Tropoelastin bridge region positions the cell-interactive C terminus and contributes to elastic fiber assembly

Abstract

The tropoelastin monomer undergoes stages of association by coacervation, deposition onto microfibrils, and cross-linking to form elastic fibers. Tropoelastin consists of an elastic N-terminal coil region and a cell-interactive C-terminal foot region linked together by a highly exposed bridge region. The bridge region is conveniently positioned to modulate elastic fiber assembly through association by coacervation and its proximity to dominant cross-linking domains. Tropoelastin constructs that either modify or remove the entire bridge and downstream regions were assessed for elastogenesis. These constructs focused on a single alanine substitution (R515A) and a truncation (M155n) at the highly conserved arginine 515 site that borders the bridge. Each form displayed less efficient coacervation, impaired hydrogel formation, and decreased dermal fibroblast attachment compared to wild-type tropoelastin. The R515A mutant protein additionally showed reduced elastic fiber formation upon addition to human retinal pigmented epithelium cells and dermal fibroblasts. The small-angle X-ray scattering nanostructure of the R515A mutant protein revealed greater conformational flexibility around the bridge and C-terminal regions. This increased flexibility of the R515A mutant suggests that the tropoelastin R515 residue stabilizes the structure of the bridge region, which is critical for elastic fiber assembly.tropoelastin assembly | protease resistance E lastic fibers confer the elastic and recoil properties required for repetitive and reversible deformation of elastic tissues during normal function (1-3). The assembly of elastic fibers from tropoelastin, the soluble precursor of elastin, is classically defined by stages of tropoelastin synthesis, coacervation, microfibrillar deposition, and cross-linking. Tropoelastin is secreted by elastogenic cells such as smooth muscle cells, endothelial cells, and fibroblasts (1, 2). At the cell surface, the monomers cluster through hydrophobic domain interactions in an aqueous environment (3-6) by the entropically driven process of coacervation (7). These tropoelastin assemblies remain attached through the C terminus to cell-surface integrin αvβ3 and glycosaminoglycans (8-10) until deposition on microfibrillar scaffolds (11), which direct the shape and orientation of elastic fibers (10, 12, 13). Microfibrillar proteins recruit lysyl oxidase (14, 15), which reacts with specific tropoelastin lysine residues to form cross-links (1, 11, 16). These cross-links occur at multiple sites in the molecule (17) and are enriched in domains 19-25 (1, 16). Cross-linking imposes expansional constraints on elastin and renders elastic fibers resilient under repetitive mechanical stretching (1,12,18).The recently solved nanostructure of tropoelastin has allowed the main functional regions of tropoelastin to be placed within a structural context (19). Most of the elasticity of the molecule is conferred by a coiled region (20) that extends from domain 2 to domain 18. A protrusion from the coil region around do...

Perspectives on the Molecular and Biological Implications of Tropoelastin in Human Tissue Elasticity

“…[30] [31] Over the past few years, we and others have found that, in vitro, these molecules are not needed for the spatial and temporal elastin assembly of tropoelastin. [9,15,20,26,32,33] This may be due to the fact that elastin is over nine times more abundant than microfibrillar components, and therefore tropoelastin-tropoelastin interactions dominate. [34] The observed tropoelastin and aggregate structures are similar to those found in natural tissues, as evidenced by electron microscopy, which supports the concept of an aggregation unit corresponding to tropoelastin assemblies.…”

“…[37] By exploring the functional roles of domains by systematically introducing mutations at selected sites across tropoelastin, in concert with SAXS and allied methods including SANS, NMR, and molecular dynamics on these constructs, we have solved the solution structures for the normal and a panel of mutant forms of tropoelastin. [9,10,20,26] In each of the mutant cases, there is altered assembly. This has facilitated mapping of these effects onto specific parts of the molecule, together with comparison of the assembly performance of wild-type and mutant forms in vitro and in concert with elastogenic cells.…”

“…We discovered that tropoelastin has a defined monomer shape in solution that is needed for assembly, [9,10,13] but also displays a large percentage of flexible, disordered regions needed for molecular elasticity. [14,15] The tertiary structure of human tropoelastin represents an ensemble of elastic conformers, [10,16] yet occasional conserved sequence elements hint at requirements for functional demands in one or more key parts of this molecule. [17][18][19] Tropoelastin is a 20-nm-long asymmetric protein monomer with a slightly curved nanospring shaft that extends from its N-terminus to a bifurcating hinged foot region towards the C-terminus.…”

“…This model of tropoelastin dynamics describes a characteristic scissors-like motion between the hinge and foot regions, as well as a twisting motion in the N-terminal coil region, in which the N-terminus experiences highest displacement. [9,10,13,20] The geometric distribution of the tropoelastin molecular volume intrinsically prompts a cohesive motion pattern between the upper and lower regions of the molecule, giving rise to local regions of conformational flexibility thought to be responsible for elastic deformation, while maintaining a defined tertiary shape with distinct functional segments for protein and cellular interactions. Despite the intrinsic flexibility of tropoelastin as a supposedly disordered elastomeric protein, [21] local changes to its native shape, manifested by a perturbed hinge region in human tropoelastin, can have a substantial impact on function, including its assembly into larger-scale structures.…”

“…[7] Although other proteins are present, elastic tissue is predominantly made of one protein, tropoelastin, [8] which dominates molecular contributions to the architecture and function of elastin. [9,10] For this reason, the present review considers the perspective of tropoelastin.…”