HotSpot Wizard: a web server for identification of hot spots in protein engineering

Abstract

HotSpot Wizard is a web server for automatic identification of 'hot spots' for engineering of substrate specificity, activity or enantioselectivity of enzymes and for annotation of protein structures. The web server implements the protein engineering protocol, which targets evolutionarily variable amino acid positions located in the active site or lining the access tunnels. The 'hot spots' for mutagenesis are selected through the integration of structural, functional and evolutionary information obtained from: (i) the databases RCSB PDB, UniProt, PDBSWS, Catalytic Site Atlas and nr NCBI and (ii) the tools CASTp, CAVER, BLAST, CD-HIT, MUSCLE and Rate4Site. The protein structure and e-mail address are the only obligatory inputs for the calculation. In the output, HotSpot Wizard lists annotated residues ordered by estimated mutability. The results of the analysis are mapped on the enzyme structure and visualized in the web browser using Jmol. The HotSpot Wizard server should be useful for protein engineers interested in exploring the structure of their favourite protein and for the design of mutations in site-directed mutagenesis and focused directed evolution experiments. HotSpot Wizard is available at http://loschmidt.chemi.muni.cz/hotspotwizard/.

Figure 1.

Flow chart of the HotSpot Wizard calculation. The input is either a PDB code or a protein structure in PDB format. The output is an annotated structure. The ‘hot spots’ for mutagenesis are selected through the integration of structural, functional and evolutionary information obtained from the bioinformatics databases (database symbol) and the tools (computational tool symbol).

Figure 2.

Graphic interface of the HotSpot Wizard results. (A) The ‘Results Browser’ includes the embedded Jmol applet enabling visualization of the annotated structure, ligands and identified tunnels. ‘Hot spots’ and functional residues are highlighted and colored according to their estimated mutability. The ‘Results Browser’ further offers the ‘Job’ panel enabling navigation through the results, ‘Control’ panel providing basic operations for manipulating the structure, annotated sequence and summary tables of ‘hot spots’, functional residues, all residues, active sites, pockets and tunnels. (B) The mutability color scale is defined in the ‘Color coding’ panel. (C) The ‘Sequence’ panel is interactively interconnected with all other sections of the ‘Results Browser’. (D) The output is summarized in the ‘Mutagenesis Hot Spots’ table listing all identified ‘hot spots’ ordered by their mutability. For individual residues, information about their mutability, structural location, functional role and annotations are provided.

Figure 3.

Comparison of the results from HotSpot Wizard with the experimental data. HotSpot Wizard identified seventeen ‘hot spots’ (balls) lining the active site pocket of the enzyme DhaA (PDB code 1BN6) and its two tunnels (tubes). All identified ‘hot spots’ could accommodate a wide range of mutations in the gene saturation experiment, except F142 (blue ball), which is adjacent to the catalytic residue. Four predicted ‘hot spots’ (red balls) were verified experimentally in three independent directed evolution experiments, showing a key role of these residues for the catalytic activity of DhaA. The picture was prepared in PyMOL using the Python script generated by the HotSpot Wizard server.