Identification and characterization of functional cation coordination sites in platelet endothelial cell adhesion molecule-1.
We have employed 45CaCl2 binding studies, terbium (Tb3+) luminescence spectroscopy, and electrospray mass spectroscopy (ESI-MS) to identify divalent metal binding properties of soluble recombinant human PECAM-1 (srPECAM-1), and to define unique cation binding domains using short, linear peptide sequences from the protein. PECAM-1 was found to directly interact with 45CaCl2, binding 2.3 nmol of Ca2+/nmol of srPECAM-1 with a Kd of 1.17 nM. PECAM-1 was found to contain high-affinity cation binding sites involving amino acids Asp443, Asp444, and Glu446 of Ig-domain 5 and residues Glu487, Glu490, Asp491, Glu538, Glu540, and Glu542 of Ig-domain 6. The PECAM cation binding sites demonstrated broad specificity for all divalent cations, with Mn2+ having a higher affinity than Ca2+ or Mg2+. Direct binding of Tb3+ to these PECAM peptides was confirmed by ESI-MS. Modeling studies predict that the six cation binding residues within Ig-domain 6 are proximal to each other in three-dimensional space, and may form a single cation coordination site. The identification of cation binding sites in PECAM-1 will direct further work in examining its cation-dependent roles in cellular signaling.
Jackson
D E
DE
Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53226-3548, USA.
Loo
R O
RO
Holyst
M T
MT
Newman
P J
PJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002412:Cations; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013057:Spectrum Analysis; D013725:Terbium / Q000378:metabolism
D002412:Cations; D010455:Peptides; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011994:Recombinant Proteins; D013725:Terbium; D002118:Calcium