Cytoskeletal-like Filaments of Ca2+-Calmodulin-Dependent Protein

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Cytoskeletal-like filaments of CaMKII are formed in a regulated and Zn2+-dependent manner Laurel Hoffman, Lin Li, Emil Alexov, Hugo Sanabria, and Melvin Neal Waxham Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00028 • Publication Date (Web): 20 Mar 2017 Downloaded from http://pubs.acs.org on March 20, 2017

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Biochemistry

Cytoskeletal-like filaments of CaMKII are formed in a regulated and Zn2+-dependent manner Laurel Hoffman1, Lin Li 2, Emil Alexov2, Hugo Sanabria2,*, M. Neal Waxham1,* 1

Department of Neurobiology and Anatomy, McGovern Medical School, Houston, Texas Department of Physics and Astronomy, Clemson University, Clemson, South Carolina

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Correspondence to: [email protected] and/or [email protected]

Primary Contact Information: Dr. Neal Waxham Department of Neurobiology and Anatomy McGovern Medical School The University of Texas Health Science Center at Houston 6431 Fannin, Room 7.254 MSB Houston, TX, 77030 Email: [email protected] Tel: 713-500-5621

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Abstract Ca2+-Calmodulin-dependent protein kinase II (CaMKII) is highly abundant in neurons, where its concentration reaches that typically found for cytoskeletal proteins. Functional reasons for such a high concentration are not known, but given the multitude of known binding partners for CaMKII, a role as a scaffolding molecule has been proposed. In this report, we provide experimental evidence that demonstrates a novel structural role for CaMKII. We discovered that CaMKII forms filaments that can extend for several microns in the presence of certain divalent cations (Zn2+, Cd2+ and Cu2+) but not with others (Ca2+, Mg2+, Co2+ and Ni2+). Once formed, depleting the divalent ion concentration with chelators completely dissociated the filaments and this process could be repeated by cyclic addition and removal of divalent ions. Using the crystal structure of the CaMKII holoenzyme, we computed an electrostatic potential map of the dodecameric complex to predict divalent ion binding sites. This analysis revealed a potential surface exposed divalent ion binding site involving amino acids that also participate in calmodulin (CaM) binding and suggested CaM binding might inhibit formation of the filaments. As predicted, Ca2+/CaM-binding both inhibited divalent induced filament formation and could disassemble pre-formed filaments. Interestingly, CaMKII within the filaments retains the capacity to autophosphorylate; however, activity towards exogenous substrates is significantly decreased. Activity is restored upon filament disassembly. We compile our results with structural and mechanistic data from the literature to propose a model of Zn2+-mediated CaMKII filament formation, where assembly and activity are further regulated by Ca2+/CaM.

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Ca2+-Calmodulin-dependent protein kinase II (CaMKII) is a ubiquitously expressed Ser/Thr protein kinase that is the product of four independent genes in mammals (α, β, γ and δ) 1, 2. While CaMKII is found in every tissue, it is particularly enriched in brain. The α and β isoforms constitute as much as 2% of total protein in rat hippocampal tissue 3 and CaMKII is further enriched at synapses where it is the most abundant protein in post-synaptic densities (PSDs) 4. CaMKII isoforms are dodecameric complexes where the association domains of 12 subunits assemble into two stacked, sixmembered rings with catalytic domains extending outward from the central hub 5-8. Each of the twelve catalytic domains can be held in an inactive state through extensive intrasubunit interactions that are disrupted when Ca2+/CaM binds to activate any given subunit 9. In addition to disrupting autoinhibition, new interactions are established that facilitate binding of Mg2+/ATP to the catalytic cleft of each subunit necessary for the phosphotransferase reaction. Once the Ca2+/CaM/CaMKII complex is formed, one subunit is rapidly autophosphorylated at Thr286 (in αCaMKII ; and Thr287 in the other isoforms) by a neighboring activated CaMKII subunit 10, generating the well-studied Ca2+-independent or autonomous activity 11. One function ascribed to the assembly of CaMKII subunits into a dodecameric complex is to dramatically increase the efficiency of intersubunit autophosphorylation 10, 12. Additionally, the multivalent nature and unique 3D structure of the holoenzyme is thought to act as a scaffold, dynamically assembling various protein complexes in both Ca2+/CaM-dependent and -independent conformations. Indeed, functional binary and tertiary protein interactions have been documented between the enzyme and other proteins 13-17. Furthermore, we have previously shown that CaMKII holoenzymes directly interact with each other under conditions mimicking ischemia in vivo, where formation of large spherical assemblies of kinase was dependent on pH, autophosphorylation, and isoform type 18. CaMKII self-association has also been documented inside neurons under ischemic conditions 19-23. In addition to regulation through Ca2+/CaM and autophosphorylation, several studies have reported that CaMKII binds to divalent ions, with binding leading to conformational changes in the enzyme. For example, Mg2+, which is crucial for the coordination of the phosphotransferase reaction, has been shown to bind CaMKII in the absence of nucleotides or CaM 24 and its binding site can accommodate Ca2+ ions as well 25. Zn2+ was also shown to bind to CaMKII and binding led to Ca2+/CaMindependent phosphorylation at low concentrations and phosphorylation inhibition at higher Zn2+ concentrations 26, 27. While these studies suggest that Zn2+-binding may produce conformational changes that partially mimic some of the rearrangements induced by Ca2+/CaM binding, the site or sites for Zn2+-binding have not yet been identified and structural changes have not been investigated. Total intracellular Zn2+ is estimated to range from 200-300 µm28 and has gained increasing attention as a regulatory ion, despite that its intracellular concentration is buffered to extremely low free levels; 100 pM or less 29, 30. In fact, intracellular metalloproteins that bind Zn2+ are second in number only to those that bind Mg2+ 29. Interestingly, PSDs that were disassembled by denaturing conditions were found to reassemble into complexes that resemble native PSDs only in the presence of Zn2+ 31. Given CaMKII’s abundance in PSDs and that its scaffolding role is mediated by its state of activation, we hypothesized that Zn2+ binding induces structural changes that influence kinase activity and higher order protein interactions, including self-assembly. By employing biochemical, electron microscopy and 3


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computational methods, we present evidence that Zn2+ induces the formation of filaments of CaMKII and we identify a potential divalent ion binding site that could mediate assembly. We found that filament assembly is reversible, regulated through Ca2+/CaM-binding, and supported by only a subset of divalent ions. We propose that this previously unrecognized divalent ion-induced assembly endows CaMKII with new structural possibilities well beyond that of assembling small di- or trivalent complexes. Since all CaMKII isoforms can undergo divalent ion induced filament formation this structural role would be relevant to all cell types. Materials and Methods Protein Preparation. CaMKII protein expression was carried out in an Sf21 insect cell expression system as previously described 32. The isoforms used in this study were expressed from the following cDNAs; the α (accession #NP_037052.1), β (accession #NP_068507) and δA (accession # NP_036651) isoforms from rat and the γB isoform from human (accession #NM_172173). The protein purification method used was an adaptation of previous work 32, 33. Briefly, insect cells containing expressed CaMKII were subjected to a freeze-thaw cycle and additionally lysed by brief sonication in buffer containing 40 mM Tris pH 7.5, 2.5% betaine, 1 mM EGTA, 1 mM EDTA, and 200 μM PMSF at 4°C. Cellular debris was separated by ultracentrifugation at 100,000 x g for 1 hr at 4°C. Supernatant was applied to a phosphocellulose column (Whatman p11) equilibrated in 50 mM PIPES pH7.1, 100 mM NaCl and 1 mM EGTA at 4°C. Protein was eluted with a salt gradient from 0 to 500 mM NaCl. Fractions containing CaMKII were further purified by gel filtration on a Superose 6 column (GE Healthcare) in buffer containing 25 mM HEPES pH 7.4, 500 mM KCl, and 1 mM EDTA at 4°C. Wild type mammalian CaM 34 and K75C mutant CaM 35 was expressed and purified exactly as previously described. Peptide substrate autocamtide-3 (AC-3) (KKALHRQETVDAL) was synthesized by LifeTein and further purified by reverse phase chromatography on a C18 Sep-Pak column (Waters). Electron Microscopy. For negative stain electron microscopy, samples were applied to glow-discharged 400 mesh, formvar carbon-coated copper grids and incubated for 1 min. Excess sample was wicked away, the grids were washed four times with water, once in 0.5% uranyl formate (Electron Microscopy Sciences), stained for 30 s in uranyl formate, blotted, and air dried. Electron micrographs were collected on a JEOL 1400 transmission electron microscope operated at 120 kV on either a 2048 х 2048 pixel Gatan UltraScan 1000XP camera or a 2000 × 1300 pixel Gatan Orius SC1000 camera. Activity Assays. Activity of CaMKII was measured using a continuous spectrophotometric assay similar to previous studies 36, 37 constructed in 96 well plates. In this assay, ADP production resulting from phosphotransferase activity to the substrate autocamtide-3 is coupled to the oxidation of NADH, which causes a reduction in its absorbance at 340 nm. To start the reaction 30 nM CaMKII was added to 1 μM CaM, 150 μM autocamtide-3, 1 mM phosphoenolpyruvate (Sigma Aldrich), 5% pyruvate kinase/lactic dehydrogenase enzyme mixture (Sigma Aldrich), and 0.28 mM NADH (Sigma Aldrich) in buffer containing 10 mM HEPES, 150 mM KCl, 0.2 mM EDTA, 0.4 mM CaCl2, and 10 mM MgCl2. To determine the effects of divalent ions, CaMKII was preincubated with 2 mM ZnCl2, or CdCl2 on ice prior to the start 4


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of the reaction. The volume of pre-incubated kinase added to the total assay volume was small, such that any added divalent ions produced negligible changes to divalent ion concentrations in the final reaction. For experiments looking at enzyme recovery following divalent treatment, the enzyme was incubated with 3 mM EDTA prior to the reaction. Absorbance was measured kinetically (time points measured every 15 sec) in a Mutliskan 340 plotted as a function of time and the slopes of the linear portions of the curves were fit to determine the velocity in OD units/sec. These values were then normalized as percentages of the apo kinase. Light Scattering. For light scattering experiments, 340 nm incident and emitted light was used with 1 nm slit widths on a PTI fluorimeter to monitor the CaMKII assembly process. Unless otherwise specified, reactions were carried out with 10 μg/mL CaMKIIα in buffer containing 25 mM HEPES pH 7.4, and 200 mM KCl, with or without 10 μg/mL CaM, 0.9 mM CaCl2, 1 mM ADP and 10 mM MgCl2. Assembly was initiated with addition of 0.1 mM ZnCl2 (final concentration). Light intensity was collected at a rate of 2 points/sec for 1000 seconds. Three separate experiments were carried out for each condition and averaged. Error bars indicate the standard deviation from the mean. Autophosphorylation. Autophosphorylation reactions were carried out on ice in the presence or absence of 0.5 mM Zn2+. Reactions consisted of 25 μg/mL CaMKIIα, 25 μg/mL CaM, 10 mM MgCl2, 1 mM ATP, 0.9 mM CaCl2 in 20 mM HEPES buffer, pH 7.4. Several time points were evaluated over the course of 90 seconds. At each time point, a sample was taken from the reaction and stopped by mixing with SDS loading dye. Samples were run on SDS PAGE and transferred to nitrocellulose. Autophosphorylation at T286 was determined by Western blot analysis with a mouse monoclonal antiphospho286 CaMKII primary antibody (Thermo Scientific previously Pierce, #MAI-047) and a secondary Alexa 488 goat anti-mouse antibody (Invitrogen, #A11029). Blots were imaged on a Typhon scanner (GE Healthcare). Electrostatic Calculations. In order to analyze the electrostatic features of CaMKII, Delphi 38 was used to calculate the electrostatic potential by solving the Poisson-Boltzmann equation. The structure of CaMKII used in the modeling was taken from Protein Data Bank (PDB ID 3SOA). In the DelPhi calculations, Amber force field parameters was used to assign atomic sizes and partial charges; the resolution was set at two grids per Å; the geometry occupancy of the CaMKII structure was 70% of the calculation box; the salt concentration was set as 0.15 mol/L. The electrostatic surface of CaMKII was generated by Chimera 39 . Ion binding sites predictions. The ion binding sites were predicted with the BION server 40, which utilizes the electrostatic potential map generated by DelPhi, and then applies geometrical considerations and clustering algorithms to predict positions of surface bound ions (details are provided in refs. http://www.ncbi.nlm.nih.gov/pubmed/26484964, http://www.ncbi.nlm.nih.gov/pubmed/22735539). Different types of ions are distinguished via their polarity, valency and size.

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Global Fitting kinetic models for protein aggregation. We used the online platform AmyloFit (http://www.amylofit.ch.cam.ac.uk) that enables robust global analysis to determine the best model to describe CaMKII association 41. This automated platform helps users to determine appropriate constraints to be placed on the aggregation mechanism based on the concentration dependence of the aggregation reaction. Several models can be chosen to describe assembly into linear aggregates. Global fitting is accomplished using advanced minimization algorithms to yield the reaction orders and rate constants. Three mechanistic models were compared. i) Nucleation and elongation 42, ii) Secondary Nucleation and elongation rate and a 43 iii) Multistep secondary nucleation 41. Results Zn2+ promotes the reversible formation of CaMKII filaments. We began our assessment of Zn2+ effects by assaying the native fluorescence of CaMKII in the presence and absence of excess Zn2+. In the presence of Zn2+, the emission spectrum of CaMKII increased in intensity and the peak was slightly red shifted, indicating that Zn2+ binds to CaMKII directly (Fig. 1A). We also assessed the time course of the reaction (Fig. 1B) and found that when Zn2+ is added to CaMKII, the emission fluorescence increases in a saturable, time dependent manner and that the rate depends on the Zn2+ concentration. Binding was completely reversible as evidenced by the fluorescence intensity quickly returning to baseline by addition of the chelator, EDTA (Fig. 1B). Native fluorescence is sensitive to changes in the local environment of Tyr and Trp residues and these results indicate that Zn2+-binding is in the vicinity of Tyr and/or Trp residues or results in conformational changes that alter their solvent accessibility/environment.

Figure 1: Fluorescent changes in CaMKII upon Zn2+ binding. A) Black trace illustrates native fluorescence of 10 μg/mL αCaMKII excited at 280 nm and emission was scanned from 300-360 nm. Addition of 100 μM ZnCl2 causes an increase in amplitude and wavelength of the peak emission (red). B) A time-based measurement of CaMKII fluorescence excited at 280 nm and monitored at 334 nm. An initial baseline was collected for CaMKII and then ZnCl2 was added (at 30 seconds). At 400 sec, 2.5 mM EDTA (chelator) was added and led to the return of the fluorescence increase to baseline. To determine if there were Zn2+-mediated structural changes in CaMKII, we evaluated the kinase with negative stain electron microscopy (EM). In the absence of Zn2+, CaMKII adopts a conformation where a central dense region is surrounded by petal-like features that extend in radial fashion (Fig. 2A). 6


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These structures are consistent with that of the previously determined 3D negative stain EM CaMKII structures 6, 7, where kinase domains extend away from the central association hub. Upon Zn2+ binding, CaMKII assembles into long ordered filament-like chains with a periodic twist (Fig. 2B). These filaments had a tendency to wrap back on themselves, forming higher order structures anecdotally resembling a “twisted phone cord”. In our experimental conditions, assembly began immediately after addition of the excess divalent ion on ice. Filaments were up to 1.5 μm in length after just 15 sec incubation, and grew to be over 7 μm with longer incubation times (mins). The approximate diameter across the chain is ~240 Å, which is slightly larger than the diameter of CaMKII. This places a constraint on the possible ways CaMKII could assemble, as the cross section of the chain diameter would be comprised of 1 or, at most, 2 holoenzymes (individual holoenzymes are ~200 Å in diameter). Remarkably, we found that addition of chelator to these filaments causes them to disassemble (Fig. 2C), consistent with the reversal of fluorescence intensity changes observed in Fig. 1B. Fig. 2 shows the impact of Zn2+-mediated filament formation with the α isoform of CaMKII; however, experiments carried-out with the three other isoforms of the kinase; β, γ, and δ, were found to behave similarly to αCaMKII in forming filaments in a Zn2+-dependent manner (Fig. 3). While all CaMKII isoforms appear to undergo Zn2+-dependent filament formation, it is possible that the assembly kinetics and/or reversibility are dependent on the isoform or splice variant expressed, which were not addressed in the current study. Overall, these results indicate that CaMKII produces reversible filamentous assemblies in response to the divalent ion Zn2+, and that the Zn2+-binding sites are shared throughout the four isoforms.

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Figure 2: Electron microscopy of Zn2+-induced filaments. These panels show negative stain micrographs of αCaMKII (A), Zn2+ assembled kinase (B), and chelator disassembled kinase (C). For these experiments, 10 μg/mL αCaMKII was assembled with 100 μM ZnCl2 for 2 min on ice, an aliquot was removed for negative stain, and the remainder of the sample disassembled with 2.5 mM EDTA for 10 min on ice. An aliquot of the disassembled kinase was then analyzed by negative stain.

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Figure 3: Electron microscopy of Zn2+-induced filaments of CaMKII Isoforms. Panels show negative stain micrographs of Zn2+-assembled rat β (A), human γΒ (B), and rat δΑ (C) isoforms of CaMKII. Assembly was accomplished by adding 10 µg/mL of each CaMKII isoform to 100 µΜ Zn2+ and incubated on ice for 30 minutes. Light scattering is a sensitive technique to monitor the size of molecules in solution particularly useful for assessing transitions from soluble to polymer forms of protein complexes. To assess the temporal dynamics of Zn2+-induced assembly, we carried out time-dependent light scattering experiments to monitor filament formation. 340 nm incident and detection wavelengths were used to monitor light scattering as a function of CaMKII assembly. As expected, the scattering amplitude was dependent on the concentration of CaMKII, with more protein corresponding to a larger signal (Fig. 4A). Additionally, we found that the rate of filament assembly was dependent on the Zn2+ concentration (Fig. 4B), with high concentrations producing faster growth.

Figure 4: Light scattering for Zn2+-induced CaMKII filaments. A) Increasing concentrations of αCaMKII were assembled with 100 μM ZnCl2 and demonstrate that the amplitude of the scattering signal is 9


Biochemistry

dependent on the initial concentration of CaMKII. B) Experiments were carried out with a constant αCaMKII concentration of 10 μg/mL and the concentration of ZnCl2 was varied to illustrate how assembly kinetics are dependent on the concentration of ZnCl2. For all experiments assembly was monitored using a fluorimeter with a 340 nm incident and detection wavelength. An initial 30 sec baseline was collected following the addition of 10 µg/ml of αCaMKII and assembly was started with addition of the indicated concentrations of ZnCl2.

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The observed filament growth resembles the polymerization reaction of cytoskeletal proteins and amyloids and we implemented the fitting strategy described by Meisl et al., 41 to investigate potential models to describe the data. We first analyzed the data in Fig. 4 by plotting on log-log scales the time to reach half maximal light scattering values versus the concentration of Zn2+ (Fig. 5A). A linear regression provides the slope of the line (the scaling factor), which serves to distinguish different filament growth models 41. The slope of -1.44, approximates the theoretical value of -1.5, indicative of a secondary nucleation model. This means that polymer growth occurs following a nucleation event involving binding between two (or more) CaMKII holoenzymes. For comparison, a single nucleation model (polymerization occurs by extension from each CaMKII holoenzyme) would produce a slope of 1.0. Having established the fitting model, we globally fit the primary data (Fig. 5B) to extract kinetic parameters. The nucleation rate (kn) was determined to be 3.8 M-1s-1, the secondary nucleation rate constant (k2) was 3.1 x 10-8 M-2s-1, and the elongation rate constant k+ was 3.6 x104 M-1s-1. From the weighted residual distribution (Fig. 4B), it is clear that the chosen model does not fully describe the data. Therefore, we tested more complicated models, however none produced a statistical improvement in the fitting. Of note is that the models all assume that CaMKII holoenzymes are permanently modified by Zn2+. One reason for the minor deviations of the fits, might be due to Zn2+ dissociating from holoenzymes during the experiment causing changes in the elongation rate and/or secondary nucleation steps.

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Figure 5: Global analysis of filament growth curves. A) A log-log plot of the half-time vs the enzyme concentration is used to calculate the scaling factor (slope of the red line); the half-time of the process (slope =-1.44) agrees best with a second order nucleation which has a theoretical scaling factor of -1.5 41. B) Normalized light scattering was plotted against time for the concentrations of Zn shown in the legend. A global fit analysis using the second order nucleation model 41, 43 was employed to calculate numeric values for the rate constants associated with the polymerization process; nucleation rate 10


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constant is kn = 3.79 M-1s-1, secondary nucleation rate constant k2= 3.1 x10-8 M-2s-1 and the elongation rate constant k+=3.58 x104 M-1s-1. Zn2+-mediated filament assembly reversibly reduces CaMKII activity but does not inhibit autophosphorylation at Thr286. To evaluate the impact that filament formation has on the autophosphorylation of CaMKII, we measured the Ca2+/CaM-dependent activity of CaMKII in the presence or absence of Zn2+ and discovered that the cation has a robust inhibitory effect on activity (Fig. 6A and 6B). The analysis revealed that pre-incubation with Zn2+ reduced activity to ~18% of CaMKII without Zn2+, indicating that CaMKII within the filaments has lost a significant fraction of its activity. Remarkably, filament disassembly by chelator was able to reverse the loss of kinase activity to ~90% of the non-Zn2+ treated enzyme, suggesting some steric or conformational change in the enzyme while in the filament form compromises the catalytic activity. To evaluate whether Zn2+ might inhibit autophosphorylation of CaMKII, we carried out autophosphorylation reactions in the presence or absence of Zn2+ and monitored Thr286 phosphorylation via Western blotting with a phospho-specific antibody. Control experiments carried out in the absence of Zn2+ showed that autophosphorylation occurs quickly and appears complete by 15-30 sec under these conditions (Fig. 6C) reasonably consistent with earlier results 12. Surprisingly, in the presence of Zn2+, the rate and extent of autophosphorylation are not different from non-Zn2+ treated reactions, indicating that CaMKII resident in filaments maintains the capacity to autophosphorylate at Thr286. Thus, we conclude that the Zn2+-mediated decrease in enzyme activity is not due to alterations in the autophosphorylation state of Thr286. Conversely, we found that when autophosphorylation was carried out first, addition of Zn2+ was unable to induce the formation of filaments. Figure 6D shows light scattering for autophosphorylated kinase in black. Upon addition of Zn2+ (red trace), the scattering signal did not increase indicating that assembly did not take place. To confirm the results, EM grids were made from each sample and structures were visualized with EM. Figure 6E shows Ca2+/CaM bound and autophosphorylated kinase and Fig. 6F shows autophosphorylated kinase incubated with Zn2+. Indeed, filaments were not evident in the Zn2+-treated sample. Under these reaction conditions, it is not possible to discern independently the role of Ca2+/CaM-binding from autophosphorylation. In fact, due to the increase in Ca2+/CaMbinding affinity when CaMKII is autophosphorylated 32, 44, it is even more probable that the images shown in panels E and F are CaMKII holoenzymes in the Ca2+/CaM bound and autophosphorylated state.

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Figure 6: Effects of Zn2+ on CaMKII activity and autophosphorylation at Thr 286. A) CaMKII activity was evaluated by monitoring phosphorylation of substrate autocamtide-3 as described in the Methods. Activity for non-Zn2+-treated (black), Zn2+ assembled αCaMKII filaments (red), and chelator disassembled αCaMKII are shown over time. Traces represent averages of triplicates. Reactions were carried out with 30 nM CaMKII, 2 mM ZnCl2 and 3 mM EDTA. B) Slopes of the activities from A are normalized and shown in the bar graph. Zn2+-assembled CaMKII filaments and chelator-disassembled kinase have ~18 and ~90% activity respectively compared to apo kinase. C) A Western blot showing time courses of autophosphorylation reactions are illustrated for αCaMKII in the absence (top) and presence (bottom) of Zn2+. No significant changes were observed. D) Light scattering experiments are shown for autophosphorylated kinase in black. Addition of 100 μM ZnCl2 to 10 μg/mL αCaMKII after a 30 sec baseline (red) did not cause an increase in light scattering signal associated with assembly. Negatively stained samples used to carry out these experiments are shown for autophosphorylated kinase (E) and autophosphorylated kinase in the presence of ZnCl2 (F). Identification of Potential Divalent Ion Binding Sites Using Computational Strategies. With the discovery that Zn2+ is binding to CaMKII to facilitate interactions between holoenzymes that lead to filament formation, we sought to identify possible Zn2+ binding sites. We took advantage of the full length human CaMKII crystal structure (PBD ID 3SOA) 8 in attempts to identify potential Zn2+-binding regions through a computational approach. The first step in this process was to determine the electrostatic surface potential map and we used Delphi for this purpose 38. The electrostatic potential distribution (Fig. 6) shows distributed but discrete regions that are electronegative (shown in red) and electropositive (blue). 12


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Figure 7: Identification of potential divalent ion binding sites. A) Side view of the electrostatic surface potential generated by calculations in Delphi based on the CaMKII crystal structure (PDB# 3SOA) B) Top view of the electrostatic surface potential; C) The predicted Zn2+ binding sites mapped on the surface of CaMKII. Zn2+ ions are represented as yellow spheres. D) The ion distribution after the symmetry elimination and clustering step. For consistency, the chain IDs for the displayed subunits are identical to the labels used in the PDB file and are also used to identify residues associated with binding sites in Table 1. The blue structure (chain i) is the monomer used in the symmetry elimination step. The red circle represents the position of the ion with lowest binding energy. E). The region identified with the red circle in (D) expanded to show the key residues that form the binding site for the top ranked Zn2+ ion. The contacting residues (distance < 10 Å) are at the interface between chains c, d and i. E216 and D217 (in red) are contributed by chain c, E360 and D363 (in green) are contributed by chain d and H388 is contributed by chain i. We then used the BION server 40 to predict bound divalent ions by probing the electrostatic map with three types of divalent cations - Zn2+, Mg2+, and Ca2+. In this step, the entire electrostatic map of CaMKII is used and the resulting positions of potential binding areas are identified. They are further refined by a clustering algorithm that keeps the positions with the lowest energy and removes all other ions within 15 Å (Fig. 7C and 7D). At this stage, the binding energy for each predicted ion is calculated and predicted positions are ranked according to their binding energies. For display purposes, Fig. 7D shows all the predicted ion binding sites in the vicinity of an arbitrarily chosen subunit (in blue) of the holoenzyme. The location of the ion position showing the lowest binging energy is shown inside the red circle in Fig 7D. A zoomed in figure showing the potential residues involved with the lowest binding energy for a Zn2+ ion is shown in Fig. 7E and identified that the site lies at the interface of 3 different 13


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subunits within the CaMKII holoenzyme. The Zn2+ contacting negatively charged residues are GLU216 and ASP217 from chain c, GLU360 and ASP363 from chain d and HIS388 of chain i. These residues along with other residues within the 10 Å “field” of the Zn2+ ion is listed in Table 1 and together contribute to binding energy calculated to be -6.2 kT. There are twelve such interfaces in each CaMKII holoenzyme leading to the potential of binding twelve Zn2+ ions. A similar procedure was employed by employing either Ca2+ or Mg2+ as the probe ion. While a number of sites were identified, none displayed the same degree of binding energy with the top-ranking site for Ca2+ and Mg2+ leading to calculated binding energies of -3.2 and -3.5 kT, respectively. Interestingly, the top Zn2+-binding site was also within the top 3 sites identified for both Ca2+ and Mg2+ and their calculated binding energy and the residues within 10 Å are also listed in Table 1. From these results, we derive two conclusions. First, while potential binding sites for Zn2+, Mg2+ and Ca2+ could be identified, the energy for binding was almost 2-fold more favorable for Zn2+. Second, the most favorable predicted binding site for Zn2+ involves residues that are also part of the Ca2+/CaM-binding domain of CaMKII and suggest that Ca2+/CaM-binding may influence the process of Zn2+-induced filament assembly. Table 1. Binding energies and residues associated with the lowest energy Zn2+ binding site and comparison to binding energies for Mg2+, and Ca2+ Ion

Binding Energy [kT]

Residues within 10 Å

Zn2+

-6.2

Chain c P177 E216 D217 Q218 H219 Y222 K300 I303 L304 T305C V306 M307 L308 A309 T310 T383 I384 L385 P387C Chain d P345 G346 M347 V359 E360 G361 L362 D363 F364 R366 Chain i H388

Mg2+

-3.3

Chain c F173 G175 P177 E216 Q218 H219 Y222 I303 L304 T305 V306 M307 L308 H381 T383 I384 L385 Chain d D344 P345 G346 M347 T348 V359 E360 G361 L362 D363 F364 R366

Ca2+

-3.1

Chain c F173 G175 P177 E216 Q218 H219 Y222 I303 L304 T305 V306 M307 L308 H381 T383 I384 L385 Chain d D344 P345 G346 M347 T348 V359 E360 G361 L362 D363 F364 R366

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Biochemistry

Ca2+/CaM inhibits Zn2+-mediated assembly. The computational work pointed towards the possibility that Ca2+/CaM binding might influence Zn2+-induced filament formation of CaMKII. To test this possibility, we monitored Zn2+-induced CaMKII light scattering in the absence or presence of Ca2+/CaM. Indeed, adding Ca2+/CaM led to a complete block in light scattering induced by Zn2+ (Fig. 8A) thereby suggesting that Ca2+/CaM prevents filament assembly. As controls for these experiments, we tested whether Ca2+ alone or Mg2+ inhibited Zn2+-induced increases in CaMKII light scattering. Neither had a detectable impact. These findings are consistent with the predictions from the computational work which indicate the energy of binding of Ca2+ or Mg2+ to the Zn2+-binding site are significantly weaker and would not compete for Zn2+-binding and this appears to be the case. Finally, we tested whether Mg2+/ADP binding to the catalytic site of the enzyme impacted Zn2+-induced light scattering. We did not utilize Mg2+/ATP in these reactions to avoid the complexities of autophosphorylation induced changes in the enzyme. We found no detectable impact of Mg2+/ADP on Zn2+-induced scattering. This experiment not only indicates that any conformational changes induced by Mg2+/ADP binding do not interfere with Zn2+-induced CaMKII assembly, but also indicate that in the cellular setting where Mg2+/ATP is presumed to be saturating, that Zn2+-induced filament formation would still occur.

Figure 8: Inhibition of Zn2+-mediated CaMKII filament assembly. Light scattering experiments were carried out for untreated CaMKII (A), Ca2+/CaM- bound (B), Ca2+-bound (C), Mg2+-bound (D), and Mg2+/ADP-bound (E) CaMKII. For all experiments, a baseline of 30 seconds was collected before ZnCl2 was added to start the assembly reaction. Experiments were carried out with 10 μg/mL αCaMKII, 10 μg/mL CaM, 0.9 mM CaCl2, 1 mM ATP or ADP, 10 mM MgCl2, and/or 0.1 mM ZnCl2. All experiments were 15


Biochemistry

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completed in triplicate. The mean of is plotted as a line graph and error bars represent the standard deviation. The plotted traces are the averages of three separate experiments and the error bars represent standard deviations. Ca2+/CaM disassembles Zn2+-assembled CaMKII filaments and recovers Zn2+-mediated loss in kinase activity. Since Ca2+/CaM binding to CaMKII prevents Zn2+-mediated filament assembly, we considered if Ca2+/CaM-binding could disassemble pre-formed filaments. For these experiments, Zn2+ was added to CaMKII and incubated on ice for 2 minutes to induce filament assembly (Fig. 9A); then, Ca2+/CaM was added and incubated for an additional 30 minutes (Fig. 9B). Filament formation was then evaluated by negative stain electron microscopy. We found that Ca2+/CaM was indeed able to completely dissociate Zn2+-induced filaments (Fig. 9B). However, if Zn2+ assembly is allowed to continue for longer times, the filaments coalesce and eventually Ca2+/CaM is unable to produced disassembly. In comparison, addition of chelator was able to disassemble kinase that had been incubated with Zn2+ for periods up to 5 minutes. (Fig. 6A and 6B). We also carried out experiments to determine if Ca2+/CaM could reverse Zn2+-mediated loss in CaMKII activity. When Zn2+ was pre-incubated with kinase for 2 min before the start of the reaction, activity was reduced to 32% that of untreated CaMKII. This result is consistent with previous activity measurement showing a reduction in kinase activity (Fig. 6B); however, here the pre-incubation time for the experiments was shortened to 2 min. The differences in activities indicate that there is a timedependence of Zn2+-mediated loss in CaMKII activity. We then measured activity under a condition where Zn2+-mediated assembly was allowed for 2 min, then Ca2+/CaM was added to the pre-incubation mix and incubated for 30 minutes prior to the start of the reaction. Remarkably, kinase activity was restored to 80% that of untreated CaMKII (Fig. 9C). A control experiment revealed that pre-incubation with Ca2+/CaM alone had no significant impact on activity. These results indicate that Ca2+/CaM can disassemble Zn2+-assembled CaMKII filaments and produce significant recovery of its activity.

Figure 9: Ca2+/CaM reversibility of Zn2+-mediated CaMKII filament assembly. Negative stain micrographs of Zn2+-assembled αCaMKII (A) and Ca2+/CaM dissociated Zn2+-assembled αCaMKII (B). C) Normalized αCaMKII activity against substrate AC-3 for untreated αCaMKII, αCaMKII pre-assembled with ZnCl2, a control with started with Ca2+/CaM, and αCaMKII pre-assembled with ZnCl2 then disassembled with Ca2+/CaM before starting the reaction (left to right bars). Reactions were carried out with 30 nM 16


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Biochemistry

αCaMKII, 2 mM ZnCl2 and 3 mM EDTA. Error bars represent standard deviation from three separate experiments. Induction of CaMKII filaments is not limited to the divalent ion Zn2+. To gain insight into the specificity of ion-induced filament formation, we evaluated other di- and tri-valent ions for their ability to induce CaMKII assembly. We showed using light scattering (Fig. 7) that Ca2+ and Mg2+ ions, known to bind in the active site of the catalytic cleft, do not promote CaMKII filament assembly nor do they compete effectively with Zn2+’s ability to induce assembly. Other divalent ions that also did not produce changes in light scattering (or filament assembly by EM) of CaMKII were Co2+, La2+, and Ni2+. However, other diand trivalent ions; Cd2+, Cu2+, Fe3+, and Tb3+, were able to induce increased light scattering and filament formation of CaMKII in a manner similar to Zn2+. Together these results indicate that the binding site(s) resident on CaMKII to induce filament assembly exhibits specificity for particular di- (or tri-) valent ions. An approximate segregation for those that promote filament assembly or do not is the ion coordination number; Ca2+, Mg2+, and Co2+ and Ni2+ do not promote assembly and have coordination numbers of 6, while Zn2+, Cu2+, and Cd2+ have preferred coordination numbers of 5 or 4 30. Discussion We present results that CaMKII can undergo reversible polymerization into unique filament-like structures through an ion-mediated (Zn2+, Cd2+, Cu2+, Fe3+, and Tb3+) mechanism. The size of these filamentous structures could be quite large (> um) and appeared to be individual CaMKII molecules (~ diameter of 200 Å) assembled into polymers with a tendency to wrap around themselves. Electron microscopy revealed that at their thinnest dimension, the polymers were one or at most two holoenzymes thick, however the density of packing prevented a clear distinction of individual holoenzymes, or how they might be oriented relative to each other. While we reported previously that CaMKII could undergo a self-association process when partially activated at lowered pH to mimic ischemic conditions 18, 45, those macromolecular assemblies were largely spherical and irreversible. The filaments described in this report are distinct morphologically and are entirely reversible, indicating they have a different mechanism of formation and presumably a different role. In fact, the Zn2+-induced polymerization kinetics show strong similarities to those described for actin polymerization 46. The simplest model that best described filament growth was a secondary nucleation model where polymerization occurred following nucleation by interactions between one or more CaMKII holoenzymes. While activity is partially decreased when CaMKII was associated into filaments, it recovered to near pre-association levels when the structures were dissociated with chelators. This important result indicates that divalent ion binding and filament formation does not lead to irreversible conformational changes in CaMKII structure. We are not aware of results reporting isolated CaMKII filamentous structures inside cells, however, our results suggest that a more systematic assessment of this possibility be undertaken. It is worth noting that the filamentous CaMKII structures described here, show a striking similarity to assemblies formed from proteins obtained by chemically dissociating postsynaptic densities 17


Biochemistry

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and then reassembling them in the presence of Zn2+ 31. While these experiments contain a host of PSD proteins, it may be that CaMKII plays a dominant role in reassembly of the PSD. Zn2+ was necessary for PSD reassembly, and the CaMKII assemblies we describe in this report might represent a crucial part of the structural scaffold that holds the PSD together in the resting state. Once activated, CaMKII interactions within the filaments would weaken or break, possibly leading to restructuring of the PSD to allow for structural changes in synapses. There is a precedent for this type of mechanism as members of the Shank family were shown to undergo Zn2+-dependent self-assembly and fluctuating Zn2+ concentrations were proposed to modify Shank-mediated protein interactions important for stabilizing proteins in the PSD 47. Additionally, loss of these Zn2+-binding forms of Shank lead to synaptic abnormalities and deficiencies in synaptic transmission 48. Given its abundance, it may also be possible for CaMKII, like Shank, to participate in buffering of Zn2+ concentrations. It is also well known that excess Zn2+, and other heavy metals, is a cellular toxin and sustained concentrations in the µM range lead to cell death49, particularly evident in neurons50. Whether CaMKII filament formation might play a role in Zn2+, or other di-(Cu2+ and Cd2+) or tri (Fe3+ and Tb3+)-valent ion mediated toxicity is a question for future investigation. We found that filament formation is inhibited by CaM activation and therefore subject to regulation by Ca2+ signaling. Once the kinase is activated, CaMKII interactions within the filaments would weaken or break, possibly leading to restructuring of the PSD to allow for structural changes in synapses. Dynamic disassembly and reassembly of CaMKII fibers could play a major role in the Ca2+dependent structural plasticity of PSDs. In addition to self-association, CaMKII has also been shown to interact with numerous synaptic proteins 4, 13, 14 and whether or not Zn2+-mediated structural alterations documented here also influence CaMKII interactions with other proteins awaits further investigation. We showed that Ca2+/CaM-binding prevents Zn2+-induced filament formation and can induce the disassembly of preformed filaments indicating that Ca2+/CaM-binding produces either structural rearrangements that disrupt the Zn2+-binding site, or perhaps sterically block the site. We favor the former possibility due to Ca2+/CaM’s capacity to reverse filament formation due to Zn2+-binding. A possible mechanism describing these transitions is illustrated in Fig. 10. Utilizing the crystal structure of CaMKII 8 a computational search for potential divalent ion binding sites identified a negatively charged interface formed between three different subunits of the holoenzyme. Due to the symmetry of the holoenzyme, this “site” is reproduced 12 times and a binding energy of ~6.2(NAkT) or 3.6 kcal/mol per site was calculated for Zn2+. Binding energies for Mg2+ and Ca2+ were significantly lower (Table 1) and may explain why these ions do not induce filament formation. Considering that there are 12 potential ion binding sites per holoenzyme, the energy contribution due to Zn2+ ion-binding would lead to a significant increase in stability of the interface within the holoenzyme. Thus, Zn2+ binding may hold CaMKII in a compact state favorable for filament formation. Contrarily, the binding energy of other ions (e.g., Mg2+ and Ca2+) is not sufficient to do so. This suggest that, in principle, the strength and tensile properties of the filaments could be regulated by modifying the relative concentrations of divalent ions; thus, providing a multitude of structural possibilities. 18


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Biochemistry

Figure 10: Cartoon model of Zn2+-mediated CaMKII fiber assembly and implications. n-Holoenzymes in presence of Zn2+ and other divalent ions can reversibly form long but collapsed fibers. However, the activation of the enzyme with Ca2+/CaM disassembles the fibers. The activation of CaMKII leads to a conformational change that disturbs the interaction surface that mediates CaMKII fiber assemblies. Zn2+ bind at the interface of two subunits in residues 216, 217 in the catalytic domain and 360, 363 in the association domain of top and bottom neighboring subunits or vice versa. Conclusion In summary, a combination of computational methods, intrinsic fluorescence, light scattering and electron microscopy revealed a novel mechanism of Zn2+-induced filament formation and its impact on the activation of CaMKII. We found that the effects of Zn2+-mediated filament formation are reversible and subject to regulation by Ca2+ signaling. Filament formation is not limited to Zn2+ and other divalent ions could lead to reversible filament formation. While the studies presented here imply substantial impacts on the scaffolding role of CaMKII, in vivo observations of CaMKII filaments are yet to be observed the implication to synaptic biology or neuronal health is worth pursuing.

Funding Information/Funding Sources This work was partially supported by a grant from the NIH/NINDS R01NS026086 (MNW). MNW also acknowledges an endowment from the William Wheless III Professorship. HS acknowledges startup funds from Clemson University. LL and EA acknowledge support from NIH R01GM093937.

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Biochemistry

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Biochemistry

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For Table of Contents Use Only Cartoon model of Zn2+-mediated CaMKII filament assembly and regulation by Ca2+/CaM.

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Cytoskeletal-like Filaments of Ca2+-Calmodulin-Dependent Protein

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