Investigating the proton-transolcating subunit of the rotary A-type ATPase

Abstract

All forms of life are dependent on proton gradients as a source of energy to power essential cellular processes, and they all store this energy in the form of the nucleotide adenosine triphosphate (ATP). The rotary ATPases are multi-subunit molecular machines that utilise a rotary catalytic mechanism to couple ATP synthesis/hydrolysis to proton translocation across biological membranes. All rotary ATPases contain membrane-embedded proton-translocating subunits, via which protons enter and exit the membrane. This is the only subunit common to all rotary ATPases for which there is no high resolution structural information, and thus the molecular understanding of mechanism by which proton translocation is coupled to ATP synthesis/hydrolysis remains incomplete. To determine this structure, twelve homologues of the proton translocating subunit from A-type rotary ATPases (subunit I) were cloned into eight different expression vectors, giving 96 expression constructs. Subunit I from the bacterium Meiothermus ruber gave the highest expression. Purification of this construct was optimised and purified protein was subjected to multi-angle laser light scattering (MALLS) analysis and crystallisation trials. The interaction between subunit I and the peripheral stalks, one of the binding partners of subunit I in the intact A-ATPase complex, was also investigated by pulldown analysis, MALLS and isothermal titration calorimetry (ITC). The X-ray crystal structure of subunit I was not able to be determined, and it was proposed that this was most likely because subunit I is most stable within the intact A ATPase complex and in the lipid bilayer of the native organism. Nevertheless, it was found that the recombinantly expressed and purified M. ruber subunit I was able to bind to one peripheral stalk heterodimer, indicating subunit I was folded. There are, however, two peripheral stalk binding sites within subunit I, suggesting the two sites have different affinities for the peripheral stalk heterodimers. This has implications for the assembly of the intact A-ATPase complex.