Microfibril-associated glycoprotein-1 binding to tropoelastin: multiple binding sites and the role of divalent cations

Eur J Biochem. 2004 Jul;271(14):3085-90. doi: 10.1111/j.1432-1033.2004.04246.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cations, Divalent / metabolism
  • Contractile Proteins / isolation & purification
  • Contractile Proteins / metabolism*
  • Extracellular Matrix Proteins / isolation & purification
  • Extracellular Matrix Proteins / metabolism*
  • Humans
  • Microfibrils / metabolism*
  • Protein Binding
  • Protein Structure, Tertiary
  • RNA Splicing Factors
  • Tropoelastin / genetics
  • Tropoelastin / metabolism*

Substances

  • Cations, Divalent
  • Contractile Proteins
  • Extracellular Matrix Proteins
  • RNA Splicing Factors
  • Tropoelastin
  • microfibrillar protein