Elucidating Functional Aspects of P-type ATPases: Structural Studies of SERCA1a and LpCopA

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandling

P-type ATPases are proteins that act to maintain ion homeostasis and electrochemical gradients through the translocation of cations across cell membranes. Underscoring their significance in humans, dysfunction of the ATPases can lead to crucial diseases. Dysfunction of the sarco(endo)plasmic reticulum Ca2+-ATPase from fast-twitch skeletal muscle (SERCA1a) is associated with skin and muscle diseases, while mutations of the Cu+-ATPase (CopA) give rise to Cu+ deficiency or excess as seen in the devastating Menkes and Wilson’s diseases. Furthermore, the essential role that these proteins have in all domains of life renders them attractive drug targets in diseases different from those that they are implicated with. For instance, SERCA1a has become an attractive target for inducing apoptosis in proliferating cancer cells while the essential role of CopA in pathogenic bacteria, fungi and parasites makes it an attractive target for novel drugs, battling the near-future threat of antimicrobial resistance. Consequently, unveiling how these two ATPases function at the atomistic level will not only advance basic bioscience, but provide a framework for designing a new generation of drugs against cancer and pathogenic microbes.

The goal of this Ph.D. dissertation was to functionally characterize SERCA1a and CopA from Legionella pneumophila (LpCopA) through a range of different methods within structural biology. Crystallographic studies of SERCA1a led to a newly determined crystal structure and helped enlighten how thapsigargin, a potent inhibitor of SERCA1a, depends on a water mediated hydrogen bond network when bound to SERCA1a. Furthermore, molecular dynamics (MD) simulations of the same P-type ATPase were used to assess a long-standing question whether cholesterol affects SERCA1a through direct or indirect interactions. The MD study supported the proposition that cholesterol regulation of SERCA1a is due to an indirect effect. Crystallization of a Wilson’s disease causing mutant inserted into LpCopA, resulted in the determination of a 5 Å resolution structure with a structural fold similar to that of the wild type (WT) protein. The discrepancy between the newly determined crystal structure of LpCopA and the functional manifestations of the missense mutation in human CopA, could indicate that LpCopA is insufficient in structurally elucidating the effect of disease-causing mutations in the human CopA proteins. MD simulations, which combine coarse-grained (CG) and atomistic procedures, were set up in order to elucidate mechanistic implications exerted by the lipid bilayer on LpCopA. The MD simulations of LpCopA corroborated previous and new in vivo activity data and showed that the bacterial, anionic phospholipids, phosphatidylglycerol (PG) and cardiolipin (CL), have an increased propensity to bind to certain areas of the transmembrane domain. Further studies are required to infer whether these observations support specific lipid-protein interactions and what their significance is for LpCopA as a model system as well as the human CopA proteins. A considerable number of crystallization experiments were set up on LpCopA in the pursuit of determining the first metal-bound state of this class of P-type ATPases. Despite various approaches, the results were predominantly discouraging; however, encouraging hits were obtained with AgNO3 and E. coli lipids, potentially underscoring the importance of utilizing native lipids. Finally, atomistic MD simulations of a Zn2+-ATPase (ZntA) were set up and computed in order to elucidate the dynamics of its ion exit pathway, and helped reveal how residues lining the pathway might drive the Zn2+ ion to exit.
Antal sider265
Rekvirerende organGraduate School of Science and Technology
StatusUdgivet - 28 jan. 2015

Note vedr. afhandling

P-type ATPases are a certain class of membrane proteins which maintains ion homeostasis and electrochemical gradients by actively translocating cations across cellular membranes. During her Ph.D. studies, Henriette has worked on two different P-type ATPases: a calcium-transporting ATPase (SERCA) and a copper-transporting ATPase (CopA), both essential for all domains of life. By employing X-ray crystallographic methods and molecular dynamics simulations, Henriette’s aim was to structurally characterize these two P-type ATPases in order to increase our current understanding of how they function at the atomistic level and to lay the groundwork for how they may be targeted by novel drugs in the future.

The PhD degree was completed at the Department of Molecular Biology and Genetics, Science and Technology, Aarhus University

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