From Protein Structure to Function with Bioinformatics.pdf

12 Prediction of Protein Function from Theoretical Models 313comprising part of the active site – the so called HxK motif (Cymerman et al. 2005).Known members of the PLD superfamily possess a bilobed structure, with a singleactive site composed of two “HxK-Xn-N-Xn-(E/Q/D)” motifs located at the interfacebetween two domains. Based on the alignments alone it was not possible todefine the remaining residues of the active site. Analysis of the placement of particularresidues in the structural model however, delivered this essential information andallowed for the selection of amino acids that potentially could serve for the formationof the catalytic centre (Fig. 12.7). The finding that DNase IIα is a remote relativeof phospholipase D was later confirmed by experimental studies (Schafer et al.2007) and explained unusual features of this nuclease, such as its resistance toEDTA. By similarity to PLD, whose mechanism has been elucidated, it was alsopossible to infer that the reaction of phosphodiester bond hydrolysis by DNase IIαwill proceed by a covalently linked reaction intermediate.The case of DNase IIα exemplifies the bioinformatics can bypass some experimentallimitations (DNase IIα is heavily glycosylated making the enzyme resistant tothe crystallization) and thereby allow further exploration of the protein properties.Fig. 12.7 Structural model of human DNase IIα. The computational analyses enabled the assignmentof DNase IIα as member of PLD family. The enzyme adopts a monomeric structure with apseudodimeric architecture. The two HxK motifs in the N (cartoon light blue representation) andin the C-terminal (cartoon grey representation) domains contain the catalytically relevant aminoacid residues (red and green sticks), which collectively form a single active site. In addition to theidentities of putative catalytic residues, the structural model accounts for the proximal positionsof cysteine residues forming disulphide bonds (orange and dark blue balls), and the exposed characterof N-glycosylated residues (represented as green balls). Putative DNA-binding loops areshown in magenta. The identification of the functionally important residues in the theoreticalmodel can greatly facilitate the process of enzyme engineering