Structure and function of the epidermal growth factor domain of P-selectin

被引:16
作者
Freedman, SJ
Sanford, DG
Bachovchin, WW
Furie, BC
Baleja, JD
Furie, B
机构
[1] TUFTS UNIV,SCH MED,DEPT BIOCHEM,BOSTON,MA 02111
[2] TUFTS UNIV,SCH MED,DEPT MED,BOSTON,MA 02111
[3] SACKLER SCH GRAD BIOMED SCI,BOSTON,MA 02111
[4] TUFTS UNIV NEW ENGLAND MED CTR,CTR HEMOSTASIS & THROMBOSIS RES,DIV HEMATOL ONCOL,BOSTON,MA 02111
关键词
D O I
10.1021/bi9610257
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
P-selectin is a multidomain adhesion protein on the surface of activated platelets and endothelial cells that functions in the recruitment of leukocytes to the site of inflammation. The amino-terminal lectin and EGF domains constitute the ligand recognition unit. We have produced a synthetic 40-residue P-selectin EGF domain (P-sel:EGF) to examine the structure and function of this domain independent of P-selectin. The peptide was folded in vitro and exhibited the same disulfide bonding pattern as other EGF-like domains. P-sel:EGF did not inhibit P-selectin-mediated cellular adhesion assays, indicating that the lectin domain is also required. We undertook the study of the P-selectin EGF by H-1 NMR to determine its structure independent of the lectin domain and to compare its structure to that of E-selectin determined crystallographically [Graves et al. (1994) Nature 367, 532]. Although the binding of P-selectin to its carbohydrate ligand is calcium dependent, and some EGF domains have calcium binding sites, addition of calcium had no effect on the NMR spectrum or on the pH-induced changes. Nearly complete resonance assignments were made from 2D H-1 NMR spectra at pH 6.0. Two sections of antiparallel beta-sheet were identified on the basis of the pattern of long-range NOEs, (3)J(HN alpha) coupling constants, and slowly exchanging amides. The solution structure of the peptide backbone was determined using distance geometry and simulated annealing calculations. The backbone RMSD to the geometric average for 19 final structures is 0.64 +/- 0.17 Angstrom. The resulting fold closely resembles that of other EGF-like peptides, including the E-selectin EGF domain (RMSD similar to 1.08 Angstrom). However, compared to the E-selectin EGF structure which also contains the lectin domain, some residues from 1-11 are less ordered, and novel contacts occur between the amino terminus and the core beta-sheet. Despite marked structural homology of the selectin polypeptide backbones, the selectin EGF surfaces show unique distributions of charged residues, a feature that likely correlates to the functional differences.
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收藏
页码:13733 / 13744
页数:12
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