Hydrophobic Domains of Human Tropoelastin Interact in a Context-dependent Manner

Abstract

Tropoelastin is the soluble precursor of elastin, the major component of the extracellular elastic fiber. Tropoelastin undergoes self-association via an inverse temperature transition termed coacervation, which is a crucial step in elastogenesis. Coacervation of tropoelastin takes place through multiple intermolecular interactions of its hydrophobic domains. Previous work has implicated those hydrophobic domains located near the center of the polypeptide as playing a dominant role in coacervation. Short constructs of domains 18, 20, 24, and a mutated form of domain 26 were largely disordered at 20°C but displayed increased order on heating that was consistent with the formation of ␤-structures. However, their conformational transitions were not sensitive to physiological temperature in contrast to the observed behavior of the native domain 26. A polypeptide consisting of domains 17-27 of tropoelastin coacervated at temperatures above 60°C, whereas individually expressed hydrophobic regions were not capable of coacervation. We conclude that coacervation depends on the hydrophobicity of the molecule and, by inference, the number of hydrophobic domains. Tropoelastin mutants were constructed to contain a Pro 3 Ala mutation in domain 26, separate deletions of domains 18 and 26, and a displacement of domain 26. These constructs displayed unequal capacities for coacervation, even when they contained the same number of hydrophobic regions and comparable levels of secondary structure. Thus, the capability for coacervation is determined by contributions from individual hydrophobic domains for which function should be considered in the context of their positions in the intact tropoelastin molecule.

New insights into elastic fiber assembly

“…Tropoelastin is capable of undergoing coacervation under physiological conditions in a process thought to facilitate selfassembly (Cox et al, 1974;Volpin et al, 1976;Clarke et al, 2006). Coacervation is the result of specific interactions between the hydrophobic domains induced by an increase in temperature (Toonkool et al, 2001). Studies suggest that coacervation is an important prerequisite for cross-linking (Narayanan et al, 1978), but it is not known at which point in assembly this occurs.…”

Conformational Transitions of the Cross-linking Domains of Elastin during Self-assembly

“…Hydrophobic domains are rich in nonpolar amino acids such as proline, glycine, valine, and leucine. These domains have been shown by experiment and molecular dynamics simulations to be highly disordered and flexible in solution (3)(4)(5)(6)(7)(8)(9). The high entropy and hydrophobic character of these domains is believed to be responsible for the extensibility and restoring force of polymeric elastin (6, 10 -12).…”

“…Coacervation is an endothermic, entropically driven process (24 -26) through which hydrophobic domain interaction has been suggested to concentrate and align cross-linking domains for polymerization (5,22,27,28). In contrast to other forms of protein aggregation that create highly ordered insoluble structures, such as ␤-strand stacking in amy-loid fiber formation, coacervation forms viscous protein-rich colloidal droplets that can interact and coalesce (29,30).…”

Direct observation of structure and dynamics during phase separation of an elastomeric protein

“…Tropoelastin is an IDP consisting of an alternating arrangement of hydrophobic domains (HDs) and cross-linking domains (CLDs) (7,19). The HDs are required for coacervation and elasticity and are disordered domains composed of pseudorepetitive sequence motifs enriched in glycine, proline, and hydrophobic amino acids (20)(21)(22)(23)(24)(25)(26). In contrast, the CLDs provide structural integrity to polymeric elastin through covalent cross-linking of lysine residues (27,28).…”