Final Report Summary - P-TYPE ATPASES (Structural studies of P-type ATPases and their characterization in native membranes)
An estimated 20-30 % of open reading frames from genomes sequences so far encode for membrane proteins (Wallin and von Heijne, 1998) and 60 % of the mass of membranes consists of proteins (Konings et al., 2002). This emphasises that a large proportion of the cells resources are used for membrane function and maintenance. Membrane proteins used for transport of substrates across biological membranes can be divided into two major functional groups: active and passive transporters. Passive transporters transport substrates down a concentration gradient, whereas active transporters use energy to transport a substrate against a concentration gradient.
The project focussed on primary, active transport, where the energy is derived from ATP hydrolysis by means of P-type ATPases. P-type ATPases belong to an enzyme family consisting of a large number of different transport ATPases, including e.g. Na+,K+-ATPases, Ca2+-ATPases, H+,K+-ATPases, and H+-ATPases (Axelsen and Palmgren, 1998) and have in common a covalent phosphoenzyme intermediate formed in the course of the reaction cycle (hence the name 'P-type' ATPase).
The ion gradients created by P-type ATPases play key roles in a wide variety of physiological functions in humans, including signalling and electric excitability through ion currents, secondary active transport, water and salt homeostasis, regulation of cell volume and intracellular pH, as well as secretion of hormones and neurotransmitters. Several P-type ATPases are of significant medical importance. Both the Na+,K+-ATPase and the Ca2+-ATPase are essential for the regulation of cardiac contractility and a number of genetic hereditary diseases involving P-type ATPases have been described (Street et al., 1998) (Odermatt et al., 1996) (Meguro et al. 2001).
The project, carried out at the Centre for Structural Biology and the Pumpkin Centre, University of Aarhus, encompassed three (sub-)projects that all aimed at elucidating the structure-function relationship of P-type ATPases:
1) structure determination of plant plasma-membrane Ca2+-ATPase (PMCA);
2) structural studies on the agrin-Na+,K+-ATPase(a3) complex;
3) topology, arrangement and function of Na+,K+-ATPases in native membranes.
Crystal structure determination of Plasma-membrane Ca2+-ATPase (PMCA)
PMCAs are calcium pumps that expel Ca2+ from eukaryotic cells to maintain overall Ca2+ homoeostasis and to provide local control of intracellular Ca2+ signalling. They are of major physiological importance with different isoforms being essential e.g. for pre- and post-synaptic Ca2+ regulation in neurons, feedback signalling in the heart and sperm motility (Strehler et al. 2007). In the resting state, PMCAs are autoinhibited by binding of their C-terminal (in mammals) or N-terminal (in plants) tail to two major intracellular loops. Activation requires binding of calcium-bound calmodulin (Ca2+-CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain (Carafoli, 1994) (Penniston and Enyedi, 1998). However, no high-resolution structural information is as yet available for any PMCA.
We have expressed and purified the plasma-membrane Ca2+-ATPase (PMCA) ACA8 form Arabidopsis thaliana as well as various truncation constructs and have set-up crystallisation trials. Activity assays confirmed autoinhibition and activation of the pump by calmodulin, however no crystals have been obtained for the full-length pump so far. In addition, we have expressed and purified the N-terminus of ACA8 (residues 40-95) in complex with calmodulin. Crystals have been obtained (Tidow et al. 2010) and structure determination is in progress.
Structural studies on interaction between Na+,K+-ATPase (a3) and agrin.
The Na+,K+-ATPase, discovered by Jens Skou in 1957 (Skou 1957) generates electrochemical gradients for sodium and potassium across the plasma membrane of animal cells that are essential for electrical excitability, cellular uptake of ions, nutrients and neurotransmitters, and regulation of cell volume and intracellular pH. The crystal structure of the sodium-potassium pump was recently solved as an aß? multi-subunit complex (Morth, Pedersen et al. 2007).
Agrin is a protein that was first identified at the vertebrate neuromuscular junction where, by signalling through a muscle specific tyrosine kinase (MuSK), it mediates the motor neuron-induced accumulation of acetylcholine receptors in the postsynaptic region of the skeletal muscle fiber membrane. It was recently shown that agrin binds to the a3 subunit isoform of the Na+,K+-ATPase in the CNS (Hilgenberg, Su et al. 2006). Agrin-a3 Na+,K+-ATPase interactions play an important role in regulating neuronal excitability: Agrin inhibits the activity of the Na+, K+-ATPase. It induces membrane depolarisation and significantly increases the frequency of spontaneous action potentials in cortical neurons.
We started to investigate the structural basis for the agrin-a3-Na+,K+-ATPase interaction by expressing various agrin laminin G3 domain constructs. Those were structurally characterised using various biophysical methods (Tidow et al. 2011). Attempts to reconstitute a functional agrin/NKA-complex are ongoing.
Topology, oligomeric arrangement and function of Na+,K+-ATPases in native membranes.
In recent years several crystal structures of SERCA and other P-type ATPases have been solved. However, their structure, topology and oligomeric arrangement in their native membrane environment are largely unknown. We were planning to investigate these features using Na+,K+-ATPases in native membranes as model system. While this subproject is still on-going, we have acquired initial cryo-electron tomography images in collaboration with Werner Kuehlbrandt (MPI, Frankfurt).
The project focussed on primary, active transport, where the energy is derived from ATP hydrolysis by means of P-type ATPases. P-type ATPases belong to an enzyme family consisting of a large number of different transport ATPases, including e.g. Na+,K+-ATPases, Ca2+-ATPases, H+,K+-ATPases, and H+-ATPases (Axelsen and Palmgren, 1998) and have in common a covalent phosphoenzyme intermediate formed in the course of the reaction cycle (hence the name 'P-type' ATPase).
The ion gradients created by P-type ATPases play key roles in a wide variety of physiological functions in humans, including signalling and electric excitability through ion currents, secondary active transport, water and salt homeostasis, regulation of cell volume and intracellular pH, as well as secretion of hormones and neurotransmitters. Several P-type ATPases are of significant medical importance. Both the Na+,K+-ATPase and the Ca2+-ATPase are essential for the regulation of cardiac contractility and a number of genetic hereditary diseases involving P-type ATPases have been described (Street et al., 1998) (Odermatt et al., 1996) (Meguro et al. 2001).
The project, carried out at the Centre for Structural Biology and the Pumpkin Centre, University of Aarhus, encompassed three (sub-)projects that all aimed at elucidating the structure-function relationship of P-type ATPases:
1) structure determination of plant plasma-membrane Ca2+-ATPase (PMCA);
2) structural studies on the agrin-Na+,K+-ATPase(a3) complex;
3) topology, arrangement and function of Na+,K+-ATPases in native membranes.
Crystal structure determination of Plasma-membrane Ca2+-ATPase (PMCA)
PMCAs are calcium pumps that expel Ca2+ from eukaryotic cells to maintain overall Ca2+ homoeostasis and to provide local control of intracellular Ca2+ signalling. They are of major physiological importance with different isoforms being essential e.g. for pre- and post-synaptic Ca2+ regulation in neurons, feedback signalling in the heart and sperm motility (Strehler et al. 2007). In the resting state, PMCAs are autoinhibited by binding of their C-terminal (in mammals) or N-terminal (in plants) tail to two major intracellular loops. Activation requires binding of calcium-bound calmodulin (Ca2+-CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain (Carafoli, 1994) (Penniston and Enyedi, 1998). However, no high-resolution structural information is as yet available for any PMCA.
We have expressed and purified the plasma-membrane Ca2+-ATPase (PMCA) ACA8 form Arabidopsis thaliana as well as various truncation constructs and have set-up crystallisation trials. Activity assays confirmed autoinhibition and activation of the pump by calmodulin, however no crystals have been obtained for the full-length pump so far. In addition, we have expressed and purified the N-terminus of ACA8 (residues 40-95) in complex with calmodulin. Crystals have been obtained (Tidow et al. 2010) and structure determination is in progress.
Structural studies on interaction between Na+,K+-ATPase (a3) and agrin.
The Na+,K+-ATPase, discovered by Jens Skou in 1957 (Skou 1957) generates electrochemical gradients for sodium and potassium across the plasma membrane of animal cells that are essential for electrical excitability, cellular uptake of ions, nutrients and neurotransmitters, and regulation of cell volume and intracellular pH. The crystal structure of the sodium-potassium pump was recently solved as an aß? multi-subunit complex (Morth, Pedersen et al. 2007).
Agrin is a protein that was first identified at the vertebrate neuromuscular junction where, by signalling through a muscle specific tyrosine kinase (MuSK), it mediates the motor neuron-induced accumulation of acetylcholine receptors in the postsynaptic region of the skeletal muscle fiber membrane. It was recently shown that agrin binds to the a3 subunit isoform of the Na+,K+-ATPase in the CNS (Hilgenberg, Su et al. 2006). Agrin-a3 Na+,K+-ATPase interactions play an important role in regulating neuronal excitability: Agrin inhibits the activity of the Na+, K+-ATPase. It induces membrane depolarisation and significantly increases the frequency of spontaneous action potentials in cortical neurons.
We started to investigate the structural basis for the agrin-a3-Na+,K+-ATPase interaction by expressing various agrin laminin G3 domain constructs. Those were structurally characterised using various biophysical methods (Tidow et al. 2011). Attempts to reconstitute a functional agrin/NKA-complex are ongoing.
Topology, oligomeric arrangement and function of Na+,K+-ATPases in native membranes.
In recent years several crystal structures of SERCA and other P-type ATPases have been solved. However, their structure, topology and oligomeric arrangement in their native membrane environment are largely unknown. We were planning to investigate these features using Na+,K+-ATPases in native membranes as model system. While this subproject is still on-going, we have acquired initial cryo-electron tomography images in collaboration with Werner Kuehlbrandt (MPI, Frankfurt).