Requirement of the hinge domain for dimerization of Ca2+-ATPase large cytoplasmic portion expressed in bacteria.
The large cytoplasmic domain of rabbit sarcoplasmic reticulum Ca2+-ATPase was overexpressed in Escherichia coli as a 48 kDa fusion protein, designated p48, containing an N-terminal hexa-His tag. Purification conditions were optimized, thus conferring long-term stability to p48. Circular dichroism spectroscopy and the pattern of limited trypsinolysis confirmed the proper folding of the domain. p48 retained 0.5 +/- 0.1 mol of high affinity 2',3'-O-(2,4,6-trinitrophenyl)adenosine-5'-triphosphate (TNP-ATP) binding sites per mol of polypeptide chain with an apparent dissociation constant of about 8 microM. Size-exclusion FPLC using protein concentrations in the range 0.03 5 mg/ml showed that p48 was essentially monodisperse with apparent molecular mass and Stokes radius (Rs) values compatible with a dimer (100 kDa and 40 A, respectively). Analysis of p48 by small-angle X-ray scattering provided an independent second proof for a dimeric p48 particle with a radius of gyration (Rg) of 39 A, suggesting that the dimer was not spherical (Rs/Rg = 1.026). When digested by proteinase K, p48 was converted to a 30 kDa fragment, designated p30, which was very resistant to further proteolysis. p30 retained high affinity TNP-ATP binding (Kd = 8 microM) and eluted as a monomer (35 kDa) in size-exclusion FPLC. As opposed to p48, the p30 fragment did not react with monoclonal antibody A52 [Clarke et al., J. Biol. Chem. 264 (1989) 11246-11251] which recognizes region E657-R672 located upstream of the hinge domain of the Ca2+-ATPase. These results indicate a requirement of the hinge domain (670-728) region for self-association of the p48 large hydrophilic domain as a dimer. We propose that this behavior points to a possible role of the hinge domain in dimerization of sarcoplasmic reticulum Ca2+-ATPase in the native membrane.
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Brazil.
P C
PC
Carvalho-Alves
V R
VR
Hering
J M
JM
Oliveira
R K
RK
Salinas
S
S
Verjovski-Almeida
D000818:Animals; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D002942:Circular Dichroism; D003593:Cytoplasm / Q000201:enzymology; D019281:Dimerization; D019286:Endopeptidase K; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D009154:Mutation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D018271:Signal Recognition Particle / Q000096:biosynthesis / Q000737:chemistry; D014961:X-Ray Diffraction / Q000379:methods
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; D018271:Signal Recognition Particle; D019286:Endopeptidase K; D000252:Calcium-Transporting ATPases