Crystal structure of Taq DNA polymerase in complex with an inhibitory Fab: the Fab is directed against an intermediate in the helix-coil dynamics of the enzyme

Abstract

We report the crystal structure of Thermus aquaticus DNA polymerase I in complex with an inhibitory Fab, TP7, directed against the native enzyme. Some of the residues present in a helical conformation in the native enzyme have adopted a gamma turn conformation in the complex. Taken together, structural information that describes alteration of helical 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 intermediate in the helix-coil dynamics of this helix by the Fab. Antibodies directed against modified helices in proteins have long been anticipated. The present structure provides direct crystallographic evidence. The Fab binds within the DNA binding cleft of the polymerase domain, interacting with several residues that are used by the enzyme in binding the primer:template complex. This result unequivocally corroborates inferences drawn from binding experiments and modeling calculations that 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 reveal the structural basis of its preference for binding to DNA polymerases 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 remarkably high relative mobility between these two domains.

Figure 1

Electron density map with refined model superimposed. Shown is a stereo representation of a 2F_o − F_c map, contoured at 1.1 σ. Phases were generated by removing residues 520–560 in TaqP and refining the structure for 40 cycles with tight geometric restraints. Final refined model for residues 540–550 in TaqP is superimposed. All residues are labeled. The figure was made by using setor (47).

Figure 2

Changes in TaqP on complex formation. (a) Superposition of the Cα drawing (gray ribbon) of native TaqP (4) with that (pink) of the enzyme in the complex. Polymerase (pol) and structure-specific exonuclease (nuc) in native structure are labeled. New orientation of the nuc domain shown is highlighted in blue. Zones of residues showing a mainchain rmsd >2.0 Å in the complex are labeled 1 through 5. Residues 527 to 552 in helix I are highlighted in red. (b) Expanded view of changes in Helix I. A Cα ribbon drawing of helix I in native TaqP (gray) is superimposed over that in this complex (red). The figures were made with grasp (48).

Figure 3

Overall View of the complex. Cα ribbon drawings of the pol domain (magenta), with the V-edit subdomain colored orange, and light (green) and heavy chains (cyan) are shown. Residues 540–550 in the helix are colored yellow. The figure was made with grasp (48).

Figure 4

Stereo view of the TaqP-TP7 interface. Parts of Cα ribbons of the pol domain and TP7, color coded as in Fig. 3, are shown. Side-chain atoms of all residues listed in Table 2 are shown as stick drawings. Carbon, nitrogen, and oxygen atoms of the sidechains are colored gray, blue, and red respectively. The figure was made with grasp (48).