A crystallizable form of RIIbeta regulatory domain obtained by limited proteolysis.
The type RIIbeta regulatory subunit of protein kinase A is primarily expressed in adipose tissue and brain. Knockout mice suggest a role for RIIbeta in regulating energy balance and adipose-tissue content, thus making it a potential target for therapeutic intervention in obesity. A truncated version of the RIalpha subunit has been used in a crystallographic study and was used here to design an analogous RIIbeta construct. Despite substantial screening, conditions were not found for the crystallization of the truncated RIIbeta subunit. However, limited proteolysis of the full-length RIIbeta subunit identified boundaries of the 'hinge' region and a fragment containing the two cAMP-binding domains which did crystallize. A recombinant version of the fragment was expressed and crystallized for X-ray diffraction studies. The crystals belong to the orthorhombic space group C222, with unit-cell parameters a = 91.6, b = 105.9, c = 85.8 A, and diffracted to at least 2.3 A.
Danley
D E
DE
Department of Exploratory Medicinal Sciences, Pfizer Central Research Division, Groton, CT 06340, USA. dennis_e_danley@groton.pfizer.com
Haggan
M E
ME
Cunningham
D
D
Fennell
K F
KF
Pauly
T A
TA
LeMotte
P K
PK
D000595:Amino Acid Sequence; D000818:Animals; D003460:Crystallization; D018360:Crystallography, X-Ray; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
D010446:Peptide Fragments; D011994:Recombinant Proteins; D017868:Cyclic AMP-Dependent Protein Kinases