Requirement of the hinge domain for dimerization of Ca 2+-ATPase large cytoplasmic portion expressed in bacteria

Abstract

The large cytoplasmic domain of rabbit sarcoplasmic reticulum Ca 2+-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 μM. 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 ( R s) values compatible with a dimer (100 kDa and 40 Å, 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 ( R g) of 39 Å, suggesting that the dimer was not spherical ( R s/ R g=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 ( K d=8 μM) 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 Ca 2+-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 Ca 2+-ATPase in the native membrane.