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摘要
Collagen prolyl 4-hydroxylase (C-P4H), an alpha(2)beta(2) heterotetramer, is a crucial enzyme for collagen synthesis. The alpha-subunit consists of an N-terminal dimerization domain, a central peptide substrate-binding (PSB) domain, and a C-terminal catalytic (CAT) domain. The beta-subunit [also known as protein disulfide isomerase (PDI)] acts as a chaperone, stabilizing the functional conformation of C-P4H. C-P4H has been studied for decades, but its structure has remained elusive. Here, we present a three-dimensional small-angle X-ray scattering model of the entire human C-P4H-I heterotetramer. C-P4H is an elongated, bilobal, symmetric molecule with a length of 290 angstrom. The dimerization domains from the two alpha-subunits form a protein-protein dimer interface, assembled around the central antiparallel coiled-coil interface of their N-terminal alpha-helices. This region forms a thin waist in the bilobal tetramer. The two PSB/CAT units, each complexed with a PDI/beta-subunit, form two bulky lobes pointing outward from this waist region, such that the PDI/beta-subunits locate at the far ends of the beta alpha alpha beta complex. The PDI/beta-subunit interacts extensively with the CAT domain. The asymmetric shape of two truncated C-P4H-I variants, also characterized in the present study, agrees with this assembly. Furthermore, data from these truncated variants show that dimerization between the alpha-subunits has an important role in achieving the correct PSB-CAT assembly competent for catalytic activity. Kinetic assays with various proline-rich peptide substrates and inhibitors suggest that, in the competent assembly, the PSB domain binds to the procollagen substrate downstream from the CAT domain.
- 出版日期2017-3-1
