Expression, purification, crystallization and preliminary X-ray diffraction analysis of a mammalian type 10 adenylyl cyclase.
The second messenger cAMP is synthesized in mammals by ten differently regulated adenylyl cyclases (AC1-10). These ACs are grouped into nucleotidyl cyclase class III based on homologies in their catalytic domains. The catalytic domain of AC10 is unique, however, in being activated through direct interaction with calcium and bicarbonate. Here, the production, crystallization and X-ray diffraction analysis of the catalytic domain of human AC10 are described as a basis for structural studies of regulator binding sites and mechanisms. The recombinant protein had high specific AC activity, and crystals of AC10 in space group P63 diffracted to ∼2.0 Å resolution on a synchrotron beamline. A complete diffraction data set revealed unit-cell parameters a = b = 99.65, c = 98.04 Å, indicating one AC10 catalytic domain per asymmetric unit, and confirmed that the obtained crystals are suitable for structure solution and mechanistic studies.
Department of Biochemistry, University of Bayreuth, 95445 Bayreuth, Germany.
Silke
S
Kleinboelting
Helmholtz-Zentrum für Infektionsforschung, 38124 Braunschweig, Germany.
Joop
J
van den Heuvel
Department of Biochemistry, University of Bayreuth, 95445 Bayreuth, Germany.
Christian
C
Kambach
Department of Biochemistry, University of Bayreuth, 95445 Bayreuth, Germany.
Michael
M
Weyand
Department of Physiological Chemistry, Ruhr-University Bochum, 44801 Bochum, Germany.
Martina
M
Leipelt
Department of Biochemistry, University of Bayreuth, 95445 Bayreuth, Germany.
Clemens
C
Steegborn
D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D011994:Recombinant Proteins; D000262:Adenylyl Cyclases
catalytic core; mammalian; soluble adenylyl cyclase; type 10