Abstract
Single molecule atomic force microscopy (smAFM) was employed to unfold transmembrane domain interactions of a unique vacuolar H(+)-pyrophosphatase (EC 3.6.1.1) from Vigna radiata. H(+)-Pyrophosphatase is a membrane-embedded homodimeric protein containing a single type of polypeptide and links PPi hydrolysis to proton translocation. Each subunit consists of 16 transmembrane domains with both ends facing the lumen side. In this investigation, H(+)-pyrophosphatase was reconstituted into the lipid bilayer in the same orientation for efficient fishing out of the membrane by smAFM. The reconstituted H(+)-pyrophosphatase in the lipid bilayer showed an authentically dimeric structure, and the size of each monomer was ∼4 nm in length, ∼2 nm in width, and ∼1 nm in protrusion height. Upon extracting the H(+)-pyrophosphatase out of the membrane, force-distance curves containing 10 peaks were obtained and assigned to distinct domains. In the presence of pyrophosphate, phosphate, and imidodiphosphate, the numbers of interaction curves were altered to 7, 8, and 10, respectively, concomitantly with significant modification in force strength. The substrate-binding residues were further replaced to verify these domain changes upon substrate binding. A working model is accordingly proposed to show the interactions between transmembrane domains of H(+)-pyrophosphatase in the presence and absence of substrate and its analog.
Highlights
Hϩ-PPase is a phosphoanhydride bond in pyrophosphate (PPi)-dependent proton translocase with 16 transmembrane domains embedded in a lipid bilayer
The patterns of Hϩ-PPase force-distance curves (F-Ds) were modified upon substrate binding, which suggested domains of the active site might undergo dynamic movements to bind its substrate during PPi hydrolysis
The reconstituted Hϩ-PPase liposome was sprayed on a freshly cleaved mica surface for atomic force microscopy (AFM) scanning (Fig. 3A) The AFM images showed that the membrane protein Hϩ-PPase was inserted into the lipid bilayer; the Hϩ-PPase-enriched liposome was adsorbed on a mica surface (Fig. 3B)
Summary
Hϩ-PPase is a PPi-dependent proton translocase with 16 transmembrane domains embedded in a lipid bilayer. H؉-Pyrophosphatase is a membrane-embedded homodimeric protein containing a single type of polypeptide and links PPi hydrolysis to proton translocation. Replacement of a conserved alanine residue at position 460 by a lysine converted the Kϩ-dependent into the Kϩ-independent form of Hϩ-PPase from Carboxydothermus hydrogenoformans [6], indicating that the Ala460 is involved in Kϩ binding. Both Hϩ-PPase subfamilies required Mg2ϩ as the cofactor, whereas Ca2ϩ, Naϩ, and FϪ could inhibit the Hϩ-PPase activities to some extent [1, 6]. Previous studies showed the structure-function relationship of Hϩ-PPase and identified the pivotal domains and residues involved in enzymatic activities and structural integrity [1, 12,13,14,15,16]
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