liu.seSearch for publications in DiVA
Change search
ReferencesLink to record
Permanent link

Direct link
Conformational Dynamics and Multimerization of Active Forms of the EphrinB Receptor 2 Kinase Domain
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.ORCID iD: 0000-0001-7004-8251
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology. (Patrik Lundström)
Molecular Structure and Function Program, Hospital for Sick Children; Department and Biochemistry, University of Toronto, Canada.
cDepartment and Biochemistry, University of Toronto; Department of Molecular Genetics, University of Toronto; Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Canada.
Show others and affiliations
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Active and autoinhibited forms of the ephrinB receptor 2 (EphB2) kinase domain have been studied using NMR spectroscopy. The project was initiated because of the finding that the crystal structures of active forms of the kinase domain and previous NMR studies suggested that a change in inter-lobe flexibility and the sampling of catalytically competent excited states conformations are responsible for activity. Using Carr-Purcell-Meiboom-Gill relaxation dispersion experiments, we have measured millisecond dynamics to identify such states. We have also performed concentration dependent relaxation experiments and analytical ultracentrifugation experiments that report on the effective protein size to look for possible differences in self-association for active and autoinhibited forms of the EphB2 kinase domain. We show that the active but not autoinhibited forms exchange between a ground state and an excited state at a rate of 1900 s-1. Similar results were found for the S677/680A mutant of the protein. The nature and importance of the excited state is still unknown. Our most important finding is that active forms of the kinase domain self-associate in a concentration dependent manner and form tetramers and possibly larger oligomers. Multimerization of the kinase domain may enable the assembly of complexes of downstream proteins and could be important for Eph signaling.

Keyword [en]
Kinase activation | Eph receptors | chemical exchange | nmr spectroscopy | protein dynamics | self-association
National Category
Chemical Sciences
URN: urn:nbn:se:liu:diva-117071OAI: diva2:805355
Available from: 2015-04-15 Created: 2015-04-15 Last updated: 2015-04-15Bibliographically approved
In thesis
1. Improved Methods for Characterization of Protein Dynamics by NMR spectroscopy and Studies of the EphB2 Kinase Domain
Open this publication in new window or tab >>Improved Methods for Characterization of Protein Dynamics by NMR spectroscopy and Studies of the EphB2 Kinase Domain
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are essential for all known forms of life and in many lethal diseases protein failure is the cause of the disease. To understand proteins and the processes they are involved in, it is valuable to know their structures as well as their dynamics and interactions. The structures may not be directly inspected because proteins are too small to be visible in a light microscope, which is why indirect methods such as nuclear magnetic resonance (NMR) spectroscopy have to be utilized. This method provides atomic information about the protein and, in contrast to other methods with similar resolution, the measurements are performed in solution resulting in more physiological conditions, enabling analysis of dynamics. Important dynamical processes are the ones on the millisecond timeframe, which may contribute to interactions of proteins and their catalysis of chemical reactions, both of significant value for the function of the proteins.

To better understand proteins, not only do we need to study them, but also develop the methods we are using. This thesis presents four papers about improved NMR techniques as well as a fifth where the kinase domain of ephrinB receptor 2 (EphB2) has been studied regarding the importance of millisecond dynamics and interactions for the activation process. The first paper presents the software COMPASS, which combines statistics and the calculation power of a computer with the flexibility and experience of the user to facilitate and speed up the process of assigning NMR signals to the atoms in the protein. The computer program PINT has been developed for easier and faster evaluation of NMR experiments, such as those that evaluate protein dynamics. It is especially helpful for NMR signals that are difficult to distinguish, so called overlapped peaks, and the soft- ware also converts the detected signals to the indirectly measured physical quantities, such as relaxation rate constants, principal for dynamics. Next are two new versions of the Carr-Purcell-Maiboom-Gill (CPMG) dispersion pulse sequences, designed to measure millisecond dynamics in a way so that the signals are more separated than in standard experiments, to reduce problems with overlaps. To speed up the collection time of the data set, a subset is collected and the entire data set is then reconstructed, by multi-dimensional decomposition co-processing. Described in the thesis is also a way to produce suitably labeled proteins, to detect millisecond dynamics at Cα positions in proteins, using the CPMG dispersion relaxation experiment at lower protein concentrations. Lastly, the kinase domain of EphB2 is shown to be more dynamic on the millisecond time scale as well as more prone to interact with itself in the active form than in the inactive one. This is important for the receptor function of the protein, when and how it mediates signals.

To conclude, this work has extended the possibilities to study protein dynamics by NMR spectroscopy and contributed to increased understanding of the activation process of EphB2 and its signaling mechanism. 

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2015. 58 p.
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1649
National Category
Chemical Sciences
urn:nbn:se:liu:diva-117076 (URN)10.3384/diss.diva-117076 (DOI)978-91-7519-103-4 (print) (ISBN)
Public defence
2015-05-22, Planck, Fysikhuset, Campus Valla, Linköping, 09:15 (English)
Available from: 2015-04-15 Created: 2015-04-15 Last updated: 2015-04-23Bibliographically approved

Open Access in DiVA

No full text

Search in DiVA

By author/editor
Ahlner, AlexandraLundström, Patrik
By organisation
ChemistryThe Institute of Technology
Chemical Sciences

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Total: 310 hits
ReferencesLink to record
Permanent link

Direct link