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Multicopy Crystallographic & Biophysical Analyses of the N-terminal Domain of NBCe1-A: Illumination of the Human R298S Mutation

The George Washington University & The GW Medical Faculty Associates, Department of Medicine, Division of Renal Diseases & Hypertension 2300 I St, NW, Ross Hall 436B, Washington, DC 20052 Tel: 202-994-4587 Background: NBCe1-A membrane-embedded macromolecules cotransport sodium and bicarbonate ions across the bilayer that serve to maintain acid-base homeostasis throughout the body. Defects are linked to a number of disorders, including proximal renal tubule acidosis, mental retardation, dental defects, and cataracts. Previously, we demonstrated the N-terminal domain of NBCe1-A (Nt) is in a pH-sensitive monomer-dimer equilibrium. At neutral pH, bicarbonate ions bind the Nt, stabilizing dimerization and intermolecular self-associations of dimers. Methods: We determine and analyze the X-ray crystal structure of the Nt as a dimer at 2.4-A resolution using molecular-replacement methods, and a multicopy crystallographic structure of the monomer using 5 atomic models and strict 4-fold NCS constraints in refinement procedures. We measure the pH-sensitivity of a truncated Nt mutant by light-scattering techniques, and bicarbonate, bisulfite, mutant self-association bindings by surface plasmon resonance techniques. Results: The structures reveal that R298 implicated in the disorders is part of a putative conduit that transverses the Nt. The conduit opens to the transmembrane domain (TMD) on one end and an apparent foyer entrance on the opposite end. The naturally occurring mutation R298S disrupts an electrostatic pocket within the conduit that disables substrate binding. We also report similar conducts in family member AE1 (Band 3) when exploring its crystal structure. Further, we identify by biophysical analyses on a truncated Nt that the autoregulatory domain (ARD) at the N-terminus of the Nt is responsible for selfassociations. Conclusions: The Nt responds to changes in pH or bicarbonate fluctuations. In proximal tubule cells, we propose a model where the ARD is a gate for the foyer. When self-associated, the foyer entrance is accessible, allowing substrate entry into the conduit. During acid loads, the gates close entry into the foyer, preventing bicarbonate from leaving the cell. The R298S defect similarly prevents bicarbonate ions from being transported to blood, giving rise to metabolic acidosis that results from the renal tubule acidosis. ABSTRACT