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. 2018 Sep;27(9):1692-1703.
doi: 10.1002/pro.3450.

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

Abhinandan V Murthy  1 , Ramita Sulu  1 , M Kristian Koski  1 , Hongmin Tu  2 , Jothi Anantharajan  1 , Shiv K Sah-Teli  1 , Johanna Myllyharju  2 , Rik K Wierenga  1
Affiliations

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

Abhinandan V Murthy et al. Protein Sci. 2018 Sep.

Abstract

The peptide-substrate-binding (PSB) domain of collagen prolyl 4-hydroxylase (C-P4H, an α2 β2 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.

Keywords: X-ray crystallography; calorimetry; collagen; extracellular matrix protein; proline-rich peptide; prolyl 4-hydroxylase; protein-peptide interactions.

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Figures

Figure 1
Figure 1
Alignment of PSB domain sequences (residues Phe144‐Ser244 of human C‐P4H‐I) from various species. The alignment includes the PSB domains from the three C‐P4H α subunit isoforms of human (Hs‐PSB‐I, Hs‐PSB‐II, Hs‐PSB‐III), rat (Rn‐PSB‐I, Rn‐PSB‐II, Rn‐PSB‐III), mouse (Mm‐PSB‐I, Mm‐PSB‐II, Mm‐PSB‐III), bovine (Bt‐PSB‐I, Bt‐PSB‐II, Bt‐PSB‐III), and chicken (Gg‐PSB‐I, Gg‐PSB‐II, Gg‐PSB‐III). The helices of the PSB domain (α7–α11) are numbered according to the full‐length C‐P4H‐α(I) subunit secondary structure16 and highlighted above the sequences (magenta). The residues in the peptide‐binding groove that interact with the bound peptide are highlighted by filled black squares and numbered according to human C‐P4H‐I α(I) subunit amino acid sequence numbering. The human PSB‐II sequence is highlighted with a red star and the first (Met142) and the last (Glu236) residues of the PSB‐II construct used in this study are highlighted by open squares.
Figure 2
Figure 2
The mode of binding of proline‐rich peptides to PSB‐I and PSB‐II. (A) The residue‐numbering scheme of the bound peptides in the different peptide complex structures of PSB‐I16 and PSB‐II (this study). The (PPG)3 mode of binding to PSB‐I has identified two proline binding pockets, being the Pro5 and Pro8 binding pocket. For each peptide, the proline residues that are bound in the Pro5 and Pro8 pockets are highlighted in yellow. For each peptide, all nine residues have been built in the electron density map and the residues of each peptide are numbered from 1 to 9. (X) and (Y) identify the X‐ and Y‐positions of the corresponding ‐X‐Y‐G‐triplet. (B) Schematic drawing showing the peptide‐binding groove (light blue) with respect to the five helices of the TPR fold of PSB‐I (gray), as known from binding studies with (PPG)3. 16 The Pro5 and Pro8 binding pockets are shown. The side chains of the six aromatic residues lining the Pro5 and Pro8 pockets, as well as Tyr233 at the C‐terminal end of the PSB peptide‐binding groove, are highlighted. The first and the last α helix (α7, α11), and the N‐ and C‐termini of the PSB fold are labeled.
Figure 3
Figure 3
The thermodynamic fingerprint (ΔH, −TΔS, ΔG) of the binding to PSB‐II of the PAG, PRG, and PEG peptides. The error bars are also shown.
Figure 4
Figure 4
Comparison of the Cα trace of the unliganded structure of the PSB‐II domain (light brown) and the structure of the PSB‐I domain (light blue, PDB code 2V5F) (standard view, stereo). The side chains of the key residues forming the peptide‐binding groove of the PSB domain are shown. Tyr196 adopts two conformations in the unliganded structure of PSB‐II, the major conformation points to Arg223.
Figure 5
Figure 5
The mode of binding of (P)9 to PSB‐II. (A) Stereo view showing the (P)9 peptide (yellow sticks) bound to the PSB‐II domain (light brown ribbon, side view). The omit 2Fo−Fc electron density map, calculated after three cycles of omit refinement (leaving the peptide out of the model) and contoured at 1.0 σ, is also drawn around the (P)9 peptide. The two key prolines, Pro4 and Pro7, of the (P)9 peptide are labeled in red. The direct hydrogen bonds between the key residues of PSB‐II (shown in light brown sticks) and (P)9 are shown. Residues from two neighboring PSB‐II molecules, which also interact with the N‐terminal region of the bound (P)9 peptide, are also shown. It concerns the C‐terminal residues Glu236′‐Glu238′ (shown in light blue sticks) of the symmetry‐related molecule 1 (shown in light blue ribbon), and Arg155′′ of the symmetry‐related molecule 2. The view is rotated ~90° clockwise around the horizontal axis from the standard view. (B) Stereo view showing the PSB‐II–(P)9 interactions in detail (standard view). Three water molecules sharing hydrogen bonds with PSB‐II and with (P)9, are shown in magenta spheres. Prolines 4 and 7 of (P)9 are labeled in red.
Figure 6
Figure 6
The mode of binding of PAG to PSB‐II. (A) Stereo view showing the peptide (yellow sticks) bound to the PSB‐II domain (light brown ribbon) (side view). The omit 2Fo−Fc electron density map, calculated after three cycles of omit refinement (leaving the peptide out of the model) and contoured at 1.0 σ, is also drawn around the PAG peptide. Pro4, Pro7, and Ala5 of the PAG peptide are labeled in red. The direct hydrogen bonds between the key residues of PSB‐II (shown in light brown sticks) and PAG are shown. (B) Stereo view showing the PSB‐II–PAG interactions in detail (standard view). The five waters (magenta) and DMSO (yellow) involved in the hydrogen bond network around PAG, are also shown. Pro4 and Pro7, as well as Ala5 of PAG are labeled in red.
Figure 7
Figure 7
Comparison of the peptide mode of binding to PSB‐II and PSB‐I. (A) The (P)9 mode of binding to PSB‐II (left) and PSB‐I (right, PDB code 4BT9). (B) The PAG mode of binding to PSB‐II (left) and the (PPG)3 mode of binding to PSB‐I (right, PDB code 4BT9). The PSB domains are shown using the surface representation (standard view), and the bound peptides are shown with yellow stick representation. Some key residues shaping the peptide‐binding groove are labeled. Also the Pro5 and Pro8 binding pockets are indicated with red P5 and P8 labels, respectively. In each of these complexes, the full‐length nine‐residue peptide has been built.
Figure 8
Figure 8
The interactions of the conserved Asn227 and Arg223‐Asp192 salt bridge in the three different modes of binding of a proline‐rich peptide to the PSB domain. (A) The loose mode of binding of (P)9 to PSB‐II, (B) The deep mode of binding of PAG to PSB‐II. (C) The water‐mediated mode of binding of (PPG)3 to PSB‐I (PDB code 4BT9). The dotted lines identify hydrogen bond interactions; the labels specify the corresponding distances (in Å).
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