Download Elucidating the complete reaction cycle for membrane

Elucidating the complete reaction cycle for membrane-bound
pyrophosphatases
Craig Wilkinson, Nita Shah and Adrian Goldman
Introduction
Pyrophosphate (PPi) is abundant in cells as a by-product of cellular anabolic processes such
as the hydrolysis of ATP. Membrane-bound pyrophosphatases (MPPases) are helical
transmembrane enzymes that couple the hydrolysis of PPi to the pumping of protons (H+)
and/or sodium ions (Na+) across a membrane, generating a chemical and electrical potential.
This potential can be used to drive other cellular reactions such as ATP synthesis and the
primary active transport of solutes. MPPases are found in bacteria, archaea, protozoans and
plants, but not in mammals. Crucially, MPPases are found in numerous bacterial and
protozoan parasites such as Plasmodium spp. (malaria), Toxoplasma gondii (toxoplasmosis),
Trypanosoma spp. (trypanosomiasis), Leishmania spp. (leishmaniasis) and Clostridium spp.
(infectious diarrhoea). In these organisms, MPPase is essential during stress such as lowenergy conditions, cold-shock and osmotic stress, and therefore has been validated as a drug
target against several of these parasites. Our work has focused on obtaining threedimensional models of Thermotoga maritima Na+-pumping MPPase (TmPPase) and the
Vigna radiata H+-pumping MPPase (VrPPase) during various stages of the reaction cycle in
order to elucidate the reaction mechanism. Understanding the mechanism of MPPases is
essential for exploiting this enzyme as a drug target, and atomic models of MPPase can be
used for structure based drug design approaches.
Results
We have recently solved the strucutres of substrate analogue (IDP) and Na+ bound TmPPase,
and single phosphate bound VrPPase. We integrated this knowledge with our previous
structures of TmPPase in resting and product-bound states and VrPPase in an IDP-bound
state to follow the structural changes in MPPases throuhgout the reaction cycle. These
structures revealed MPPases are homodimeric, and each monomer has a novel 16
transmembrane-helical structure (Fig. 1). This integral-membrane protein consists of a
IDP
Mg2+
Cell
inner-membrane
Cytoplasm
Na+
Figure 1. Structure of Thermotoga maritima MPPase
(TmPPase). Dimeric TmPPase bound with the PPi
2+
+
analogue IDP (orange), Mg (green) and Na (purple).
One monomer of TmPPase has been coloured light
blue, the other dark blue. The position of the lipid
membrane is represented by a bilayer assembled using
course grained MD simulations. The top of the enzyme
(bound to PPi) is facing the cytoplasm whereas the
bottom of the enzyme is facing the periplasm.
Periplasm
continuous active site with four distinct parts: the cytoplasmic facing hydrolytic centre, the
coupling funnel, the ion gate, and the periplasmic facing exit channel (Fig. 1). By analysing
the different MPPase strucutres, we have identified the major steps during the PPi cleavage
and ion-pumping in the reaction cycle. When substrate binds and the loops on the
cytoplasmic side close, the transported ion (Na+ or H+) binds to the ion-binding site at the
ionic-gate, displacing a lysine (K16.50) from the gate (Fig. 2A). This is either driven by, or
drives, the ‘downward’ movement of tranmembrane helix (TMH) 6. The downward motion
of TMH 6 activates the enzyme by placing an aspartic acid (D6.43) into position, which
(a)
(b)
Figure 2: Transitions in the coupling
funnel of TmMPPase during the
catalytic cycle. (a) TmMPPase in the
substrate analogue-bound state,
+
revealing the position of bound Na
(purple) coordinated by acidic
residues
(blue
dashes).
(b)
Comparison of TmPPase in the
resting state with no product or
substrate bound (light blue) with a
formed
salt-bridge
network
(magenta dashes) against substrate
+
analogue and Na bound TmPPase
(dark blue). This highlights the
changes in the position of D6.43 and
D16.39 between resting and
substrate bound states.
coordinates a water nucleophile in the coupling funnel (Fig. 2B). Concomitantly, there is a
constriction of the active site cavity by the movement of the cytoplasmic ends of TMHs 5, 6,
11, 12, 15 and 16, and capping of the active site by the helix 5-6 loop. The hydrolysis of PPi
then drives the enzyme into an occluded state, open neither to the cytoplasm nor the
periplasm. We posit that ion-pumping requires a short-lived transition state in which the iongate and the exit channel open in sequence, which likely involves the ‘downward’ movement
of TMH 12. There may be a second, loosely-bound state for the pumped ion on the
periplasmic side of a glutamic acid residue (E6.53 in TmPPase); in such a state, K16.50
would swing back in to close the ionic gate from the cytoplasmic side. Only after these steps
have occured can the active site reopen on cytoplasmic side, allowing the leaving group
phosphate to diffuse away, and the enzyme to return to the resting state.
Publications
Saarenpää T.J., Jaakola V-P & Goldman A. (2015) Baculovirus mediated expression of
GPCRs in insect cells. Methods Enzymol. 556:185-218.
Rosti K., Goldman A. & Kajander T. (2015) Solution structure and biophysical
characterization of the multifaceted signalling effector protein growth arrest specific-1. BMC
Biochem. 16: 8.
Bhattacharjee A., Reuter S., Trojnar E., Kolodziejczyk R., Seeberger H., Hyvarinen S.,
Uzonyi B., Szilagyi A., Prohaszka Z., Goldman A., Jozsi M., Jokiranta T.S. (2015) The major
autoantibody epitope on Factor H in atypical Hemolytic Uremic Syndrome is structurally
different from its homologous site in Factor H related protein 1 supporting a novel model for
induction of autoimmunity in this disease. J. Biol. Chem. 10:9500-9510.
Funding
This work was funded by the BBSRC and the H2020 program: Nita Shah is a Marie Curie
fellow.
Collaborators
University of Leeds: S. Harris, L. Jeuken and R. Tuma.
External: J. Kellosalo, T. Kajander, Y-J. Sun, J. Yli-Kauhaluoma, H. Xhaard, S. Meri, R.
Lahti.