Electrostatic Interactions Involving Lysine Make Major Contributions to Collagen Triple-Helix Stability

Abstract

Important stabilizing features for the collagen triple helix include the presence of Gly as every third residue, a high content of imino acids, and interchain hydrogen bonds. Host-guest peptides have been used previously to characterize triple-helix propensities of individual residues and Gly-X-Y triplets. Here, comparison of the thermal stabilities of host-guest peptides of the form (Gly-Pro-Hyp)3-Gly-X-Y-Gly-X'-Y'-(Gly-Pro-Hyp)3 extends the study to adjacent tripeptide sequences, to encompass the major classes of potential direct intramolecular interactions. Favorable hydrophobic interactions were observed, as well as stabilizing intrachain interactions between residues of opposite charge in the i and i + 3 positions. However, the greatest gain in triple-helix stability was achieved in the presence of Gly-Pro-Lys-Gly-Asp/Glu-Hyp sequences, leading to a T(m) value equal to that seen for a Gly-Pro-Hyp-Gly-Pro-Hyp sequence. This stabilization is seen for Lys but not for Arg and can be assigned to interchain ion pairs, as shown by molecular modeling. Computational analysis shows that Lys-Gly-Asp/Glu sequences are present at a frequency much greater than expected in collagen, suggesting this interaction is biologically important. These results add significantly to the understanding of which surface ion pairs can contribute to protein stability.

Solution Structure of an ABC Collagen Heterotrimer Reveals a Single-register Helix Stabilized by Electrostatic Interactions

“…The salt bridges observed in our solution structure are able to explain the surprisingly high thermal stability of homotrimeric triple helices with the KG(D/E) sequence (21), as well as our own heterotrimer (16). The inter-chain charge pairs that can be formed in those systems, homotrimers or heterotrimers, are equivalent and occur from the K in triplet n of one chain and interact with the D in triplet n ϩ 1 of the next chain.…”

“…For this reason, their role in the stabilization of the triple helix (38), the supramolecular assembly of collagen (39), and its interactions with other macromolecules (10) has been actively researched in the past few years. Experiments focused on determining the relationship between sequence and thermal stability of the triple helix found that model homotrimeric peptides containing the motif XKGDYЈG or XKGEYЈG are particularly stable (21). Changing the order of the charged amino acids or replacing Lys with Arg results in a decrease in the melting temperature of the assembly.…”

“…Recently, the structure of a homotrimer composed of a sequence from collagen type III that included the motif NRGERG showed that oppositely charged residues in that arrangement within the triple helix are able to form a network of intra-and inter-chain hydrogen bonds (40). The extent to which those interactions are present in solution, and not exclusively as a result of crystal packing, is unclear as model peptides with the RGE sequence do not exhibit any unusual stabilization (21).…”

“…We hypothesized that the high thermal stability of these systems comes from the formation of charge pairs between lysine and aspartic acid. Homotrimeric model peptides that contain the sequence KGD, which occurs both in mammalian collagen (21) and bacterial collagen-like proteins (22), apparently also use this charge pairing. However, no structural information has been available to confirm the nature of the interactions.…”

Predicting the Clinical Lethality of Osteogenesis Imperfecta from Collagen Glycine Mutations

“…The related peptide GFASPAGPO was synthesized which reverses the location of the low-stability GFA triplet and the high-stability GPO triplet (Table 6). Previous studies suggested that in the absence of a mutation, the interchange of triplets located at different positions does not affect thermal stability unless favorable charge interactions are created (31,32). The CD spectra of both peptides exhibit a maximum at 225 nm, which is characteristic of the triple-helix conformation ( Figure 4A), but peptide GFASPAGPO (3700 deg cm 2 dmol -1 ) exhibited a slightly higher mean residue ellipticity (MRE 225 ) compared to peptide GPOSPAGFA (3570 deg cm 2 dmol -1 ).…”