Identification and Characterization of Functional Cation Coordination Sites in Platelet Endothelial Cell Adhesion Molecule-1

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• 作者：Denise E. Jackson ; Rachel O. Loo ; M. Trudy Holyst ; and Peter J. Newman
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：August 5, 1997
• 年：1997
• 卷：36
• 期：31
• 页码：9395 - 9404
• 全文大小：359K
• 年卷期：v.36,no.31(August 5, 1997)
• ISSN：1520-4995

文摘

We have employed ⁴⁵CaCl₂binding studies, terbium (Tb³⁺) luminescencespectroscopy, andelectrospray mass spectroscopy (ESI-MS) to identify divalent metalbinding properties of solublerecombinant human PECAM-1 (srPECAM-1), and to define unique cationbinding domains using short,linear peptide sequences from the protein. PECAM-1 was found todirectly interact with ⁴⁵CaCl₂,binding2.3 nmol of Ca²⁺/nmol of srPECAM-1 with aK_d of 1.17 nM. PECAM-1 was found to containhigh-affinity cation binding sites involving amino acids Asp₄₄₃,Asp₄₄₄, and Glu₄₄₆ of Ig-domain 5 andresiduesGlu₄₈₇, Glu₄₉₀, Asp₄₉₁,Glu₅₃₈, Glu₅₄₀, and Glu₅₄₂ ofIg-domain 6. The PECAM cation binding sitesdemonstrated broad specificity for all divalent cations, withMn²⁺ having a higher affinity than Ca²⁺orMg²⁺. Direct binding of Tb³⁺ to thesePECAM peptides was confirmed by ESI-MS. Modelingstudiespredict that the six cation binding residues within Ig-domain 6 areproximal to each other inthree-dimensional space, and may form a single cation coordinationsite. The identification of cationbinding sites in PECAM-1 will direct further work in examining itscation-dependent roles in cellularsignaling.