The Glutamate Switch of Bacteriophage T7 DNA Helicase: ROLE IN COUPLING NUCLEOTIDE TRIPHOSPHATE (NTP) AND DNA BINDING TO NTP HYDROLYSIS

Ajit K Satapathy,Charles C Richardson

doi:10.1074/jbc.m111.218651

Abstract

The DNA helicase encoded by gene 4 of bacteriophage T7 forms a hexameric ring in the presence of dTTP, allowing it to bind DNA in its central core. The oligomerization also creates nucleotide-binding sites located at the interfaces of the subunits. DNA binding stimulates the hydrolysis of dTTP but the mechanism for this two-step control is not clear. We have identified a glutamate switch, analogous to the glutamate switch found in AAA+ enzymes that couples dTTP hydrolysis to DNA binding. A crystal structure of T7 helicase shows that a glutamate residue (Glu-343), located at the subunit interface, is positioned to catalyze a nucleophilic attack on the γ-phosphate of a bound nucleoside 5'-triphosphate. However, in the absence of a nucleotide, Glu-343 changes orientation, interacting with Arg-493 on the adjacent subunit. This interaction interrupts the interaction of Arg-493 with Asn-468 of the central β-hairpin, which in turn disrupts DNA binding. When Glu-343 is replaced with glutamine the altered helicase, unlike the wild-type helicase, binds DNA in the presence of dTDP. When both Arg-493 and Asn-468 are replaced with alanine, dTTP hydrolysis is no longer stimulated in the presence of DNA. Taken together, these results suggest that the orientation of Glu-343 plays a key role in coupling nucleotide hydrolysis to the binding of DNA.

Highlights

Helicases are molecular motors that translocate unidirectionally along single-stranded nucleic acids using energy derived from nucleotide hydrolysis [1,2,3]
This study suggests that hydrogen bonding between Arg493 and Asn-468 from adjacent subunits is critical for the DNA binding ability of the T7 helicase [16]
Biochemical characterization of the altered helicases along with the structural interpretation of the results provides evidence that Arg-493 acts as a glutamate switch to control singlestranded DNA (ssDNA)-dependent nucleotide hydrolysis, and nucleotide-dependent ssDNA binding

Summary

Introduction

Helicases are molecular motors that translocate unidirectionally along single-stranded nucleic acids using energy derived from nucleotide hydrolysis [1,2,3]. Like the SF IV group of helicases, AAAϩ ATPases (ATPases associated with diverse cellular activities) form oligomeric structures (often hexamers) that form a ring-shaped structure with a central pore These proteins function as a molecular motor that couples ATP binding and hydrolysis to changes in conformational states. Analysis of the crystal structures of several AAAϩ proteins containing ATP or ADP with or without DNA enabled a comparison of the position of the glutamate in different states [13]. The nucleotide-binding sites in the structure reveals three different states of nucleotide occupancy designated as NTP-binding competent, NDP-binding competent, and empty state In such a crystal structure model it became possible to locate the position of the catalytic glutamate in response to the presence or absence of nucleotide (AMPPNP) (Fig. 1A). Arg-493 may play a crucial role in nucleotide hydrolysis by controlling the positioning of Glu-343 between active and inactive status

Methods

Results

Conclusion