How electrostatic networks modulate specificity and stability of collagen

Abstract

One-quarter of the 28 types of natural collagen exist as heterotrimers. The oligomerization state of collagen affects the structure and mechanics of the extracellular matrix, providing essential cues to modulate biological and pathological processes. A lack of high-resolution structural information limits our mechanistic understanding of collagen heterospecific self-assembly. Here, the 1.77-Å resolution structure of a synthetic heterotrimer demonstrates the balance of intermolecular electrostatics and hydrogen bonding that affects collagen stability and heterospecificity of assembly. Atomistic simulations and mutagenesis based on the solved structure are used to explore the contributions of specific interactions to energetics. A predictive model of collagen stability and specificity is developed for engineering novel collagen structures.

Keywords: cooperativity; molecular dynamics; protein design; self-assembly; triple helix.

Fig. 1.

Sequence-based design (18) (A) and atomic-resolution structure (B) of abc (PDB ID code 5YAN). Salt bridges with distance d between Asp side-chain carboxylate oxygen and Lys side-chain amine nitrogen d <6 Å are denoted by black dashed lines. Salt bridges that are possible from a discrete sequence model, but are found to be weak (d > 6 Å) in the structure are denoted with yellow dashed lines. See SI Appendix for the crystallography statistics and structure details.

Fig. 2.

Favorable charge–pair distance distributions determined by MD simulations of abc for (A) axial KD interactions, (B) lateral KD interactions, (C) axial DK interactions, and (D) lateral DK interactions. Distributions are averaged over all occurrences of a particular class of interactions. Distances are between the closest side-chain carboxylate oxygen on Asp and the terminal side-chain amine on Lys. Residue-pair-specific distributions are presented in SI Appendix, Fig. S2.

Fig. 3.

Mutagenesis of the three-residue network c:K19-a:D21/c:K19-a:D24 between chains c and a, comprising both lateral and axial KD salt bridges. (A) Three-residue network depicted in both sequence and structure. Thermal denaturation observed by CD spectroscopy at 225 nm for abc, a:D21 (B) or a:D24 (C) substituted with Ala or Lys, and combined with peptides b and c.

Fig. 4.

Three-residue c:D7-a:K12/c:D10-a:K12 network containing axial and lateral DK salt bridges. (A) Salt bridges are depicted in both sequence and structure. (B and C) c:D7 (B) and c:D10 (C) were mutated to Ala and Lys, mixed with peptides a and b, and characterized by thermal denaturation, monitored at 225 nm by CD spectroscopy.

Fig. 5.

Stability measurements for 10 experimentally characterized synthetic collagen peptide heterotrimer systems were taken from various studies (18, 37, 39). Corresponding predicted stabilities were calculated by using Eq. 1 and the original scoring function used to design abc (18, 35). Where registry of a heterotrimer was not determined, the most stable computed association state was assumed. A detailed breakdown of favorable and unfavorable electrostatic interactions is presented in SI Appendix, Table S5.

Fig. 6.

(A) Relative orientation of two triple helices in an asymmetric unit. (B) CH-π interaction between a’:Hyp19 and a:Tyr1. The geometric center of the aromatic ring is represented with a dot. (C and D) Two complex salt-bridge networks. To show atomic-level details of the CH-π interaction, the stick representations in B are further enlarged compared with those in C and D.