Disulfide-Bridged Heterotrimeric Collagen Peptides Containing the Collagenase Cleavage Site of Collagen Type I. Synthesis and Conformational Properties

Abstract

Collagenous peptides containing the collagenase cleavage site α1(772−784) and α2(772−784) of collagen type I were synthesized and assembled into heterotrimers via regioselective C-terminal interchain-disulfide bridging in a defined α1α2α1‘ staggered register of the three peptide strands. Various approaches were attempted to induce and stabilize the collagen-characteristic triple-helical fold even in the sequence portion of the collagenase cleavage site with its weak triple-helix propensity. By N-terminal chain elongation with (Gly-Pro-Hyp) n tripeptide repeats, particularly with n = 5, and in an even more pronounced manner, by incorporation of an additional tripeptide repeat adjacent to the cystine knot, a collagenous heterotrimer was obtained which was found to exhibit dichroic properties fully consistent with the triple-helical fold. Thermal denaturation revealed a remarkable stability with a melting temperature of 41 °C. Although the complex cystine knot of natural collagen was reduced in these synthetic heterotrimers to two interchain-disulfide bridges, it showed not only the expected entropic contribution to the refolding process by keeping the three chains assembled, but more importantly a triple-helix nucleation was induced. In fact, temperature jump experiments clearly revealed two-phase refolding kinetics very similar to those of the disulfide-bridged natural collagen fragment of Col 1−3, where refolding without nucleation difficulty was obtained followed by a slower process dominated by the cis → trans isomerization for triple-helix propagation. These results would indicate that even the simplified artificial cystine knot is capable of aligning the three peptide chains in the defined α1α2α1‘ one-residue shift register. Moreover, the synthetic heterotrimers were cleaved by interstitial collagenases in a single cut through all three chains without release of intermediates during the relatively slow enzymatic digestion process. This observation confirms that, with the de novo designed heterotrimers, functional collagen epitopes were mimicked in highly efficient manner; it also strongly suggests that the preselected α1α2α1‘ register may indeed represent the correct staggered alignment of the α subunits at least in collagen type I.

Self-assembly of synthetic collagen triple helices

“…1 A). Cleavage from the resin afforded fragments 1 and 2 in 12% and 28% overall yields, respectively, which significantly exceed those reported for analogous solution-phase syntheses (27). The N and C termini in 1 and 2 were left unprotected to mimic natural collagen.…”

“…2) (27). Briefly, the thiol of a deprotected ␣1 strand was reacted with the p-Npys-activated thiol of a resin-bound ␣2 strand in aqueous buffer to afford an ␣1␣2 heterodimer.…”

“…Hence, we relied on covalent bonds to tether the strands of our collagen fragments. Specifically, we prepared fragments 1 and 2 in which three helicogenic strands (7) are linked by a pair of disulfide bonds (27) that both offset the strands and set their register (Fig. 1A).…”

The 1.8-Å Crystal Structure of a Matrix Metalloproteinase 8-Barbiturate Inhibitor Complex Reveals a Previously Unobserved Mechanism for Collagenase Substrate Recognition

“…For the collagenases, considerable flexibility near the active site environment appears physiologically necessary for triple helical peptide processing (47). The flexibility observed in the present structure suggests that the loop Ala 206 -Asn 218 will provide much of the flexibility necessary for collagen substrate recognition (48), along with the additional plasticity seen at the catalytic Zn 2ϩ and its ligating residues.…”

Specific Recognition of the Collagen Triple Helix by Chaperone HSP47

“…3A). Here, we used cystine knots to tether the three peptide strands, since cystine knots were reported to be structurally compatible with the collagen triple helix and a one-residue stagger between the adjacent chains can be unambiguously introduced (17). Only one of the three N-terminal amino groups was left free to enable immobilization onto aminereactive Sepharose beads in a fixed molecular orientation.…”