Design and chemical synthesis of a neoprotein structural model for the cytoplasmic domain of a multisubunit cell-surface receptor: integrin alpha IIb beta 3 (platelet GPIIb-IIIa).
Integrins are a class of heterodimeric cell adhesion receptors involved in cell migration, cell anchorage, and cell-cell interactions. The cytoplasmic domains of integrins are of key importance in these activities. We have designed and chemically synthesized a 126 amino acid model protein (MP-1) containing both cytoplasmic tails of the platelet-derived integrin alpha IIb beta 3 covalently linked via a helical coiled coil. The coiled-coil tertiary structure was incorporated to mimic the membrane-spanning domain of the integrin and to act as a topological constraint fixing the two cytoplasmic tails in a parallel arrangement. This molecule, which contains two C-termini, was constructed by chemical dovetailing. The bromoacetylated and cysteinyl peptide synthons were unambiguously ligated through the formation of a thioether linkage. Ultraviolet circular dichroism (CD) spectroscopy has been performed on MP-1 and related compounds, confirming that a helical coiled coil is present within the MP-1 molecule. Significantly, the helicity apparently extends beyond the predicted amphiphilic region of MP-1. Fluorescence measurements suggest that a defined tertiary structure has formed by the association of the two cytoplasmic domains. We conclude that this is a practical design strategy for the study of the cytoplasmic domain of multisubunit cell-surface receptors.
Muir
T W
TW
Scripps Research Institute, La Jolla, California 92037.
Williams
M J
MJ
Ginsberg
M H
MH
Kent
S B
SB
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003593:Cytoplasm / Q000737:chemistry*; D015195:Drug Design; D005813:Genes, Synthetic; D016023:Integrins / Q000737:chemistry*; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins / Q000138:chemical synthesis* / Q000737:chemistry; D017433:Protein Structure, Secondary; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D016023:Integrins; C087981:MP-1 protein, synthetic; D046911:Macromolecular Substances; D010446:Peptide Fragments; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins; D011506:Proteins