Identification of a critical ankyrin-binding loop on the cytoplasmic domain of erythrocyte membrane band 3 by crystal structure analysis and site-directed mutagenesis

Seon Hee Chang, Philip S. Low

Research output: Contribution to journalArticle

69 Scopus citations

Abstract

The cytoplasmic domain of erythrocyte membrane band 3 (cdb3) serves as a center of membrane organization, interacting with such proteins as ankyrin, protein 4.1, protein 4.2, hemoglobin, several glycolytic enzymes, a tyrosine phosphatase, and a tyrosine kinase, p72syk. The crystallographic structure of the cdb3 dimer has revealed that residues 175-185 assume β-hairpin loop similar to a putative ankyrin-binding motif at the cytoplasmic surface of the Na+/K+-ATPase. To test whether this hairpin loop constitutes an ankyrin-binding site on cdb3, we have deleted amino acids 175-185 and substituted the 11-residue loop with a Gly-Gly dipeptide that bridges the deletion without introducing strain into the structure. Although the deletion mutant undergoes the same native conformational changes exhibited by wild type cdb3 and binds other peripheral proteins normally, the mutant exhibits no affinity for ankyrin. This suggests that the exposed β-hairpin turn indeed constitutes a major ankyrin-binding site on cdb3. Other biochemical studies suggest that ankyrin also docks at the NH2 terminus of band 3. Thus, antibodies to the NH2 terminus of cdb3 block ankyrin binding to the cdb3, and ankyrin binding to cdb3 prevents p72syk phosphorylation of cdb3 at its NH2 terminus (predominantly at Tyr-8). However, a truncation mutant of cdb3 lacking the NH2-terminal 50 residues displays the same binding affinity as wild type cdb3. These data thus suggest that the NH2 terminus of cdb3 is proximal to but not required for the cdb3. ankyrin interaction.

Original languageEnglish (US)
Pages (from-to)6879-6884
Number of pages6
JournalJournal of Biological Chemistry
Volume278
Issue number9
DOIs
StatePublished - Feb 28 2003

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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