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. 2018 Dec 18;115(12):2327-2335.
doi: 10.1016/j.bpj.2018.11.012. Epub 2018 Nov 16.

Enzymatic Phosphorylation of Ser in a Type I Collagen Peptide

Yimin Qiu  1 , Erik Poppleton  1 , Arya Mekkat  2 , Hongtao Yu  2 , Sourav Banerjee  3 , Sandra E Wiley  3 , Jack E Dixon  3 , David L Kaplan  4 , Yu-Shan Lin  2 , Barbara Brodsky  5
Affiliations

Enzymatic Phosphorylation of Ser in a Type I Collagen Peptide

Yimin Qiu et al. Biophys J. .

Abstract

Phosphoproteomics studies have reported phosphorylation at multiple sites within collagen, raising the possibility that these post-translational modifications regulate the physical or biological properties of collagen. In this study, molecular dynamics simulations and experimental studies were carried out on model peptides to establish foundational principles of phosphorylation of Ser residues in collagen. A (Gly-Xaa-Yaa)11 peptide was designed to include a Ser-containing sequence from type I collagen that was reported to be phosphorylated. The physiological kinase involved in collagen phosphorylation is not known. In vitro studies showed that a model kinase ERK1 (extracellular signal-regulated protein kinase 1) would phosphorylate Ser within the consensus sequence if the collagen-like peptide is in the denatured state but not in the triple-helical state. The peptide was not a substrate for FAM20C, a kinase present in the secretory pathway, which has been shown to phosphorylate many extracellular matrix proteins. The unfolded single chain (Gly-Xaa-Yaa)11 peptide containing phosphoSer was able to refold to form a stable triple helix but at a reduced folding rate and with a small decrease in thermal stability relative to the nonphosphorylated peptide at neutral pH. These biophysical studies on model peptides provide a basis for investigations into the physiological consequences of collagen phosphorylation and the application of phosphorylation to regulate the properties of collagen biomaterials.

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Figures

Figure 1
Figure 1
Reported phosphorylation sites in the triple-helical domain (residues 179–1192) of α1 chain of human type I collagen. Phosphorylation of S543 and S546 was found in the recombinant human gelatin expressed by yeast (9); pSer at 184, 291, and 787 sites was reported in rat organs and tissues (11); phosphorylation of S271, S291, T445, S543, S546, S1063, and S1125 was observed in human tumor tissues (12, 13, 15); and phosphorylation of S513, S522, S543, S546, T766, S1023, S1029, and S1125 was reported in HeLa cell lines (10, 14). The sequence around residue Ser546 is shown, together with the design of peptides to model the Ser546 site. To see this figure in color, go online.
Figure 2
Figure 2
(A) Molecular dynamics (MD) simulation results of the α1(I)3 wild-type (WT) Ser546 peptide at neutral pH, (B) α1(I)3 pSer546 at neutral pH, and (C) α1(I)3 pSer546 at acidic pH. Top: A side view of the triple helices in the end of the simulations is shown. The GPO triplets are colored white, and human sequence triplets are colored gray; Ser546/pSer546 and Asp are drawn in sticks. Middle: A diagram of the average occupancy of interchain NH···CO hydrogen bonds within the peptide is shown. In the sequences, phosphorylated serines are designated as J. Arrows are used to indicate hydrogen bonds, defined as a donor-acceptor distance <3.5 Å and a hydrogen-donor-acceptor angle <30° and based on the average of two 100 ns MD runs. Bottom: Occupancy of the interchain NH···CO hydrogen bonds in the human sequence region as a function of simulation time is shown. Hydrogen bond occupancies as a function of time during each of the two MD runs are depicted as dashed and dotted, respectively; the average of the two runs is shown in a solid line. A running average with a sliding window of 1 ns is used. To see this figure in color, go online.
Figure 3
Figure 3
(A) Circular dichroism (CD) spectra of Ser546 (dashed blue line) and pSer546 (solid red line) peptides in neutral pH phosphate buffer at 0°C, indicating their triple-helical conformation, and at 70°C (Ser546, dotted blue line; pSer546, dashed red line), showing their unfolded state. CD temperature scans of Ser546 (dashed blue line) and pSer546 (solid red line) at (B) neutral pH and (C) pH 2.9 are shown. (D) Refolding profiles of Ser546 (dotted blue line) and pSer546 (solid red line) at 0°C (pH 7.4). Data are representative of three independent experiments and depict mean ± SEM. To see this figure in color, go online.
Figure 4
Figure 4
Establishment of conditions for the native and denatured states of peptide Ser546 for phosphorylation assays. (A) CD spectra of peptide Ser546 after 3 h of incubation at 30°C (green solid line), showing it is indistinguishable from the spectrum at 0°C (Fig. 3A) and maintains the native state; CD spectrum of peptide Ser546 after heating to 70°C for 20 min followed by 3 h of incubation at 30°C (pink dashed line), showing the peptide remains in the denatured state. (B) The refolding profiles of peptide Ser546 heated to 70°C for 20 min, followed by refolding at 0°C (blue solid line), 30°C (green dashed line), at 34°C (orange dash-dotted line), and 37°C (dark orange dotted line); the ellipticity at 225 nm was monitored with time to follow folding. To see this figure in color, go online.
Figure 5
Figure 5
Mass spectroscopy of (A) Ser546 control peptide; (B) pSer546 control peptide; (C) incubation of native triple-helical Ser546 peptide with ERK1 kinase for 3 h at 30°C; and (D) incubation of denatured peptide Ser546 with ERK1 for 3 h at 30°C.
Figure 6
Figure 6
Phosphorylation efficiency of Ser546 peptide using ERK1 kinase was assessed by determination of the amount of ADP generated in the reaction with the ADP-Glo assay method. (A) Titration of Ser546 peptide contained 20 ng of ERK1 kinase, 100 μM ATP, and a serial dilution of Ser546 substrate. (B) The optimal ERK1 amount for phosphorylation of Ser546 (100 μM ATP, 40 μM Ser546 substrate). Data are representative of three independent experiments and presented as mean ± SD of six replicates.
Figure 7
Figure 7
Mass spectroscopy of phosphorylation products of Ser546 peptides by Fam20C. (A) The incubation of native triple-helical Ser546 peptide with Fam20C kinase for 1 h at 30°C; (B) the incubation of denatured peptide Ser546 with Fam20C for 1 h at 30°C.
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