HotSpot Wizard: web server for identification of mutagenesis hot spots in enzyme structures

 

A. Pavelka$, E. Chovancová, J. Damborský

 

Loschmidt Laboratories, Institute of Experimental Biology and National Centre for Biomolecular Research
Faculty of Science, Masaryk University, Kamenice 5/A4, 625 00 Brno, Czech Republic

$Email: tonda@chemi.muni.cz

 

      HotSpot Wizard is a web server for identification of hot spots for rational engineering of substrate specificity, activity and enantioselectivity of enzymes. Rational design usually requires considerable knowledge of structure-function relationships of a target protein. Mutations targeting the residues located in the active sites or lining the access tunnels, have a better chance to produce enzyme variants with novel catalytic properties, than mutations localized in other parts of the protein structure. On the other hand, replacement of the amino acid residues that are highly conserved through evolution due to their essential role for the structural stability or functionality, may lead to the loss of enzymatic activity. Mutagenesis of variable sites, which contributes to the substrate binding, transition state stabilization or product release, represents a useful concept in protein engineering [1]. To follow this concept, the users need to have experiences with analyses of protein se­quences and structures. Furthermore, these analyses are time-consuming even for experienced users.

      HotSpot Wizard automates and speeds-up the analysis by integration of structural and evolutionary information obtained from selected bioinformatics databases and tools. HotSpot Wizard requires a structure of the query protein in PDB format and an e-mail address as the only obligatory inputs. In the first step, Catalytic Site Atlas [2] and UniProt [3] databases are used to determine the residues indispensable for enzyme function. HotSpot Wizard then searches for potentially important residues by CASTp [4], identifying the active site pocket, and by CAVER [5, 6], calculating tunnels connecting buried cavities with the outside solvent. Finally, evolutionary conservation of individual positions in the query structure is estimated by ConSurf [7] from the multiple sequence alignment of closely related proteins. In the output, HotSpot Wizard lists residues ordered by predicted suitability for mutagenesis together with information on their conservation level, potential structural and functional importance, available mutagenesis data and existing sequence variants. Results are mapped on the enzyme structure and can be visualized directly in a web browser using Jmol [8] or downloaded to the local computer as input for PyMOL [9] or text files.

      The primary application of HotSpot Wizard is in the rational design of the hot spots for site directed mutagenesis or focused directed evolution. Alternatively, HotSpot Wizard can serve as a structure annotation tool. Altogether, using HotSpot Wizard one can perform several structural and evolutionary analyses at once with minimal demands on a user, making this server potentially useful for experimentalists with no prior knowledge of structural and bioinformatics analyses.

      HotSpot Wizard is freely available at http://loschmidt.chemi.muni.cz/hotspotwizard/.

 

References

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