Structural studies of protein : protein complexes in the early secretory pathway
Author: Svärd, Maria
Location: Samuelssonsalen, Tomtebodavägen 6, Karolinska Institutet, Stockholm
Department: Inst för medicinsk biokemi och biofysik / Dept of Medical Biochemistry and Biophysics
In this thesis, components of three different protein-protein complexes in the early secretory pathway have been studied by NMR spectroscopy and X-ray crystallography, with the aim of giving detailed structural information on each protein and gaining a deeper insight into the molecular mechanisms by which each protein performs its function and interactions. The work has resulted in the three-dimensional structures of MCFD2, a protein involved in transport of coagulation factors V and VIII, Erv41p, a vesicle protein proposed to play a role in glycoprotein processing and transport, and the co-chaperone P58IPK.
NMR spectroscopy showed MCFD2 to be disordered in the absence of Ca2+ ions, but to adopt a predominantly ordered conformation upon binding of Ca2+. MCFD2 forms a Ca2+-dependent complex with the glycoprotein transport receptor ERGIC-53, and our data suggest that the requirement of Ca2+ for folding of MCFD2 is the mechanism behind the Ca2+-dependence of complex formation. In addition, we could explain the mechanism by which two missense mutations in MCFD2 cause the bleeding disorder combined deficiency of factor V and factor VIII.
The first structure of Erv41p or any of its homologues was determined by X-ray crystallography. The protein was shown to form a β-sandwich with only limited structural homology to other proteins and this structure, in combination with the suggested role of Erv41p in glycoprotein processing, provided a starting point for our further studies to gain a better understanding of its function in the cell.
X-ray crystallography was used to determine the structure of the co-chaperone P58IPK. P58IPK had been shown to interact with the chaperone BiP and stimulate its ATPase activity, and a conserved HPD motif in the J domain of P58IPK is known to mediate interactions between these types of proteins. The structure provided some initial insights into how the interaction with the chaperone BiP could be mediated. Further investigations of this interaction, using surface plasmon resonance biosensor technology, showed that additional interactions, beyond the J domain and the HPD motif, are important for the stimulating effect of P58IPK on BiP.
I. Guy, J.E., Wigren, E., Svärd, M., Härd, T., Lindqvist, Y., 2008. New Insights into Multiple Coagulation Factor Deficiency from the Solution Structure of Human MCFD2. Journal of Molecular Biology 381, 941-955.
II. Biterova, E.I., Svärd, M., Possner, D.D.D., Guy, J.E., 2013. Purification, crystallization and preliminary X-ray crystallographic analysis of the lumenal domain of the ER-vesicle protein Erv41p from Saccharomyces cerevisiae. Acta Crystallographica Section F - Structural Biology and Crystallization Communications 69, 544-546.
III. Biterova, E.I., Svärd, M., Possner, D.D.D., Guy, J.E., 2013. The Crystal Structure of the Lumenal Domain of Erv41p, a Protein Involved in Transport between the Endoplasmic Reticulum and Golgi Apparatus. Journal of Molecular Biology 425, 2208-2218.
IV. Svärd, M., Biterova, E.I., Bourhis, J.M., Guy, J.E., 2011. The Crystal Structure of the Human Co-Chaperone P58IPK. PLoS ONE 6, e22337.
V. Svärd, M., Danielson, U.H., Guy, J.E., 2013. Studies of the Complex between the Chaperone BiP and the Co-Chaperone P58IPK Suggest a Dynamic Interaction. [Manuscript].
Supervisor: Guy, Jodie
Doktorsavhandlingar / Doctoral Theses - C2 (MBB)
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Thesis_Maria_Svärd.pdf 294
Spikblad_Maria_Svärd.pdf 186
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