Structural studies of Ca2+-ATPase ligand and regulatory complexes

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

  • Nikolaj Düring Drachmann, Danmark
The Ca2+-ATPase (sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA is a part of the vital P-type ATPase
family, which was first discovered in 1957 by Professor Jens Christian Skou. He was the first to describe the
Na+,K+-ATPase and its role in generating the membrane potential across the axonal membrane. Now, more
members of the P-type ATPase family are described and it consists of membrane proteins from all living
organisms, and with different substrate specificities. A common feature of P-type ATPases is the
translocation of charged substrates, mainly cations, across biological membranes against their concentration
gradient upon ATP hydrolysis. The ion gradients are used to drive several key cellular processes, like the
action potential in nerve tissue, acidification of the gastric juice, cell signalling and muscle contraction.
The Ca2+-ATPase is an important part of mammalian cells, and is expressed in different isoforms with
different cellular locations. SERCA1a is mainly expressed in fast twitch muscle tissue, SERCA2a in cardiac
tissue, SERCA2b is ubiquitously expressed, while SERCA3 isoforms are co-expressed with SERCA2b in nonmuscular
cells. In general, SERCA is responsible for the re-uptake of Ca2+-ions to the sarcoplasmic reticulum.
In cardiac and muscle tissue, SERCA is the main determinant of the rate of muscle contraction and is
responsible for the muscle relaxation by removal of Ca2+-ions from the cytosol. SERCA1a is regulated by the
endogenous regulator Sarcolipin, while SERCA2a is regulated by the Sarcolipin homologue Phospholamban.
They act upon binding by increasing the apparent Ca2+ affinity of the ATPases, thus regulating the activity in
the physiologically relevant Ca2+ concentrations. In the first part of the thesis, a purification protocol of
native SERCA2a from pig hearts is presented. The purified protein was used for X-ray crystallographic studies
aiming at determining the three dimensional structure of the SERCA2a isoform in a Ca2+-free conformation.
Crystals of the Ca2+ free state of SERCA2a stabilised by the inhibitor cyclopiazonic acid was obtained and a
dataset was collected scaling to 3.26 Å resolution, allowing a preliminary structural analysis. The overall
crystal structure is very similar to SERCA1a. Additionally, co-crystallisation studies have been initiated of
SERCA2a and recombinantly expressed Phospholamban.
Besides the above mentioned regulatory peptides, the surrounding membrane itself has a huge influence on
SERCA structure and function. Changes in the membrane thickness can alter the activity of the ATPase
significantly, and even cause changes in the stoichiometry of ion transport. Structural studies on SERCA in
the presence of four different phosphatidyl choline lipids with different aliphatic chain length and saturation
show three specific lipid binding sites. The four different lipids analysed bind to the same binding sites with
varying degrees of disorder. The study contributes to understanding the complex interplay between the
surrounding membrane and SERCA.
Small molecule inhibitors regulate SERCA function by direct interactions. Studying SERCA:ligand interactions
are important for understanding the molecular mechanism of regulation, and ligands have been extensively
used for structural and physiological characterisation of SERCA. In order to explore the possibilities for an
efficient screening of ligand-bound SERCA structures, serial femtosecond crystallography experiments of
microcrystals of SERCA1a in the Ca2+ bound state and in a vanadate stabilised E2 state was conducted. A
structure obtained at 2.8 Å maximum resolution of the proof-of-concept Ca2+ bound crystal form, indicated
that the information content of SFX data is higher than synchrotron data, and ligands and ions can be
detected with low redundant data. The data of the E2 stabilised form was processed to 5 Å resolution, and it
was possible to extract useful anomalous data showing vanadate binding in the proposed binding site near
the catalytic aspartate residue. These studies show that this method can be particularly valuable for the
purpose of efficient ligand screening with membrane proteins, circumventing the need to grow and optimize
large, well-diffracting macrocystals.
Antal sider115
Rekvirerende organGraduate School of Science and Technology
StatusUdgivet - 23 jan. 2015

Note vedr. afhandling

During his PhD studies, Nikolaj Düring Drachmann has determined structures of Ca2+-ATPases also known as calcium pumps, which are vital membrane proteins involved in muscle and heart function. These pumps determine for example heart contractility and are regulated by another protein called phospholamban that responds to e.g. adrenaline signaling. Nikolaj has provided new information on how calcium pumps interact with lipids in the membrane, and also determined the first structures of the heart-specific isoform of the calcium pump. He also contributed to pioneering studies with ultra intensive X-ray pulses using a so-called X-ray free-electron laser (XFEL) at the Stanford Linear Accelerator in California, where nanocrystals of the calcium pump were investigated.
These findings further our understanding of the complex regulation of the calcium pump, and enable future drug development targeting for example cardiovascular diseases.
The PhD degree was completed at Department of Molecular Biology and Genetics, Science and Technology, Aarhus University.

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