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Kinetic and mechanistic differences in the interactions between caldendrin and calmodulin with AKAP79 suggest different roles in synaptic function
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
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2012 (English)In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 25, no 10, 495-503 p.Article in journal (Refereed) Published
Abstract [en]

The kinetic and mechanistic details of the interaction between caldendrin, calmodulin and the B-domain of AKAP79 were determined using a biosensor-based approach. Caldendrin was found to compete with calmodulin for binding at AKAP79, indicating overlapping binding sites. Although the AKAP79 affinities were similar for caldendrin (KD?=?20?n m) and calmodulin (KD?=?30?n m), their interaction characteristics were different. The calmodulin interaction was well described by a reversible one-step model, but was only detected in the presence of Ca2+. Caldendrin interacted with a higher level of complexity, deduced to be an induced fit mechanism with a slow relaxation back to the initial encounter complex. It interacted with AKAP79 also in the absence of Ca2+, but with different kinetic rate constants. The data are consistent with a similar initial Ca2+-dependent binding step for the two proteins. For caldendrin, a second Ca2+-independent rearrangement step follows, resulting in a stable complex. The study shows the importance of establishing the mechanism and kinetics of proteinprotein interactions and that minor differences in the interaction of two homologous proteins can have major implications in their functional characteristics. These results are important for the further elucidation of the roles of caldendrin and calmodulin in synaptic function.

Place, publisher, year, edition, pages
2012. Vol. 25, no 10, 495-503 p.
Keyword [en]
postsynaptic density, A-kinase anchoring protein, caldendrin, calmodulin, surface plasmon resonance, induced fit
National Category
Natural Sciences
URN: urn:nbn:se:uu:diva-183563DOI: 10.1002/jmr.2215ISI: 000309067200002OAI: oai:DiVA.org:uu-183563DiVA: diva2:574884
Available from: 2012-12-06 Created: 2012-10-29 Last updated: 2014-04-29Bibliographically approved
In thesis
1. Revealing Secrets of Synaptic Protein Interactions: A Biosensor based Strategy
Open this publication in new window or tab >>Revealing Secrets of Synaptic Protein Interactions: A Biosensor based Strategy
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein interactions are the basis of synaptic function, and studying these interactions on a molecular level is crucial for understanding basic brain function, as well as mechanisms underlying neurological disorders. In this thesis, kinetic and mechanistic characterization of synaptic protein interactions was performed by using surface plasmon resonance biosensor technology. Fragment library screening against the reverse transcriptase of HIV was included, as it served as an outlook for future drug discovery against ligand-gated ion channels.

The protein-protein interaction studies of postsynaptic Ca2+ -binding proteins revealed caldendrin as a novel binding partner of AKAP79. Caldendrin and calmodulin bind and compete at similar binding sites but their interactions display different mechanisms and kinetics. In contrast to calmodulin, caldendrin binds to AKAP79 both in the presence and absence of Ca2+ suggesting distinct in vivo functional properties of caldendrin and calmodulin.

Homo-oligomeric β3 GABAA receptors, although not yet identified in vivo, are candidates for a histamine-gated ion channel in the brain. To aid the identification of the receptor, 51 histaminergic ligands were screened and a unique pharmacology was determined. A further requirement for identifying β3 receptors in the brain, is the availability of specific high-affinity ligands. The developed biosensor assay displayed sufficient sensitivity and throughput for screening for such ligands, as well as for being employed for fragment-based drug discovery.

AMPA receptors are excitatory ligand-gated ion channels, involved in synaptic plasticity, and modulated by auxiliary proteins. Previous results have indicated that Noelin1, a secreted glycoprotein, interacts with the AMPA receptor. By using biochemical methods, it was shown that Noelin1 interacts directly with the receptor. The kinetics of the interaction were estimated by biosensor analysis, thereby confirming the interaction and suggesting low nanomolar affinity. The results provide a basis for functional characterization of a novel AMPA receptor protein interaction.

The results demonstrate how secrets of synaptic protein interactions and function were revealed by using a molecular based approach. Improving the understanding of such interactions is valuable for basic neuroscience. At the same time, the technical advancements that were achieved to study interactions of ligand-gated ion channels by surface plasmon resonance technology, provide an important tool for discovery of novel therapeutics against these important drug targets.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 73 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1133
Surface plasmon resonance, biosensor, AMPA receptor, GABAA receptor, ligand-gated ion channel, A-kinase anchoring protein, caldendrin, calmodulin, HIV, fragment based drug discovery
National Category
Biochemistry and Molecular Biology Neurosciences
Research subject
Biochemistry; Neuroscience
urn:nbn:se:uu:diva-220879 (URN)978-91-554-8916-8 (ISBN)
Public defence
2014-05-16, B42, Husargatan 3, 751 23 Uppsala, 13:15 (English)
EU, FP7, Seventh Framework Programme
Available from: 2014-04-25 Created: 2014-03-22 Last updated: 2014-04-29

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