it is challenging to unambiguously identify the boundaries of the functional regions. METHODS: We present a new methodology, called PatchFinder, that automatically identifies patches of conserved residues that are located in close proximity to each other on the protein surface. PatchFinder is based on the following steps: (1) Assignment of conservation scores to each amino acid position on the protein surface. (2) Assignment of a score to each putative patch, based on its likelihood to be functionally important. The patch of maximum likelihood is considered to be the main functionally important region, and the search is continued for non-overlapping patches of secondary importance. RESULTS: We examined the accuracy of the method using the IGPS enzyme, the SH2 domain and a benchmark set of 112 proteins. These examples demonstrated that PatchFinder is capable of identifying both the main and secondary functional patches. AVAILABILITY: The PatchFinder program is available at: http://ashtoret.tau.ac.il/~nimrodg/