CRYSTAL-STRUCTURE OF TAQ DNA-POLYMERASE IN COMPLEX WITH AN INHIBITORYFAB - THE FAB IS DIRECTED AGAINST AN INTERMEDIATE IN THE HELIX-COIL DYNAMICS OF THE ENZYME

We report the crystal structure of Thermus aquaticus DNA polymerase I in complex with an inhibitory Fab, TP7, directed against the native en zyme. Some of the residues present in a helical conformation in the na tive enzyme have adopted a gamma turn conformation in the complex. Tak en together, structural information that describes alteration of helic al structure and solution studies that demonstrate the ability of TP7 to inhibit 100% of the polymerase activity of the enzyme suggest that the change in conformation is probably caused by trapping of an interm ediate in the helix-coil dynamics of this helix by the Fab, Antibodies directed against modified helices in proteins have long been anticipa ted. The present structure provides direct crystallographic evidence, The Fab binds within the DNA binding cleft of the polymerase domain, i nteracting with several residues that are used by the enzyme in bindin g the primer:template complex. This result unequivocally corroborates inferences drawn from binding experiments and modeling calculations th at the inhibitory activity of this Fab is directly attributable to its interference with DNA binding by the polymerase domain of the enzyme, The combination of interactions made by the Fab residues in both the polymerase and the vestigial editing nuclease domain of the enzyme rev eal the structural basis of its preference for binding to DNA polymera ses of the Thermus species. The orientation of the structure-specific nuclease domain with respect to the polymerase domain is significantly different from that seen in other structures of this polymerase. This reorientation does not appear to be antibody-induced and implies rema rkably high relative mobility between these two domains.