title: A negatively charged residue stabilizes the tropoelastin N-terminal region for elastic fiber assembly.
abstract: 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.
authors.affiliation: From the School of Molecular Bioscience and Charles Perkins Centre, University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Giselle C
authors.initials: GC
authors.lastname: Yeo
authors.affiliation: the Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.
authors.forename: Clair
authors.initials: C
authors.lastname: Baldock
authors.affiliation: the Heart Research Institute, Sydney, New South Wales 2042, Australia, and the Sydney Medical School and.
authors.forename: Steven G
authors.initials: SG
authors.lastname: Wise
authors.affiliation: From the School of Molecular Bioscience and Charles Perkins Centre, University of Sydney, Sydney, New South Wales 2006, Australia, Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia tony.weiss@sydney.edu.au.
authors.forename: Anthony S
authors.initials: AS
authors.lastname: Weiss
mesh_terms: D001224:Aspartic Acid; D004548:Elasticity; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012867:Skin / Q000166:cytology; D014333:Tropoelastin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D014333:Tropoelastin; D001224:Aspartic Acid
keywords: Aspartate (Aspartic Acid); Domain 6; Elastic Fiber Assembly; Elastin; N-terminal Region; Protein Self-assembly; Small Angle X-ray Scattering; Tertiary Structure; Tropoelastin