Crystal structures of the clock protein EA4 from the silkworm Bombyx mori.
Many insects pass the winter in an arrested developmental stage called diapause, either as eggs, as pupae, or even as adults. Exposure to the prolonged cold of winter is required to permit awakening from diapause in the spring. In the diapause eggs of the silkworm Bombyx mori, a metalloglycoprotein, esterase A4 (EA4), has been suggested to serve as a cold-duration clock because its characteristic ATPase activity is transiently elevated at the end of the necessary cold period. This timer property of EA4 is known to start with the dissociation of an inhibitory peptide (called "peptidyl inhibitory needle") under cold conditions, but its time-measuring mechanism is completely unknown. Here we present the crystal structures and functional properties of EA4 with and without glycosylation. We show that EA4 is a homodimeric ATPase, with each subunit consisting of a copper-zinc superoxide dismutase fold. There is an additional short N-terminal region that is capable of binding one more copper ion, suggesting a timer mechanism in which this ion is involved. The sugar chain appears to reinforce the binding of peptidyl inhibitory needle, which may in turn stabilize the initial conformation of the N-terminal domain, explaining the requirement for glycosylation and for the peptide to set the clock.
Hiraki
Toshiki
T
Protein Design Laboratory, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama 230-0045, Japan.
Shibayama
Naoya
N
Akashi
Satoko
S
Park
Sam-Yong
SY
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000378:metabolism*; D018360:Crystallography, X-Ray; D019281:Dimerization; D004950:Esterases / Q000737:chemistry*; D006031:Glycosylation; D019476:Insect Proteins / Q000737:chemistry*; D008667:Metalloproteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D021122:Protein Subunits / Q000737:chemistry; D013482:Superoxide Dismutase / Q000737:chemistry
D019476:Insect Proteins; D008667:Metalloproteins; D010455:Peptides; D021122:Protein Subunits; D013482:Superoxide Dismutase; D004950:Esterases; D000251:Adenosine Triphosphatases