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Biophysical analysis of the dynamics of calmodulin interactions with neurogranin and Ca2+/calmodulin-dependent kinase II
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC. Beactica AB, Uppsala, Sweden.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.ORCID iD: 0000-0002-1135-2744
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC. Uppsala University, Science for Life Laboratory, SciLifeLab. Beactica AB, Uppsala, Sweden.ORCID iD: 0000-0003-2728-0340
2017 (English)In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 30, p. 1-11, article id e2621Article in journal (Refereed) Published
Abstract [en]

Calmodulin (CaM) functions depend on interactions with CaM-binding proteins, regulated by Ca2+. Induced structural changes influence the affinity, kinetics, and specificities of the interactions. The dynamics of CaM interactions with neurogranin (Ng) and the CaM-binding region of Ca2+/calmodulin-dependent kinase II (CaMKII290-309) have been studied using biophysical methods. These proteins have opposite Ca2+ dependencies for CaM binding. Surface plasmon resonance biosensor analysis confirmed that Ca2+ and CaM interact very rapidly, and with moderate affinity (KDSPR=3M). Calmodulin-CaMKII290-309 interactions were only detected in the presence of Ca2+, exhibiting fast kinetics and nanomolar affinity (KDSPR7.1nM). The CaM-Ng interaction had higher affinity under Ca2+-depleted (KDSPR480nM,3.4x105M-1s-1 and k(-1) = 1.6 x 10(-1)s(-1)) than Ca2+-saturated conditions (KDSPR19M). The IQ motif of Ng (Ng(27-50)) had similar affinity for CaM as Ng under Ca2+-saturated conditions (KDSPR=14M), but no interaction was seen under Ca2+-depleted conditions. Microscale thermophoresis using fluorescently labeled CaM confirmed the surface plasmon resonance results qualitatively, but estimated lower affinities for the Ng (KDMST890nM) and CaMKII290-309(KDMST190nM) interactions. Although CaMKII290-309 showed expected interaction characteristics, they may be different for full-length CaMKII. The data for full-length Ng, but not Ng(27-50), agree with the current model on Ng regulation of Ca2+/CaM signaling.

Place, publisher, year, edition, pages
2017. Vol. 30, p. 1-11, article id e2621
Keywords [en]
calmodulin, calmodulin-dependent kinase, surface plasmon resonance, microscale thermophoresis, neurogranin
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-329023DOI: 10.1002/jmr.2621ISI: 000405095400006OAI: oai:DiVA.org:uu-329023DiVA, id: diva2:1138991
Funder
Swedish Research Council, D0571301Available from: 2017-09-06 Created: 2017-09-06 Last updated: 2018-10-16Bibliographically approved
In thesis
1. Interaction kinetic analysis in drug design, enzymology and protein research
Open this publication in new window or tab >>Interaction kinetic analysis in drug design, enzymology and protein research
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The work presented here is focused on the phenomenon of molecular recognition – the mutual ability of biological molecules to recognize each other through their chemical signatures. Here, the kinetic aspects of recognition were evaluated, as interaction kinetics reveal valuable dimensions in the description of molecular events in biological systems. The primary objects studied in this thesis were human proteins and their interaction partners. Proteins serve a fundamental role in living organisms, supporting the biochemical machinery by means of catalysis, signalling and transport; additionally, proteins are the main targets for drugs.

In the first study, carbonic anhydrase (CA) isozymes were employed as a model system to address the problem of drug selectivity. Kinetic signatures preferable for the design of selective sulphonamide-based inhibitors were identified. In a follow up study, the recognition between CA and sulphonamides was separated into two parts, uncovering intrinsic recognition features that genuinely reflect the interaction mechanism. For the first time, the concept of intrinsic interaction kinetics was applied to a drug-target system.

Another model protein studied in this thesis was calmodulin (CaM), as its interactions with other proteins should have specific kinetic signatures to support the dynamics of calcium-dependent signalling. The study evolved around calcium-dependent CaM interactions with the neuronal protein neurogranin (Ng), and revealed its complex nature. Ng was found to interact with CaM both in presence and absence of calcium, but with different kinetics and affinity. This finding supports development of a mechanistic model of calcium sensitivity regulation.

The last two projects were more applied, exploring the druggability of an emerging class of pharmaceutical targets – epigenetic enzymes. Expertise and methodology for biophysically guided drug discovery towards histone demethylase LSD1 and histone methyltransferase SMYD3 were developed. For LSD1, the project assisted the rational design of active site-targeting macrocyclic peptides, and resulted in the development of competitive inhibitors with a well described mechanism of action. A novel biophysical platform for screening was developed for SMYD3. It proved to be successful, as it identified previously unknown allosteric ligand binding site. Both projects were supported by structural studies, expanding the druggable space of epigenetic targets.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 51
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1735
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-363323 (URN)978-91-513-0479-3 (ISBN)
Public defence
2018-12-05, B42, BMC, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2018-11-09 Created: 2018-10-16 Last updated: 2018-11-09

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Seeger, ChristianTalibov, Vladimir ODanielson, U. Helena

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