In erythrocytes, the reversible deformability needed to accomplish passage through capillaries is largely controlled by a vast network of proteins attached to the membrane. Spectrin is a key element of this network. The attachment between the membrane and the spectrin cytoskeleton is accomplished through an adaptor protein, ankyrin, which bridges the interaction between 2-spectrin and the membrane bound anion- exchange transporter band 3. The ankyrin-spectrin interaction has important implications for normal cell function and for some diseases. Hereditary spherocytosis and hereditary elliptocytosis are human blood disorders where mutations have been mapped to spectrin and ankyrin. Ankyrin defects are also responsible for the cardiac death syndrome, long-QT cardiac arrhythmia. In addition, ankyrin, and in particular its spectrin binding site, has been involved in malaria red blood cell infection. This proposal is concerned with biochemical and structural studies of human erythroid 2-spectrin and ankyrin with the goal of providing an atomic understanding of this complex. In the past few years we have made substantial progress towards this goal, including solving the atomic structure of the individual binding domains of 2-spectrin and ankyrin R, and the complex formed by these two domains. These structures are starting to provide important structural information on these two critical molecules and allowing us to relate the structure to the wealth of existing functional, biochemical, and clinical data. For the next project period we propose to continue and expand our studies of ankyrin and spectrin. The specific aims for this proposal are: 1) to continue our work on the three dimensional structure of a complex of the spectrin binding domain of ankyrin and the ankyrin binding domain of spectrin and to study the role of different amino acids and different regions in the spectrin/ankyrin recognition process and, 2) to expand our biophysical and structural studies to include other regions of spectrin and ankyrin that have not been characterized structurally. The work is based on a combination of molecular biological, biophysical, and biochemical methods to produce and characterize the molecules that we require for our work, X-ray crystallography to solve their atomic structures, and cellular studies to correlate our in vitro findings to observations in erythrocytes. The knowledge of the structure of the complex formed by spectrin and ankyrin promises to provide important and relevant information on this critical protein complex involved in a central cellular process and with important implications to human well-being.