Systematic study of the functions for the residues around the nucleotide pocket in simian virus 40 AAA+ hexameric helicase

Abstract

The high-resolution structural data for simian virus 40 large-T-antigen helicase revealed a set of nine residues bound to ATP/ADP directly or indirectly. The functional role of each of these residues in ATP hydrolysis and also the helicase function of this AAA+ (ATPases associated with various cellular activities) molecular motor are unclear. Here, we report our mutational analysis of each of these residues to examine their functionality in oligomerization, DNA binding, ATP hydrolysis, and double-stranded DNA (dsDNA) unwinding. All mutants were capable of oligomerization in the presence of ATP and could bind single-stranded DNA and dsDNA. ATP hydrolysis was substantially reduced for proteins with mutations of residues making direct contact with the gamma-phosphate of ATP or the apical water molecule. A potentially noncanonical "arginine finger" residue, K418, is critical for ATP hydrolysis and helicase function, suggesting a new type of arginine finger role by a lysine in the stabilization of the transition state during ATP hydrolysis. Interestingly, our mutational data suggest that the positive- and negative-charge interactions in the uniquely observed residue pairs, R498/D499 and R540/D502, in large-T-antigen helicase are critically involved in the transfer of energy of ATP binding/hydrolysis to DNA unwinding.

FIG. 1.

Nine residues around the nucleotide binding pocket of LTAg which interact with ATP or ADP (Protein Data Bank accession no. 1svl and 1svm). (A) Residues that bind ATP at the nucleotide binding cleft between two monomers of LTAg. The backbone of the cis subunit, defined by the location of the ATP binding pocket, is highlighted in gray. The backbone of the trans subunit is highlighted in red. (B) Residues that bind ADP.

FIG. 2.

Relative levels of ATP hydrolysis by WT and mutated LTAgs in the presence of 1 μM poly(deoxyribosyladenine)₅₀ and 1 mM ATP. The presence of helicase activity is indicated below the designation for each protein (+, present; −, absent). The data represent an average of three or more experiments.

FIG. 3.

Results of helicase assay of LTAg mutations. Each lane in all panels contains 15 fmol dsDNA substrate. Three protein concentrations (50, 100, 200 ng protein or 0.85, 1.7, 3.4 pmol monomer) for each protein sample (labeled above the gels) were used in the assay. Lane 1, boiled dsDNA substrate; lane 2, dsDNA substrate alone. The circle labeled with an asterisk indicates radiolabeled ssDNA annealed to nonradiolabeled M13 ssDNA. The horizontal line labeled with an asterisk indicates unwound radiolabeled ssDNA.

FIG. 4.

ssDNA binding curves for WT LTAg in the presence (•) and absence (▪) of 1 mM ATP. ssDNA binding experiments contained 50 nM 80-nucleotide, 5-carboxyfluorescein-labeled ssDNA, 20 mM Tris (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 0.1 mg/ml bovine serum albumin, and the indicated concentration of hexameric LTAg. The ssDNA binding curve with 1 mM ATP fits equation 1, where r_max is 0.24 ± 0.01, apparent K_d (K_d,app) is (84 ± 3) nM, n is 2.1 ± 0.1, and c is 0.144 ± 0.001. The ssDNA binding curve without ATP fits equation 1, where r_max is 0.22 ± 0.01, K_d,app is (180 ± 10) nM, n is 1.8 ± 0.1, and c is 0.131 ± 0.001.