Identification of the β1-integrin binding site on α-actinin by cryoelectron microscopy

Deb Kelly, Kenneth A. Taylor

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Cell-matrix adhesions in migrating cells are usually mediated by integrins, α-β heterodimeric transmembrane proteins that link extracellular matrix molecules such as fibronectin to the cytoskeleton. We have synthesized the cytoplasmic domain of the β1-integrin (residues H738-K778) with a histidine tag at its N-terminus. The binding of this peptide to a lipid monolayer containing a chelated-nickel group (dimyristoylphosphatidyl choline-suberimide-nitriloacetic acid:nickel salt) mimics the native environment at the cytoplasmic leaflet of the plasma membrane. A Nanogold particle was covalently linked to cysteines introduced at the C-terminus and after residue T757 on the integrin peptide, and co-crystallized with chicken smooth muscle α-actinin. The 2-D arrays of the β1-integrin-α-actinin complex were examined by cryoelectron microscopy, with and without the gold label. Averaged projections were calculated for each specimen along with a difference map to determine the relative position of the gold-labeled β1-integrin peptide. The difference maps indicate that the β1-integrin cytoplasmic domain binds α-actinin between the first and second, 3-helix motifs in the central rod domain.

Original languageEnglish (US)
Pages (from-to)290-302
Number of pages13
JournalJournal of Structural Biology
Volume149
Issue number3
DOIs
StatePublished - Jan 1 2005

Fingerprint

Cryoelectron Microscopy
Actinin
Integrins
Binding Sites
Nickel
Gold
Peptides
Cell-Matrix Junctions
Extracellular Matrix Proteins
Choline
Cytoskeleton
Fibronectins
Histidine
Cysteine
Smooth Muscle
Chickens
Salts
Cell Membrane
Lipids
Acids

All Science Journal Classification (ASJC) codes

  • Structural Biology

Cite this

@article{913b9c478ec547a997be2d9577ce693a,
title = "Identification of the β1-integrin binding site on α-actinin by cryoelectron microscopy",
abstract = "Cell-matrix adhesions in migrating cells are usually mediated by integrins, α-β heterodimeric transmembrane proteins that link extracellular matrix molecules such as fibronectin to the cytoskeleton. We have synthesized the cytoplasmic domain of the β1-integrin (residues H738-K778) with a histidine tag at its N-terminus. The binding of this peptide to a lipid monolayer containing a chelated-nickel group (dimyristoylphosphatidyl choline-suberimide-nitriloacetic acid:nickel salt) mimics the native environment at the cytoplasmic leaflet of the plasma membrane. A Nanogold particle was covalently linked to cysteines introduced at the C-terminus and after residue T757 on the integrin peptide, and co-crystallized with chicken smooth muscle α-actinin. The 2-D arrays of the β1-integrin-α-actinin complex were examined by cryoelectron microscopy, with and without the gold label. Averaged projections were calculated for each specimen along with a difference map to determine the relative position of the gold-labeled β1-integrin peptide. The difference maps indicate that the β1-integrin cytoplasmic domain binds α-actinin between the first and second, 3-helix motifs in the central rod domain.",
author = "Deb Kelly and Taylor, {Kenneth A.}",
year = "2005",
month = "1",
day = "1",
doi = "10.1016/j.jsb.2004.11.010",
language = "English (US)",
volume = "149",
pages = "290--302",
journal = "Journal of Structural Biology",
issn = "1047-8477",
publisher = "Academic Press Inc.",
number = "3",

}

Identification of the β1-integrin binding site on α-actinin by cryoelectron microscopy. / Kelly, Deb; Taylor, Kenneth A.

In: Journal of Structural Biology, Vol. 149, No. 3, 01.01.2005, p. 290-302.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Identification of the β1-integrin binding site on α-actinin by cryoelectron microscopy

AU - Kelly, Deb

AU - Taylor, Kenneth A.

PY - 2005/1/1

Y1 - 2005/1/1

N2 - Cell-matrix adhesions in migrating cells are usually mediated by integrins, α-β heterodimeric transmembrane proteins that link extracellular matrix molecules such as fibronectin to the cytoskeleton. We have synthesized the cytoplasmic domain of the β1-integrin (residues H738-K778) with a histidine tag at its N-terminus. The binding of this peptide to a lipid monolayer containing a chelated-nickel group (dimyristoylphosphatidyl choline-suberimide-nitriloacetic acid:nickel salt) mimics the native environment at the cytoplasmic leaflet of the plasma membrane. A Nanogold particle was covalently linked to cysteines introduced at the C-terminus and after residue T757 on the integrin peptide, and co-crystallized with chicken smooth muscle α-actinin. The 2-D arrays of the β1-integrin-α-actinin complex were examined by cryoelectron microscopy, with and without the gold label. Averaged projections were calculated for each specimen along with a difference map to determine the relative position of the gold-labeled β1-integrin peptide. The difference maps indicate that the β1-integrin cytoplasmic domain binds α-actinin between the first and second, 3-helix motifs in the central rod domain.

AB - Cell-matrix adhesions in migrating cells are usually mediated by integrins, α-β heterodimeric transmembrane proteins that link extracellular matrix molecules such as fibronectin to the cytoskeleton. We have synthesized the cytoplasmic domain of the β1-integrin (residues H738-K778) with a histidine tag at its N-terminus. The binding of this peptide to a lipid monolayer containing a chelated-nickel group (dimyristoylphosphatidyl choline-suberimide-nitriloacetic acid:nickel salt) mimics the native environment at the cytoplasmic leaflet of the plasma membrane. A Nanogold particle was covalently linked to cysteines introduced at the C-terminus and after residue T757 on the integrin peptide, and co-crystallized with chicken smooth muscle α-actinin. The 2-D arrays of the β1-integrin-α-actinin complex were examined by cryoelectron microscopy, with and without the gold label. Averaged projections were calculated for each specimen along with a difference map to determine the relative position of the gold-labeled β1-integrin peptide. The difference maps indicate that the β1-integrin cytoplasmic domain binds α-actinin between the first and second, 3-helix motifs in the central rod domain.

UR - http://www.scopus.com/inward/record.url?scp=13844253573&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=13844253573&partnerID=8YFLogxK

U2 - 10.1016/j.jsb.2004.11.010

DO - 10.1016/j.jsb.2004.11.010

M3 - Article

VL - 149

SP - 290

EP - 302

JO - Journal of Structural Biology

JF - Journal of Structural Biology

SN - 1047-8477

IS - 3

ER -