Assembly of the elongated collagen prolyl 4-hydroxylase α2β2 heterotetramer around a central α2 dimer

Abstract

Collagen prolyl 4-hydroxylase (C-P4H), an αβ heterotetramer, is a crucial enzyme for collagen synthesis. The α-subunit consists of an N-terminal dimerization domain, a central peptide substrate-binding (PSB) domain, and a C-terminal catalytic (CAT) domain. The β-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 Å. The dimerization domains from the two α-subunits form a protein-protein dimer interface, assembled around the central antiparallel coiled-coil interface of their N-terminal α-helices. This region forms a thin waist in the bilobal tetramer. The two PSB/CAT units, each complexed with a PDI/β-subunit, form two bulky lobes pointing outward from this waist region, such that the PDI/β-subunits locate at the far ends of the βααβ complex. The PDI/β-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 α-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.

Structural enzymology binding studies of the peptide‐substrate‐binding domain of human collagen prolyl 4‐hydroxylase (type‐II): High affinity peptides have a PxGP sequence motif

“…C‐P4H is a processive enzyme and it has been proposed that the PSB domain anchors the unhydroxylated procollagen in such a way that the catalytic domain of the α subunit can do several successive hydroxylations without being released from its substrate . Therefore, the PSB domain is expected to have an important role in the substrate specificity of C‐P4Hs .…”

“…C‐P4H‐I is the most abundant isoform and it is found in all tissues studied, whereas C‐P4H‐II is enriched in chondrocytes, osteoblasts and endothelial cells . Small‐angle X‐ray scattering (SAXS) studies have shown that the four subunits in C‐P4H‐I are assembled into a rod‐shaped molecule in solution …”

“…The exact role of the PSB domain in the C‐P4H reaction mechanism is currently unknown . Its sequence characteristics are correlated with the different substrate binding properties of C‐P4H‐I and C‐P4H‐II .…”

“…11 Small-angle X-ray scattering (SAXS) studies have shown that the four subunits in C-P4H-I are assembled into a rod-shaped molecule in solution. 12 C-P4H enzymes have a small PRD in the middle of their α subunit (preceding the C-terminal catalytic region). This domain, referred to as peptide-substratebinding (PSB) domain, is around 100 amino acid residues long, being a helical protein composed of five helices that fold into two repeats of the tetratricopeptide repeat (TPR) motif plus a C-terminal extra helix.…”

“…16 The exact role of the PSB domain in the C-P4H reaction mechanism is currently unknown. 9,12 Its sequence characteristics are correlated with the different substrate binding properties of C-P4H-I and C-P4H-II. [13][14][15] The affinity and the mode of peptidebinding of human PSB-II have been studied here, using biophysical characterization methods and X-ray crystallography, and are compared with the corresponding PSB-I properties.…”