Systematic study of the functions for the residues around the nucleotide pocket in simian virus 40 AAA+ hexameric helicase.
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.
Greenleaf
William B
WB
Department of Molecular and Computational Biology, University of Southern California, Los Angeles, California 90089-1340, USA.
Shen
Jingping
J
Gai
Dahai
D
Chen
Xiaojiang S
XS
D000251:Adenosine Triphosphatases / Q000032:analysis; D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D000957:Antigens, Viral, Tumor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases / Q000032:analysis / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013539:Simian virus 40 / Q000235:genetics*
D000957:Antigens, Viral, Tumor; D004277:DNA, Single-Stranded; D009711:Nucleotides; D010761:Phosphorus Radioisotopes; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D000409:Alanine