A Negatively Charged Residue Stabilizes the Tropoelastin N-terminal Region for Elastic Fiber Assembly

作者：Yeo Giselle C; Baldock Clair; Wise Steven G; Weiss Anthony S^*

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2014, 289(50): 34815-34826.

DOI：10.1074/jbc.M114.606772

• Aspartate (Aspartic Acid)
• Elastin
• Protein Self-assembly
• Small Angle X-ray Scattering
• Tertiary Structure
• Domain 6
• Elastic Fiber Assembly
• N-terminal Region
• Tropoelastin

Background: Negative residues are rare in human tropoelastin, and their contributions to protein structure and function are unknown. Results: Mutating aspartate 72 reduces coacervation, cross-linking, and elastogenesis and alters the N-terminal conformation. Conclusion: Aspartate 72 residue stabilizes the N-terminal structure for functional assembly into elastic fibers. Significance: This work provides the first direct evidence of the importance of the tropoelastin N-terminal structure in elastic fiber assembly.
Tropoelastin is an extracellular matrix protein that assembles into elastic fibers that provide elasticity and strength to vertebrate tissues. Although the contributions of specific tropoelastin regions during each stage of elastogenesis are still not fully understood, studies predominantly recognize the central hinge/bridge and C-terminal foot as the major participants in tropoelastin assembly, with a number of interactions mediated by the abundant positively charged residues within these regions. However, much less is known about the importance of the rarely occurring negatively charged residues and the N-terminal coil region in tropoelastin assembly. The sole negatively charged residue in the first half of human tropoelastin is aspartate 72. In contrast, the same region comprises 17 positively charged residues. We mutated this aspartate residue to alanine and assessed the elastogenic capacity of this novel construct. We found that D72A tropoelastin has a decreased propensity for initial self-association, and it cross-links aberrantly into denser, less porous hydrogels with reduced swelling properties. Although the mutant can bind cells normally, it does not form elastic fibers with human dermal fibroblasts and forms fewer atypical fibers with human retinal pigmented epithelial cells. This impaired functionality is associated with conformational changes in the N-terminal region. Our results strongly point to the role of the Asp-72 site in stabilizing the N-terminal segment of human tropoelastin and the importance of this region in facilitating elastic fiber assembly.

• 出版日期2014-12-12