Microfibril-associated glycoprotein-1 binding to tropoelastin: multiple binding sites and the role of divalent cations.
Microfibrils and elastin are major constituents of elastic fibers, the assembly of which is dictated by multimolecular interactions. Microfibril-associated glycoprotein-1 (MAGP-1) is a microfibrillar component that interacts with the soluble elastin precursor, tropoelastin. We describe here the adaptation of a solid-phase binding assay that defines the effect of divalent cations on the interactions between MAGP-1 and tropoelastin. Using this assay, a strong calcium-dependent interaction was demonstrated, with a dissociation constant of 2.8 +/- 0.3 nm, which fits a single-site binding model. Manganese and magnesium bestowed a weaker association, and copper did not facilitate the protein interactions. Three constructs spanning tropoelastin were used to quantify their relative contributions to calcium-dependent MAGP-1 binding. Binding to a construct spanning a region from the N-terminus to domain 18 followed a single-site binding model with a dissociation constant of 12.0 +/- 2.2 nm, which contrasted with the complex binding behavior observed for fragments spanning domains 17-27 and domain 27 to the C-terminus. To further elucidate binding sites around the kallikrein cleavage site of domains 25/26, MAGP-1 was presented with constructs containing C-terminal deletions within the region. Construct M1659, which spans a region from the N-terminus of tropoelastin to domain 26, inclusive, bound MAGP-1 with a dissociation constant of 9.7 +/- 2.0 nm, which decreased to 4.9 +/- 1.0 nm following the removal of domain 26 (M155n), thus displaying only half the total capacity to bind MAGP-1. These results demonstrate that MAGP-1 is capable of cumulative binding to distinct regions on tropoelastin, with different apparent dissociation constants and different amounts of bound protein.
Clarke
Adam W
AW
School of Molecular and Microbial Biosciences, University of Sydney, NSW, Australia.
Weiss
Anthony S
AS
D002413:Cations, Divalent / Q000378:metabolism; D003285:Contractile Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D020894:Microfibrils / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D000072260:RNA Splicing Factors; D014333:Tropoelastin / Q000235:genetics / Q000378:metabolism*
D002413:Cations, Divalent; D003285:Contractile Proteins; D016326:Extracellular Matrix Proteins; D000072260:RNA Splicing Factors; D014333:Tropoelastin; C016598:microfibrillar protein