Structure and function of H+/K+ pump mutants reveal Na+/K+ pump mechanisms

Abstract

Ion-transport mechanisms evolve by changing ion-selectivity, such as switching from Na+ to H+ selectivity in secondary-active transporters or P-type-ATPases. Here we study primary-active transport via P-type ATPases using functional and structural analyses to demonstrate that four simultaneous residue substitutions transform the non-gastric H+/K+ pump, a strict H+-dependent electroneutral P-type ATPase, into a bona fide Na+-dependent electrogenic Na+/K+ pump. Conversion of a H+-dependent primary-active transporter into a Na+-dependent one provides a prototype for similar studies of ion-transport proteins. Moreover, we solve the structures of the wild-type non-gastric H+/K+ pump, a suitable drug target to treat cystic fibrosis, and of its Na+/K+ pump-mimicking mutant in two major conformations, providing insight on how Na+ binding drives a concerted mechanism leading to Na+/K+ pump phosphorylation.

Crystal structures of Na+,K+‐ATPase reveal the mechanism that converts the K+‐bound form to Na+‐bound form and opens and closes the cytoplasmic gate

“…Thus, NKA utilizes the change in inclination of M5C as the central part of the mechanism [3,32], in particular in the E2 → E1 transition (Movie S1). This mechanism is shared with SERCA.…”

“…6) because a Pro residue (Pro778) disrupts the helix into cytoplasmic (M5C) and extracellular (M5E) halves. The inclination of M5C is determined primarily by bound metal ions at the unwound part [3,32]. The M5 helix is bent by ~12°between M5C and M5E in E2Á2K + towards M1 but becomes straight in E1ÁMg 2+ and even slightly (~3°) bent in the opposite direction in E1Á3Na + .…”

“…Comparison with SERCA: Same principle but different realization Thus, NKA utilizes the change in inclination of M5C as the central part of the mechanism [3,32], in particular in the E2 ? E1 transition (Movie S1).…”

ATP12A Proton Pump as an Emerging Therapeutic Target in Cystic Fibrosis and Other Respiratory Diseases

“…ATP12A proton pumps exist as heterodimers composed of a catalytic α-subunit and an auxiliary β-subunit [3]. The molecular structure of the rat ATP12A was recently resolved by cryo-EM analysis [13]. The α-subunit consists of a transmembrane domain (TMD) with ten membrane-spanning helices, and N-and C-terminal cytosolic tails [13].…”

“…The molecular structure of the rat ATP12A was recently resolved by cryo-EM analysis [13]. The α-subunit consists of a transmembrane domain (TMD) with ten membrane-spanning helices, and N-and C-terminal cytosolic tails [13]. The TMD contains the ion-binding site residues (between transmembrane helices 4 and 8), whereas the cytosolic portion forms the actuator (A), the phosphorylation (P), and the nucleotide-binding (N) domains responsible for auto-phosphorylation and ATP hydrolysis [13].…”