Homodimeric quaternary structure is required for the in vivo function and thermal stability of Saccharomyces cerevisiae and Schizosaccharomyces pombe RNA triphosphatases.
Saccharomyces cerevisiae Cet1 and Schizosaccharomyces pombe Pct1 are the essential RNA triphosphatase components of the mRNA capping apparatus of budding and fission yeast, respectively. Cet1 and Pct1 share a baroque active site architecture and a homodimeric quaternary structure. The active site is located within a topologically closed hydrophilic beta-barrel (the triphosphate tunnel) that rests on a globular core domain (the pedestal) composed of elements from both protomers of the homodimer. Earlier studies of the effects of alanine cluster mutations at the crystallographic dimer interface of Cet1 suggested that homodimerization is important for triphosphatase function in vivo, albeit not for catalysis. Here, we studied the effects of 14 single-alanine mutations on Cet1 activity and thereby pinpointed Asp280 as a critical side chain required for dimer formation. We find that disruption of the dimer interface is lethal in vivo and renders Cet1 activity thermolabile at physiological temperatures in vitro. In addition, we identify individual residues within the pedestal domain (Ile470, Leu519, Ile520, Phe523, Leu524, and Ile530) that stabilize Cet1 in vivo and in vitro. In the case of Pct1, we show that dimerization depends on the peptide segment 41VPKIEMNFLN50 located immediately prior to the start of the Pct1 catalytic domain. Deletion of this peptide converts Pct1 into a catalytically active monomer that is defective in vivo in S. pombe and hypersensitive to thermal inactivation in vitro. Our findings suggest an explanation for the conservation of quaternary structure in fungal RNA triphosphatases, whereby the delicate tunnel architecture of the active site is stabilized by the homodimeric pedestal domain.
Hausmann
Stéphane
S
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Pei
Yi
Y
Shuman
Stewart
S
D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D020134:Catalytic Domain / Q000235:genetics; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D006358:Hot Temperature; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012568:Schizosaccharomyces / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D004268:DNA-Binding Proteins; D029702:Schizosaccharomyces pombe Proteins; C087021:res2 protein, S pombe; D001224:Aspartic Acid; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase