The Structural Motifs for Substrate Binding and Dimerization of the α Subunit of Collagen Prolyl 4-Hydroxylase

SummaryCollagen prolyl 4-hydroxylase (C-P4H) catalyzes the proline hydroxylation of procollagen, an essential modification in the maturation of collagens. C-P4H consists of two catalytic α subunits and two protein disulfide isomerase β subunits. The assembly of these subunits is unknown. The α subunit contains an N domain (1–143), a peptide-substrate-binding-domain (PSB, 144–244) and a catalytic domain (245–517). Here, we report the dimeric structure of the N-terminal region (1–244) of the α subunit. It is shown that the N domain has an important role in the assembly of the C-P4H tetramer, by forming an extended four-helix bundle that includes an antiparallel coiled-coil dimerization motif between the two α subunits. Complexes of this construct with a C-P4H inhibitor and substrate show the mode of peptide-binding to the PSB domain. Both peptides adopt a poly-(L)-proline-type-II helix conformation and bind in a curved, asymmetric groove lined by conserved tyrosines and an Arg-Asp salt bridge

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

Abstract Collagen prolyl 4-hydroxylase (C-P4H), an α2β2 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

Abstract The peptide‐substrate‐binding (PSB) domain of collagen prolyl 4‐hydroxylase (C‐P4H, an α2β2 tetramer) binds proline‐rich procollagen peptides. This helical domain (the middle domain of the α subunit) has an important role concerning the substrate binding properties of C‐P4H, although it is not known how the PSB domain influences the hydroxylation properties of the catalytic domain (the C‐terminal domain of the α subunit). The crystal structures of the PSB domain of the human C‐P4H isoform II (PSB‐II) complexed with and without various short proline‐rich peptides are described. The comparison with the previously determined PSB‐I peptide complex structures shows that the C‐P4H‐I substrate peptide (PPG)3, has at most very weak affinity for PSB‐II, although it binds with high affinity to PSB‐I. The replacement of the middle PPG triplet of (PPG)3 to the nonhydroxylatable PAG, PRG, or PEG triplet, increases greatly the affinity of PSB‐II for these peptides, leading to a deeper mode of binding, as compared to the previously determined PSB‐I peptide complexes. In these PSB‐II complexes, the two peptidyl prolines of its central P(A/R/E)GP region bind in the Pro5 and Pro8 binding pockets of the PSB peptide‐binding groove, and direct hydrogen bonds are formed between the peptide and the side chains of the highly conserved residues Tyr158, Arg223, and Asn227, replacing water mediated interactions in the corresponding PSB‐I complex. These results suggest that PxGP (where x is not a proline) is the common motif of proline‐rich peptide sequences that bind with high affinity to PSB‐II

Strategic Design of Catalytic Lysine-Targeting Reversible Covalent BCR-ABL Inhibitors

Targeted covalent inhibitors have re-emerged as validated drugs to overcome acquired resistance in cancer treatment. Herein, by using a carbonyl boronic acid warhead, we report the structure-based design of BCR-ABL inhibitors via reversible covalent targeting of the catalytic lysine with improved single-digit nanomolar potency against both wild-type and mutant ABL kinases, especially ABLT315I bearing the gatekeeper residue mutation. We show that, by using techniques including mass spectrometry, time-dependent biochemical assays and X-ray crystallography, the evolutionarily conserved lysine can be targeted selectively. Furthermore, we show that the selectivity depends largely on molecular recognition of the non-covalent pharmacophore in this class of inhibitors, probably due to the moderate reactivity of the warhead. We report the first co-crystal structures of covalent inhibitor-ABL kinase domain complexes, providing insights into the interaction of this warhead with the catalytic lysine. We also employed label-free mass spectrometry to evaluate potential off-targets of our compounds at proteome-wide level in different cancer cell lines