Pactolus I-domain: functional switching of the Rossmann fold.
Murine Pactolus is a neutrophil-specific single chain glycoprotein that plays a role as an apoptosis marker for macrophages. The extracellular region of the protein shows strong sequence similarities to integrin beta-subunits. Critical sequence modifications differentiate its function when compared to the integrin family. We show experimentally that Pactolus I-domain does not bind divalent metal ions, indicating that ligand binding is not mediated through a metal ion-dependent adhesion site (MIDAS). NMR data was used to map secondary structure and the strand pairing within the beta-sheet to confirm an overall Rossmann fold topology. Homology modeling enhanced by the NMR data was used to determine the overall structure, with two key loop insertions/deletions (insertion 2 and SDL) that distinguish the Pactolus I-domain from the integrin alpha I-domain and beta I-domains. NMR peak exchange broadening is observed due to dimerization, correlating to the beta I-domain and beta propeller heterodimerization region within the integrin headpiece. Two unique N-linked glycosylation sites (Asn151 and Asn230) within this region disrupt dimerization and may account for why Pactolus is not found to associate with an alpha-subunit. These changes in quaternary structure, ligand binding loops, glycosylation, and metal sites illustrate how evolution has rapidly and effectively altered key aspects of the integrin beta-subunit to derive a protein of novel function on an existing protein scaffold.
Sen
Mehmet
M
Department of Biology and Biochemistry, University of Houston, 4800 Calhoun, Houston, Texas 77204-5001, USA.
Legge
Glen B
GB
D002118:Calcium / Q000737:chemistry; D002850:Chromatography, Gel; D019281:Dimerization; D006031:Glycosylation; D006860:Hydrogen Bonding; D019012:Integrin beta1 / Q000737:chemistry* / Q000235:genetics; D008274:Magnesium / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding
D019012:Integrin beta1; C111859:pactolus; D008274:Magnesium; D002118:Calcium