Threonine Phosphorylation Is a Bioreversible Surrogate of Proline Hydroxylation in a Collagen Triple Helix

Collagen, the most abundant protein in the human body and structural foundation of connective tissue, derives its remarkable stability from repeating (Xaa-Yaa-Gly) n sequences rich in (2 S )-proline (Pro) and (2 S,4 R )-4-hydroxyproline (Hyp), the product of a post-translational modification. Here, we demonstrate that another such modification, phosphorylation, yields a bioreversible mimic of Hyp. Using synthetic collagen-mimetic peptides, we substituted the Xaa or Yaa position of a central Pro-Hyp-Gly triplet with (2 S )-serine (Ser), (2 S,3 R )-threonine (Thr), or their O -phosphorylated derivatives, p Ser or p Thr. All substitutions were destabilizing─except p Thr in the Yaa position, which preserved the triple-helical stability conferred by Hyp. Moreover, p Thr within a collagen triple helix is a substrate for a mammalian secretory phosphatase. Notably, Thr is enriched at the Yaa position in mammalian fibrillar collagens, consistent with p Thr being a linchpin of collagen remodeling in the extracellular matrix. Thus, the phosphorylation of threonine (which is bioreversible) mimics the hydroxylation of proline (which is not), enabling dynamic modulation of collagen architecture in development and disease.