Elucidation of the interactions between band 3 and ankyrin
Abstract
The erythrocyte membrane is primarily linked to the spectrin-based cytoskeleton through association of band 3 with both ankyrin and adducin. Both interactions are critical for membrane stability and erythrocyte morphology. Although previous studies have identified regions on the cytoplasmic domain of band 3 (cdB3) that are at least partly responsible for this essential bridge, several studies suggest that additional ankyrin binding sites exist. 1) A second loop, comprising residues 63-74, adjacent to the first that is highly conserved, 2) in vivo effects of the first deletion mutant, residues 175-185, in mice were surprisingly mild, 3) antibodies to regions outside of this primary binding loop block ankyrin binding and 4) multiple domains of ankyrin bind band 3, suggesting multiple binding sites on band 3 must be available for binding intact ankyrin. This research is dedicated to further understanding the interactions between band 3 and ankyrin. We provide evidence that implicates another loop on cdB3 in ankyrin binding, as well as residues on ankyrin that bind to cdB3. First, a synthetic peptide comprising residues 63-74 of cdB3 abrogated ankyrin binding. Next, expressed mutant cdB3 lacking residues 63-74 failed to bind ankyrin in pull-down experiments. Then, to gain insight on how cdB3 and ankyrin dock, molecular simulations were conducted and several residues on ankyrin were identified as potential interaction sites. We decided to create a mutant of ankyrin in which these residues were changed to amino acids of opposite charge. This mutant of ankyrin did not bind to cdB3 in pull-down experiments. We now have a molecular understanding of the interactions between the cytoplasmic domain of band 3 and ankyrin. With this knowledge we have defined the molecular interactions between these critical proteins in the red cell.
Degree
Ph.D.
Advisors
Low, Purdue University.
Subject Area
Biochemistry|Physical chemistry
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