Periodic Reporting for period 1 - NaKStruc (Structural studies of Na,K-ATPase isoforms and mutants) Reporting period: 2018-06-01 to 2020-05-31 Summary of the context and overall objectives of the project This project focuses on the investigation of disease mutants of Na,K-ATPase. The Na,K-ATPase is a protein found in the cell membrane of virtually every animal cell that maintains the unequal distribution of sodium and potassium between the inside and outside of cells. It is thus vital for numerous functions such as import of other solute, e.g. sugars, neurotransmitters and other ions, cell volume regulation as well as maintaining the electric excitability of neurons and muscle. Mutations of single amino acids have recently been identified as the cause of several neurological diseases. These include Alternating hemiplegia of childhood (AHC), which is characterized by an early onset in infancy, motor impairments such as hemiplegia, i.e. episodic weakness of one half of the body, epileptic seizures and developmental delay. Since there are more than 200 individual mutations causing the disease symptoms vary from milder outcomes to early death. The disease does not only affect individuals, yet depending on severity also their family and caretakers. On of the most severe mutations causing AHC is the E815K mutant, a change of a negatively charged glutamate to a positively charged lysine, which accounts for more than 10% of cases. We aim at studying this mutant and its affect on pump function and the impact on the structure of the protein. We hope that a better understanding of mutations such as E815K will help to pave the way towards cures and that structural insights will lead to drug developments in the near future.We aimed at studying the underlying kinetic mechanism of the E815K-mutation and characterize its impact on the structure of the protein. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We have generated the E815K mutant version of the Na,K-ATPase in yeast, purified the protein and characterized the impact of the mutation on the pump cycle. The single amino acid replacement renders the pump inactive. In order to transport ions the protein has to go through a sequence of ion binding, phosphorylation, conformational transition and ion release on the other side of the membrane, then followed by a second half-cycle for the second ion. We found that the mutant only retained its ability to bind Na-ions, yet is unable to undergo phosphorylateion, resulting in its inactivity. To further elucidate the impact of the mutation we attempted to characterize the structure of the mutant. We have established a reconstitution protocol for Na,K-ATPase in nanodiscs for cryo transmission electron microscopy using the wild type protein as a reference. However, during these studies we found that the mutant exhibits strong oligomeriation/aggregation propensities not shown by the wild type protein. These could not be overcome by any of the tested parameters yet unfortunately severely impede any attempts of structure determination. This has not been described for the E815K mutant yet similar observations were made for other mutants causing severe forms of AHC. The L924P mutant was found to be missfolded and cause an unfolded protein response. In agreement with this we were unable to express the L924P mutant.It should be noted that although not successful for the E815K mutant, the established protocol will widen the possibility to study wild type protein and other versions of the Na,K-ATPase that can not be obtained from native sources. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) This work represents the first characterization of a purified AHC variant of the Na,K-ATPase. Unfortunately, no structure could be obtained due to the strong propensity of the mutant to oligomerize/aggregate. However, the established platform may allow the study and characterization of other disease variants and will hopefully lead to a better understanding of Na,K-ATPase associated diseases and may ultimately pave the way towards compound screening and drug development. Properties of the E815K mutant and its location on the Na,K-ATPase.
