Breakage of the oligomeric CaMKII hub by the regulatory segment of the kinase

Figures

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  Figure 1.. CaMKII architecture and peptides used in mass spectrometry analysis. (A) The architecture of dodecameric CaMKII, with subunits. The kinase domains are followed by the regulatory segments, which are connected to the hub domains by linkers. The hub domains (in darker grey) associate to form a ring-like hub assembly. For clarity, only a single kinase domain, regulatory segment, and linker are shown in colors, while the remainder are shown in shades of grey. In this subunit, the kinase domain is colored pale blue, the regulatory segment is colored yellow and pale pink, and the unstructured kinase-hub linker is colored green. (B) Domains of a CaMKII-α subunit. The color scheme is the same as in (A). The calmodulin-binding element and the β-clip segment are labeled. The positively charged residues of the calmodulin-binding element are colored deep blue. Phosphorylation sites are shown as empty red circles. (C) Peptides used in the mass spectrometry analyses. All the peptides are derived from the sequence of CaMKII-α, except Peptide H, a peptide that inhibits the kinase domain of CaMKII. The color schemes denote the region of the CaMKII-α subunit that the peptide is derived from and are…

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  Figure 2.. Native mass spectra of the hub incubated with peptides derived from the calmodulin-binding element of CaMKII-α. The masses (in Daltons) of the respective species are noted. (A) The isolated hub assembly is stable as dodecamers and tetradecamers in the absence of peptides. (B) The hub assembly (120 µM subunit concentration) incubated with 100 µM of Peptide A, shows disassembly within 3 min of initiation ionization (left). In addition to dodecamers and tetradecamers, monomers, dimers, tetramers, hexamers and octamers are observed. At ~ 4 min (right), the smaller oligomers are predominant. (C) The hub assembly (120 µM subunit concentration) incubated with 10 µM of Peptide A shows some release of smaller oligomeric species at ~2 min (left). This becomes more pronounced at 3–4 min (right), although the dodecameric and tetradecameric hub assemblies are also present. In all the spectra of the hub incubated with peptides, species corresponding to dodecameric or tetradecameric hub assemblies with one or two peptides bound are present.

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  Figure 2—figure supplement 1.. Native mass spectra of the hub incubated with peptides derived from the calmodulin-binding element of CaMKII-α. (A) Mass spectrum of the isolated hub with a sample cone voltage of 150 V. Monomeric species of the hub assembly are observed at this high sample cone voltage. (B) Incubation of the hub assembly with 1 µM of Peptide A shows no disassembly, even up to 10 min after the start of ionization, and only dodecamers and tetradecamers are observed. (C) Incubation of the hub assembly with 1 mM of Peptide A shows significant protein and peptide aggregation. In all the spectra of the hub incubated with peptides, species corresponding to dodecameric or tetradecameric hub assemblies with one or two peptides bound are present, even when no hub disassembly is observed. The masses (in Daltons) of the respective species are noted.

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  Figure 3.. Role of different regions of the calmodulin-binding element of CaMKII-α in inducing hub disassembly. The masses (in Daltons) of the respective species are noted. (A) Peptide B, which contains the β-clip segment of the calmodulin-binding element, but not the positively charged residues, induces disassembly, but at a slower rate than Peptide A. When 100 µM of Peptide B is incubated with the hub assembly, no smaller species are observed up to ~2.5 min after initiation of ionization (left). At 3.5–4.5 min, smaller oligomeric species corresponding to monomers, dimers, tetramers and hexamers are observed (right). (B) Incubation of the hub assembly with 1 mM of Peptide C, in which three positively charged residues of Peptide A are replaced with negatively charged residues (colored red in sequence), does not result in the release of smaller oligomeric species up to 6 min after ionization, even though the peptide binds to the hub assembly. (C) Modifying the β-clip segment of the calmodulin-binding element abrogates disassembly. Incubation of the hub assembly with 1 mM of Peptide D, in which the β-clip segment of Peptide A is replaced with glycine and serine residues (colored purple) does…

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  Figure 3—figure supplement 1.. Role of different regions of the calmodulin-binding element of CaMKII-α in inducing hub disassembly. (A) Incubation of the hub assembly with 1 mM of Peptide E, in which a portion of the β-clip segment of Peptide A is replaced with glycine and serine residues (colored purple) results in the release of some smaller oligomeric species up to 6 min after ionization. (B) Incubation of the hub assembly with 1 mM of Peptide F, in which a portion of the β-clip segment of Peptide A is replaced with glycine and serine residues (colored purple) results in the release of some smaller oligomeric species up to 4 min after ionization. (C) Incubation of the hub assembly with 100 µM of Peptide H (CaMKIINTide), an inhibitor that binds to the substrate-recognition site of the kinase domain of CaMKII, does not result in the release of smaller oligomeric species up to 4 min after the start of ionization. The CAMKII dodecamers and tetradecamers are not resolved in this mass spectra due to the soft instrument source conditions used, and peak broadening that resulted from nonvolatile salts present in the peptide solution. The masses (in Daltons) of the respective species are noted.

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  Figure 4.. Native mass spectra of the hub incubated with phosphorylated peptides derived from the calmodulin-binding element of CaMKII-α. Due to the rapid rate at which the hub is destabilized in the presence of phosphorylated peptide, the 5-min incubation step was dispensed which and the sample was injected into the mass spectrometer as soon as the hub was mixed with the peptide. The phosphorylated residue (Thr 305) is indicated with a filled red circle. The masses (in Daltons) of the respective species are noted. (A) The hub assembly (120 µM subunit concentration) incubated with 100 µM of Peptide Aphos shows disassembly within 1–2 min after the start of ionization (left). In addition to dodecamers and tetradecamers, monomers, dimers, tetramers and hexamers are observed. At ~2–3 min (right), the spectra consist primarily of protein and peptide aggregates. (B) The hub assembly (120 µM subunit concentration) incubated with 10 µM of Peptide Aphos also shows disassembly within 1–2 min of ionization (left). In addition to dodecamers and tetradecamers, monomers, dimers, tetramers and hexamers are observed. At ~2–3 min (right), the spectra consist primarily of protein and peptide aggregates. (C)…

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  Figure 4—figure supplement 1.. Analytical gel filtration of the CaMKII-α hub with and without Peptide A. (A) Analytical gel filtration of the isolated hub shows one peak with a retention volume corresponding to the dodecameric/tetradecameric hub assembly. (B) Gel filtration of the hub incubated with 500 µM of Peptide A also shows a single peak with a retention volume that corresponds to the dodecameric/tetradecameric hub assembly.

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  Figure 5.. Calmodulin-binding elements from two of the twelve regulatory segments dock on the hub in the simulation of the dodecameric CaMKII-α hub, with considerable distortions at the interfaces where they bind. The color scheme for the regions of the subunits are the same as in Figure 1A. (A) Instantaneous snapshot from the start (top) and end (bottom) of the simulation showing interface where Docking 1 occurs. The calmodulin-binding element of subunit L docks at the interface between the hub domains of subunits B and L. The positively-charged residues of the regulatory segment (shown in blue) form interactions with the negatively-charged residues (shown in red) that line the interface between subunits B and L. The hub domain of subunit B rotates away from the hub domain of subunit L. (B) Instantaneous snapshot from the start (top) and end (bottom) of the simulation showing interface where Docking 2 occurs. The calmodulin-binding element of subunit F docks at the interface between the hub domains of subunits F and H. The positively-charged residues of the regulatory segment (shown in blue) form interactions with the negatively-charged residues (shown in red) that line the interface…

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  Figure 5—figure supplement 1.. Docking of regulatory segments onto hub assembly. (A) A schematic representation of the hub assembly with the subunit labeling convention used in this study. The two regulatory segments that dock on the hub assembly are shown, and colored using the same scheme as in Figure 1A. (B) Presence of interactions between residues 298-306 of the calmodulin-binding element of each subunits and the corresponding interfacial β-sheet (residues 410–416) over the course of the simulation. An interaction is considered to be present if at least two backbone hydrogen bonds (distance between donor and acceptor of less than or equal to 3 Å) are formed between the calmodulin-binding element and the β-sheet. (C) Representative sample of distances between residues of the calmodulin-binding element and residues at the interface of the hub assembly from the two docking events. Docking 1 is on the left and Docking 2 is on the right. The darker traces are the time-averaged values of the distances calculated using a moving window of 470 ns while the lighter shades are the actual distances. The two figures on top show distances between positively-charged residues from the calmodulin-binding element and…

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  Figure 5—figure supplement 2.. Close-up of two docking events in the simulation, in comparison with a crystal structure where a peptide docks at the interface. The calmodulin-binding element is colored pale pink, with the positively charged residues colored blue. The interfacial β-sheet is shown in white. (A) Close-up of Docking 1 showing backbone interactions between residues 298–300 of the calmodulin-binding element of subunit L, and residues 412–416 of the edge of the β-sheet of subunit L. (B) Close-up of Docking two showing backbone interactions between residues 301-305 of the calmodulin-binding element of subunit F, and residues 412–416 at the edge of the β-sheet of subunit F. (C) Interface from the crystal structure of CaMKII-B hub from Nematostella vectensis (PDB ID: 5IG5) at pH 4.2 (Bhattacharyya et al., 2016). In this structure, the linker folds back to interact with the hub, extending the interfacial β-sheet. Here, we have replaced the residues of the linker in the crystal structure with residues from the calmodulin-binding element, while maintaining the backbone of the linker, to show how the positively-charged residues from the calmodulin-binding element (in pale blue) might interact with the…

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  Figure 6.. Interfaces at which docking occur are stabilized in an open form, while remaining interfaces close. (A) Schematic showing the naming scheme used to identify subunits. The interhelix angle between axes of αA helices are used to measure the rotation of each hub domain with respect to the adjacent hub domain. (B) Variation of the angle between the axes of the αA helices of adjacent subunits in the simulation with the regulatory segment. The darker traces are the time-averaged values of the interhelix angles calculated using a moving window of 240 ns, while the lighter shades are the actual distances. The two interfaces where regulatory segments dock between subunits L and B (colored blue), and between subunits F and H (colored pink) open and the remaining interfaces close. In the lower ring the interfaces mirror the behavior of the interfaces in the upper ring. The interhelix angle between the αA helices of adjacent subunits from the crystal structure of an autoinhibited, dodecameric holoenzyme (PDB ID - 3SOA) (Chao et al., 2011), which is perfectly symmetrical, is indicated by the horizontal black line. (C) Variation in the angle between the axes of the αA helices of hub domains…

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  Figure 6—figure supplement 1.. Close-up of hub interfaces from the molecular dynamics simulation of CaMKII-α. (A) Close-up of interface at which Docking 1 occurs, showing instantaneous snapshots from the end of the simulation overlaid with the start of the simulation, while aligning on the subunit to the right (subunit L). Subunit B (to the left) rotates away from subunit L over the course of the simulation. For clarity, only the hub domains are shown. (B) Hydrogen bond interactions between subunits B and L (same interface as (A)) at the start (top) and end (bottom) of the simulation. (C) Close-up of interface at which a regulatory segment does not dock, showing instantaneous snapshots from the end of the simulation overlaid with the start of the simulation, while aligning on the subunit to the right (subunit J). Subunit L (to the left) rotates toward from subunit J over the course of the simulation. For clarity, only the hub domains are shown. (D) Hydrogen bond interactions between subunits L and J (same interface as (C)) at the start (top) and end (bottom) of the simulation. The hydrogen bonds are intact in both the interface that opens and the interface that closes.