Analysis of the AAA sensor-2 motif in the C-terminal ATPase domain of Hsp104 with a site-specific fluorescent probe of nucleotide binding.
Hsp104 from Saccharomyces cerevisiae is a hexameric protein with two AAA ATPase domains (N- and C-terminal nucleotide-binding domains NBD1 and NBD2, respectively) per monomer. Our previous analysis of the Hsp104 ATP hydrolysis cycle revealed that NBD1 and NBD2 have very different catalytic properties, but each shows positive cooperativity in hydrolysis. There is also communication between the two domains, in that ATP hydrolysis at NBD1 depends on the nucleotide that is bound to NBD2. Here, we extend our understanding of the Hsp104 ATP hydrolysis cycle through mutagenesis of the AAA sensor-2 motif in NBD2. To do so, we took advantage of the lack of tryptophan residues in Hsp104 to place a single tryptophan in the C-terminal domain (Y819W). The Y819W substitution has no significant effects on folding stability of the C-terminal domain or on ATP hydrolysis by NBD1 or NBD2. The fluorescence of this tryptophan changes in response to ATP and ADP binding, allowing the K(d) and Hill coefficient to be determined for each nucleotide. By using this site-specific probe of binding, we analyze the effect of mutating the conserved arginine residue in the sensor-2 motif in Hsp104 NBD2. An R826M mutation causes nearly equal decreases in affinity of NBD2 for both ATP and ADP, indicating that at this site, the sensor-2 provides binding energy, but does not act to sense the difference between these nucleotides. In addition, the rate of ATP hydrolysis at NBD1 is decreased by the R826M mutation, providing further evidence for interdomain communication in the Hsp104 ATP hydrolysis cycle.
Hattendorf
Douglas A
DA
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA.
Lindquist
Susan L
SL
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D005456:Fluorescent Dyes; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D005456:Fluorescent Dyes; D005656:Fungal Proteins; D006360:Heat-Shock Proteins; D029701:Saccharomyces cerevisiae Proteins; C070972:HsP104 protein, S cerevisiae; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases