A powerful method to gain biological insights in the functioning of a protein is to use data available for the protein (super) family and transfer this data to the protein of interest. Systematically correlating the sheer amount of data available is difficult and time consuming. Therefore protein superfamily-specific databases are needed. It is shown that such systems can help to predict protein interaction sites, active site residues, residues important in enzyme specificity, protein stabilizing residues, etc. These systems are therefore powerful tools in drug design and protein engineering studies, but building and keeping such systems op to date is time consuming. Here we describe 3DM (ref. 1), a system that can automatically build such protein superfamily databases. The system starts with building a large accurate structure based multiple sequence alignment of a class of proteins (superfamily). Secondly, it collects and stores amino acid associated data, such as mutational information, ligand- and substrate contacts, etc. together with data derived form the alignment such as correlated mutations and conservation information. These information types are linked to the alignment making it possible to easily transfer information from well studied members of the family to the protein of interest. Navigating between the alignment and associated data is done via interactive HTML pages. A 3DM built for the nuclear receptor superfamily predicted a role in cofactor binding for 15 amino acid positions. A literature search revealed that mutating eleven of these positions indeed has an effect on cofactor binding. Two of the remaining four are located in the cofactor binding site, suggesting a direct role in cofactor binding and mutating a third residue indeed showed a profound effect on cofactor binding. The PEP/ICL enzyme superfamily 3DM database predicted an important function for an oxaloacetate hydrolase subfamily specific serine (ref. 2). Site directed mutagenesis of this serine changed substrate specificity which revealed the unknown reaction mechanism (ref. 3).

references

1. Joosten HJ, Folkertsma S, Zimmeren F, Lutje Hulsik DJ , Kuipers R, Ittmann E, Roijen E, Vriend G, Schaap PJ. 3DM: A new generation of molecular-class-specific information systems applied to four protein super-families. Publication in progress.

2. Joosten HJ, Han Y, Niu W, Du J, Vervoort J, Dunaway-Mariano D, Schaap PJ. Identification of fungal oxaloacetate hydrolyase within the isocitrate lyase/PEP mutase enzyme superfamily using a sequence marker based method. Publication in progress

3. Han Y, Joosten HJ, Niu W, Zhao Z, Mariano PS, McCalman MT, Kan JAL, Schaap PJ, Dunaway-Mariano D. oxaloacetate hydrolase: the C-C bond lyase of oxaloacetate of oxalate secreting fungi. Publication in progress

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