CaMKII autophosphorylation can occur between holoenzymes without subunit exchange
- Humboldt-Universität zu Berlin, Germany
- Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Germany
- Freie Universität Berlin, Germany
Abstract
The dodecameric protein kinase CaMKII is expressed throughout the body. The alpha isoform is responsible for synaptic plasticity and participates in memory through its phosphorylation of synaptic proteins. Its elaborate subunit organization and propensity for autophosphorylation allow it to preserve neuronal plasticity across space and time. The prevailing hypothesis for the spread of CaMKII activity, involving shuffling of subunits between activated and naïve holoenzymes, is broadly termed subunit exchange. In contrast to the expectations of previous work, we found little evidence for subunit exchange upon activation, and no effect of restraining subunits to their parent holoenzymes. Rather, mass photometry, crosslinking mass spectrometry, single molecule TIRF microscopy and biochemical assays identify inter-holoenzyme phosphorylation (IHP) as the mechanism for spreading phosphorylation. The transient, activity-dependent formation of groups of holoenzymes is well suited to the speed of neuronal activity. Our results place fundamental limits on the activation mechanism of this kinase.
Data availability
All data generated during this study are included in the manuscript, supporting figures and supporting tables.
Article and author information
Author details
Andrew J R Plested
Funding
HORIZON EUROPE Marie Sklodowska-Curie Actions (798696)
- Iva Lučić
Deutsche Forschungsgemeinschaft (272140445)
- Andrew J R Plested
Deutsche Forschungsgemeinschaft (323514590 & 446182550)
- Andrew J R Plested
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2023, Lučić et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 168
- downloads
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Further reading
-
- Biochemistry and Chemical Biology
The protein ligase Connectase can be used to fuse proteins to small molecules, solid carriers, or other proteins. Compared to other protein ligases, it offers greater substrate specificity, higher catalytic efficiency, and catalyzes no side reactions. However, its reaction is reversible, resulting in only 50% fusion product from two equally abundant educts. Here, we present a simple method to reliably obtain 100% fusion product in 1:1 conjugation reactions. This method is efficient for protein-protein or protein-peptide fusions at the N- or C-termini. It enables the generation of defined and completely labeled antibody conjugates with one fusion partner on each chain. The reaction requires short incubation times with small amounts of enzyme and is effective even at low substrate concentrations and at low temperatures. With these characteristics, it presents a valuable new tool for bioengineering.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
African trypanosomes are the causative agents of neglected tropical diseases affecting both humans and livestock. Disease control is highly challenging due to an increasing number of drug treatment failures. African trypanosomes are extracellular, blood-borne parasites that mainly rely on glycolysis for their energy metabolism within the mammalian host. Trypanosomal glycolytic enzymes are therefore of interest for the development of trypanocidal drugs. Here, we report the serendipitous discovery of a camelid single-domain antibody (sdAb aka Nanobody) that selectively inhibits the enzymatic activity of trypanosomatid (but not host) pyruvate kinases through an allosteric mechanism. By combining enzyme kinetics, biophysics, structural biology, and transgenic parasite survival assays, we provide a proof-of-principle that the sdAb-mediated enzyme inhibition negatively impacts parasite fitness and growth.
