New insights into the role of the N terminus in conformational transitions of the Na,K-ATPase.
The deletion of 32 residues from the N terminus of the alpha1 catalytic subunit of the rat Na,K-ATPase (mutant alpha1M32) shifts the E(1)/E(2) conformational equilibrium toward E(1), and the combination of this deletion with mutation E233K in the M2-M3 loop acts synergistically to shift the conformation further toward E(1) (Boxenbaum, N., Daly, S. E., Javaid, Z. Z., Lane, L. K., and Blostein, R. (1998) J. Biol. Chem. 273, 23086-23092). To delimit the region of the cytoplasmic N terminus involved in these interactions, the consequences of a series of N-terminal deletions of alpha1 beyond Delta32 were evaluated. Criteria to assess shifts in conformational equilibrium were based on effects of perturbation of the entire catalytic cycle ((i) sensitivity to vanadate inhibition, (ii) K(+) sensitivity of Na-ATPase measured at micromolar ATP, (iii) changes in K'(ATP), and (iv) catalytic turnover), as well as estimates of the rates of the conformational transitions of phospho- and dephosphoenzyme (E(1)P --> E(2)P and E(2)(K(+)) --> E(1) + K(+)). The results show that, compared with alpha1M32, the deletion of up to 40 residues (alpha1M40) further shifts the poise toward E(1). Remarkably, further deletions (mutants alpha1M46, alpha1M49, and alpha1M56) reverse the effect, such that these mutants increasingly resemble the wild type alpha1. These results suggest novel intramolecular interactions involving domains within the N terminus that impact the manner in which the N terminus/M2-M3 loop regulatory domain interacts with the M4-M5 catalytic loop to effect E(1) <--> E(2) transitions.
Department of Biochemistry, McGill University, Quebec H3G 1A4, Canada.
Laura
L
Segall
Lois K
LK
Lane
Rhoda
R
Blostein
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000254:Sodium-Potassium-Exchanging ATPase