Structure–function relationships in membrane segment 6 of the yeast plasma membrane Pma1 H+-ATPase

Abstract

The crystal structures of the Ca2+- and H+-ATPases shed light into the membrane embedded domains involved in binding and ion-translocation. Consistent with site-directed mutagenesis, these structures provided additional evidence that membrane-spanning segments M4, M5, M6 and M8 are the core through which cations are pumped. In the present study, we have used alanine/serine scanning mutagenesis to study the structure-function relationships within M6 (Leu-721-Pro-742) of the yeast plasma membrane ATPase. Of the 22 mutants expressed and analyzed in secretory vesicles, alanine substitutions at two well conserved residues (Asp-730 and Asp-739) led to a complete block in biogenesis; in the mammalian P-ATPases, residues corresponding to Asp-730 are part of the cation-binding domain. Two other mutants (V723A and I736A) displayed a dramatic 20 fold increase in the IC50 for inorganic orthovanadate compared to the wild-type control, accompanied by a significant reduction in the Km for Mg-ATP, and an alkaline shift in the pH optimum for ATP hydrolysis. This behavior is apparently due to a shift in equilibrium from the E2 conformation of the ATPase towards the E1 conformation. By contrast, the most striking mutants lying toward the extracellular side in a helical structure (L721A, I722A, F724A, I725A, I727A and F728A), were expressed in secretory vesicles but had a severe reduction of ATPase activity. Moreover, all of these mutants but one (F728A) were unable to support yeast growth when the wild-type chromosomal PMA1 gene was replaced by the mutant allele. Surprisingly, in contrast to M8 where mutations S800A and E803Q (Guerra, G. et al., Biochim. Biophys. Acta 1768: 2383–2392, 2007) led to a dramatic increase in the apparent stoichiometry of H+ transport, three substitutions (A726S, A732S and T733A) in M6 showed a reduction in the apparent coupling ratio. Taken together, these results suggest that M6 residues play an important role in protein stability and function, and probably are responsible for cation binding and stoichiometry of ion transport as suggested by homology modeling.

Ser/Thr Motifs in Transmembrane Proteins: Conservation Patterns and Effects on Local Protein Structure and Dynamics

“…A recent example of a serine-scanning investigation in which Ser/Thr Signatures have been introduced is the investigation by Miranda et al (2011) of the structure–function relationship in TM6 of the yeast H + ATPase Pma1. The yeast Pma1 has a Thr at position 733; the A732S and A735S mutations, which introduce ST and, respectively, TxS Signatures, led to a reduction in the proton pumping activity (Miranda et al 2011). In the absence of detailed information about the structure, dynamics, and water interactions of the mutant proteins, the molecular origin of the mutation effect is unclear.…”

Structure of the hexameric fungal plasma membrane proton pump in its autoinhibited state

“…3 ). This residue corresponds to Asp 684 in AHA2 and the Ca 2+ -coordinating residue Asp 800 in SERCA and has been shown to be essential for proton transport and E 1→ E 2 transitions in Pma1 ( 20 , 21 ). Asp 730 is situated adjacent to the internal cavity and opposite the equally highly conserved Asn 154 in M2, at a distance suitable for capturing a proton within a hydrogen bond (2.9 Å between the aspartate carboxyl and the asparagine amide).…”

Biophysical comparison of ATP-driven proton pumping mechanisms suggests a kinetic advantage for the rotary process depending on coupling ratio

“…Therefore, at least based on our paradigm, kinetics would not be a criterion in the evolutionary selection of a specific mechanism for the proton pump when the coupling ratio is one. The plant/fungal plasma membrane proton pumps, for example, with a coupling ratio of one (based on currently available kinetic and structural evidence [ 3 , 8 , 10 , 38 , 39 ]) employs the alternating access mechanism (P-ATPase). We speculate that the single protein P-ATPase [ 10 ], compared to the 25–39 protein complex for the V-ATPase [ 9 ], was selected for its simplicity.…”