Abstract
We have employed echistatin, a 5.4 kDa snake venom disintegrin, as a model protein to investigate the paradox that small ligand-mimetics can bind to the resting αIIbβ3 integrin while adhesive macromolecules cannot. We characterized the interactions between purified human αIIbβ3 and two recombinant echistatin variants: rEch (1-49) M28L, chosen for its selectivity toward β3-integrins, and rEch (1-40) M28L, a carboxy-terminal truncation mutant. While both contain an RGD integrin targeting sequence, only rEch (1-49) M28L was an effective inhibitor of αIIbβ3 function. Electron microscopy of rotary shadowed specimens yielded a variety of αIIbβ3 conformers ranging from compact, spherical particles (maximum dimension 22 nm) to the classical "head with two tails" forms (32 nm). The population of larger particles (42-56 nm) increased from 17% to 28% in the presence of rEch (1-49) M28L, indicative of ligand-induced oligomerization. Sedimentation velocity measurements demonstrated that both full length and truncated echistatin perturbed αIIbβ3's solution structure, yielding slower-sedimenting open conformers. Dynamic light scattering showed that rEch (1-49) M28L protected αIIbβ3 from thermal aggregation, raising its transition mid-point from 46°C to 69°C; a smaller shift resulted with rEch (1-40) M28L. Sedimentation equilibrium demonstrated that both echistatin ligands induced substantial αIIbβ3 dimerization. van't Hoff analysis revealed a pattern of entropy/enthalpy compensation similar to tirofiban, a small RGD ligand-mimetic that binds tightly to αIIbβ3, but yields smaller conformational perturbations than echistatin. We propose that echistatin may serve as a paradigm for understanding multidomain adhesive macromolecules because its ability to modulate αIIbβ3's structure resides on an RGD loop, while full disintegrin activity requires an auxiliary site that includes the carboxy-terminal nine amino acid residues.
| Original language | English |
|---|---|
| Pages (from-to) | 1625-1636 |
| Number of pages | 12 |
| Journal | Journal of Molecular Biology |
| Volume | 342 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - Oct 1 2004 |
Fingerprint
Keywords
- conformation
- disintegrin
- integrin
- oligomerization
- thermodynamics
ASJC Scopus subject areas
- Virology
Cite this
The disintegrin echistatin stabilizes integrin αIIbβ3's open conformation and promotes its oligomerization. / Hantgan, Roy R.; Stahle, Mary C.; Connor, John H.; Lyles, Douglas S.; Horita, David A.; Rocco, Mattia; Nagaswami, Chandrasekaran; Weisel, John W.; McLane, Mary Ann.
In: Journal of Molecular Biology, Vol. 342, No. 5, 01.10.2004, p. 1625-1636.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - The disintegrin echistatin stabilizes integrin αIIbβ3's open conformation and promotes its oligomerization
AU - Hantgan, Roy R.
AU - Stahle, Mary C.
AU - Connor, John H.
AU - Lyles, Douglas S.
AU - Horita, David A.
AU - Rocco, Mattia
AU - Nagaswami, Chandrasekaran
AU - Weisel, John W.
AU - McLane, Mary Ann
PY - 2004/10/1
Y1 - 2004/10/1
N2 - We have employed echistatin, a 5.4 kDa snake venom disintegrin, as a model protein to investigate the paradox that small ligand-mimetics can bind to the resting αIIbβ3 integrin while adhesive macromolecules cannot. We characterized the interactions between purified human αIIbβ3 and two recombinant echistatin variants: rEch (1-49) M28L, chosen for its selectivity toward β3-integrins, and rEch (1-40) M28L, a carboxy-terminal truncation mutant. While both contain an RGD integrin targeting sequence, only rEch (1-49) M28L was an effective inhibitor of αIIbβ3 function. Electron microscopy of rotary shadowed specimens yielded a variety of αIIbβ3 conformers ranging from compact, spherical particles (maximum dimension 22 nm) to the classical "head with two tails" forms (32 nm). The population of larger particles (42-56 nm) increased from 17% to 28% in the presence of rEch (1-49) M28L, indicative of ligand-induced oligomerization. Sedimentation velocity measurements demonstrated that both full length and truncated echistatin perturbed αIIbβ3's solution structure, yielding slower-sedimenting open conformers. Dynamic light scattering showed that rEch (1-49) M28L protected αIIbβ3 from thermal aggregation, raising its transition mid-point from 46°C to 69°C; a smaller shift resulted with rEch (1-40) M28L. Sedimentation equilibrium demonstrated that both echistatin ligands induced substantial αIIbβ3 dimerization. van't Hoff analysis revealed a pattern of entropy/enthalpy compensation similar to tirofiban, a small RGD ligand-mimetic that binds tightly to αIIbβ3, but yields smaller conformational perturbations than echistatin. We propose that echistatin may serve as a paradigm for understanding multidomain adhesive macromolecules because its ability to modulate αIIbβ3's structure resides on an RGD loop, while full disintegrin activity requires an auxiliary site that includes the carboxy-terminal nine amino acid residues.
AB - We have employed echistatin, a 5.4 kDa snake venom disintegrin, as a model protein to investigate the paradox that small ligand-mimetics can bind to the resting αIIbβ3 integrin while adhesive macromolecules cannot. We characterized the interactions between purified human αIIbβ3 and two recombinant echistatin variants: rEch (1-49) M28L, chosen for its selectivity toward β3-integrins, and rEch (1-40) M28L, a carboxy-terminal truncation mutant. While both contain an RGD integrin targeting sequence, only rEch (1-49) M28L was an effective inhibitor of αIIbβ3 function. Electron microscopy of rotary shadowed specimens yielded a variety of αIIbβ3 conformers ranging from compact, spherical particles (maximum dimension 22 nm) to the classical "head with two tails" forms (32 nm). The population of larger particles (42-56 nm) increased from 17% to 28% in the presence of rEch (1-49) M28L, indicative of ligand-induced oligomerization. Sedimentation velocity measurements demonstrated that both full length and truncated echistatin perturbed αIIbβ3's solution structure, yielding slower-sedimenting open conformers. Dynamic light scattering showed that rEch (1-49) M28L protected αIIbβ3 from thermal aggregation, raising its transition mid-point from 46°C to 69°C; a smaller shift resulted with rEch (1-40) M28L. Sedimentation equilibrium demonstrated that both echistatin ligands induced substantial αIIbβ3 dimerization. van't Hoff analysis revealed a pattern of entropy/enthalpy compensation similar to tirofiban, a small RGD ligand-mimetic that binds tightly to αIIbβ3, but yields smaller conformational perturbations than echistatin. We propose that echistatin may serve as a paradigm for understanding multidomain adhesive macromolecules because its ability to modulate αIIbβ3's structure resides on an RGD loop, while full disintegrin activity requires an auxiliary site that includes the carboxy-terminal nine amino acid residues.
KW - conformation
KW - disintegrin
KW - integrin
KW - oligomerization
KW - thermodynamics
UR - http://www.scopus.com/inward/record.url?scp=4444378872&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=4444378872&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2004.08.009
DO - 10.1016/j.jmb.2004.08.009
M3 - Article
C2 - 15364586
AN - SCOPUS:4444378872
VL - 342
SP - 1625
EP - 1636
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 5
ER -