Laurel Hoffman

Calcium-Calmodulin-Dependent Protein Kinase II Isoforms Differentially Impact the Dynamics and Structure of the Actin Cytoskeleton

Calcium-calmodulin-dependent protein kinase II (CaMKII) has been implicated in a wide variety of cellular processes, which include a critical regulatory role in actin cytoskeletal assembly. CaMKII is ubiquitous in cells, expressed as one of four isoforms termed α, β, γ, and δ. Characterization of the CaMKII-actin interaction has mainly focused on the β isoform, which has been shown to bundle actin filaments and sequester actin monomers in an activity-dependent manner. Much less is known about the interactions of other CaMKII isoforms with actin. In this work, isoform specific interactions of CaMKII with actin are described and reveal that the δ isoform of CaMKII bundles F-actin filaments like the β isoform while the γ isoform induces a novel layered structure in filaments. Using electron tomography, CaMKII holoenzymes are clearly identified in the complexes bridging the actin filaments, allowing direct visualization of the interactions between CaMKII isoforms and actin. In addition, we determined the isoform specificity of CaMKII-mediated inhibition of actin polymerization and discovered that all isoforms inhibit polymerization to varying degrees: β > γ ≈ δ > α (from most to least effective). Ca(2+)/CaM activation of all kinase isoforms produced a robust increase in actin polymerization that surpassed the rates of polymerization in the absence of kinase inhibition. These results indicate that diversity exists between the types of CaMKII-actin interactions mediated by the different isoforms and that the CaMKII isoform composition differentially impacts the formation and maintenance of the actin cytoskeleton.

Protein recognition and selection through conformational and mutually induced fit

Significance Protein–protein interactions drive most every biological process, but in many instances the domains mediating recognition are disordered. How specificity in binding is attained in the absence of defined structure is a fascinating problem but not well understood. Calmodulin presents a unique opportunity to investigate mechanisms for selectivity given that it interacts with several hundred different potential targets. In our work, a combined experimental and theoretical approach is taken to define how target selectivity occurs at the molecular level. Our study shows that the binding interactions require mutually induced conformational changes in both calmodulin and the target protein, and broadly informs how intrinsically disordered proteins can achieve both high affinity and high specificity. Protein–protein interactions drive most every biological process, but in many instances the domains mediating recognition are disordered. How specificity in binding is attained in the absence of defined structure contrasts with well-established experimental and theoretical work describing ligand binding to protein. The signaling protein calmodulin presents a unique opportunity to investigate mechanisms for target recognition given that it interacts with several hundred different targets. By advancing coarse-grained computer simulations and experimental techniques, mechanistic insights were gained in defining the pathways leading to recognition and in how target selectivity can be achieved at the molecular level. A model requiring mutually induced conformational changes in both calmodulin and target proteins was necessary and broadly informs how proteins can achieve both high affinity and high specificity.

Conformational changes underlying calcium/calmodulin-dependent protein kinase II activation

Calcium/calmodulin‐dependent protein kinase II (CaMKII) interprets information conveyed by the amplitude and frequency of calcium transients by a controlled transition from an autoinhibited basal intermediate to an autonomously active phosphorylated intermediate (De Koninck and Schulman, 1998). We used spin labelling and electron paramagnetic resonance spectroscopy to elucidate the structural and dynamic bases of autoinhibition and activation of the kinase domain of CaMKII. In contrast to existing models, we find that autoinhibition involves a conformeric equilibrium of the regulatory domain, modulating substrate and nucleotide access. Binding of calmodulin to the regulatory domain induces conformational changes that release the catalytic cleft, activating the kinase and exposing an otherwise inaccessible phosphorylation site, threonine 286. Autophosphorylation at Thr286 further disrupts the interactions between the catalytic and regulatory domains, enhancing the interaction with calmodulin, but maintains the regulatory domain in a dynamic unstructured conformation following dissociation of calmodulin, sustaining activation. These findings support a mechanistic model of the CaMKII holoenzyme grounded in a dynamic understanding of autoregulation that is consistent with a wealth of biochemical and functional data.

Neurogranin Alters the Structure and Calcium Binding Properties of Calmodulin

Background: Neurogranin is an IQ motif protein that binds to both apo- and calcium-saturated calmodulin. Results: Neurogranin makes contacts with both the N- and C-domains of calmodulin that functionally leads to altered calcium binding kinetics. Conclusion: The availability and calcium saturation of calmodulin is determined by neurogranin. Significance: Neurogranin plays a major role in controlling the intracellular calcium-calmodulin-signaling pathway. Neurogranin (Ng) is a member of the IQ motif class of calmodulin (CaM)-binding proteins, and interactions with CaM are its only known biological function. In this report we demonstrate that the binding affinity of Ng for CaM is weakened by Ca2+ but to a lesser extent (2–3-fold) than that previously suggested from qualitative observations. We also show that Ng induced a >10-fold decrease in the affinity of Ca2+ binding to the C-terminal domain of CaM with an associated increase in the Ca2+ dissociation rate. We also discovered a modest, but potentially important, increase in the cooperativity in Ca2+ binding to the C-lobe of CaM in the presence of Ng, thus sharpening the threshold for the C-domain to become Ca2+-saturated. Domain mapping using synthetic peptides indicated that the IQ motif of Ng is a poor mimetic of the intact protein and that the acidic sequence just N-terminal to the IQ motif plays an important role in reproducing Ng-mediated decreases in the Ca2+ binding affinity of CaM. Using NMR, full-length Ng was shown to make contacts largely with residues in the C-domain of CaM, although contacts were also detected in residues in the N-terminal domain. Together, our results can be consolidated into a model where Ng contacts residues in the N- and C-lobes of both apo- and Ca2+-bound CaM and that although Ca2+ binding weakens Ng interactions with CaM, the most dramatic biochemical effect is the impact of Ng on Ca2+ binding to the C-terminal lobe of CaM.

Conformational frustration in calmodulin–target recognition

Calmodulin (CaM) is a primary calcium (Ca2+)‐signaling protein that specifically recognizes and activates highly diverse target proteins. We explored the molecular basis of target recognition of CaM with peptides representing the CaM‐binding domains from two Ca2+‐CaM‐dependent kinases, CaMKI and CaMKII, by employing experimentally constrained molecular simulations. Detailed binding route analysis revealed that the two CaM target peptides, although similar in length and net charge, follow distinct routes that lead to a higher binding frustration in the CaM–CaMKII complex than in the CaM–CaMKI complex. We discovered that the molecular origin of the binding frustration is caused by intermolecular contacts formed with the C‐domain of CaM that need to be broken before the formation of intermolecular contacts with the N‐domain of CaM. We argue that the binding frustration is important for determining the kinetics of the recognition process of proteins involving large structural fluctuations. Copyright

Domain Contributions to Signaling Specificity Differences Between Ras-Guanine Nucleotide Releasing Factor (Ras-GRF)1 and Ras-GRF2

Background: Ras-GRF1 and Ras-GRF2 are similar exchange factors with different functions in synaptic plasticity. Results: Chimeras between Ras-GRF proteins reveal that IQ, pleckstrin homology, coiled-coil, and CDC25 domains are most important for signaling specificity. Conclusion: Signaling specificity of GRF proteins is encoded in a surprisingly small number of their common domains. Significance: Domains of Ras-GRF proteins have been identified for future studies on signaling specificity. Ras-GRF1 (GRF1) and Ras-GRF2 (GRF2) constitute a family of similar calcium sensors that regulate synaptic plasticity. They are both guanine exchange factors that contain a very similar set of functional domains, including N-terminal pleckstrin homology, coiled-coil, and calmodulin-binding IQ domains and C-terminal Dbl homology Rac-activating domains, Ras-exchange motifs, and CDC25 Ras-activating domains. Nevertheless, they regulate different forms of synaptic plasticity. Although both GRF proteins transduce calcium signals emanating from NMDA-type glutamate receptors in the CA1 region of the hippocampus, GRF1 promotes LTD, whereas GRF2 promotes θ-burst stimulation-induced LTP (TBS-LTP). GRF1 can also mediate high frequency stimulation-induced LTP (HFS-LTP) in mice over 2-months of age, which involves calcium-permeable AMPA-type glutamate receptors. To add to our understanding of how proteins with similar domains can have different functions, WT and various chimeras between GRF1 and GRF2 proteins were tested for their abilities to reconstitute defective LTP and/or LTD in the CA1 hippocampus of Grf1/Grf2 double knock-out mice. These studies revealed a critical role for the GRF2 CDC25 domain in the induction of TBS-LTP by GRF proteins. In contrast, the N-terminal pleckstrin homology and/or coiled-coil domains of GRF1 are key to the induction of HFS-LTP by GRF proteins. Finally, the IQ motif of GRF1 determines whether a GRF protein can induce LTD. Overall, these findings show that for the three forms of synaptic plasticity that are regulated by GRF proteins in the CA1 hippocampus, specificity is encoded in only one or two domains, and a different set of domains for each form of synaptic plasticity.

Relative Cosolute Size Influences the Kinetics of Protein-Protein Interactions

Protein signaling occurs in crowded intracellular environments, and while high concentrations of macromolecules are postulated to modulate protein-protein interactions, analysis of their impact at each step of the reaction pathway has not been systematically addressed. Potential cosolute-induced alterations in target association are particularly important for a signaling molecule like calmodulin (CaM), where competition among >300 targets governs which pathways are selectively activated. To explore how high concentrations of cosolutes influence CaM-target affinity and kinetics, we methodically investigated each step of the CaM-target binding mechanism under crowded or osmolyte-rich environments mimicked by ficoll-70, dextran-10, and sucrose. All cosolutes stabilized compact conformers of CaM and modulated association kinetics by affecting diffusion and rates of conformational change; however, the results showed that differently sized molecules had variable effects to enhance or impede unique steps of the association pathway. On- and off-rates were modulated by all cosolutes in a compensatory fashion, producing little change in steady-state affinity. From this work insights were gained on how high concentrations of inert crowding agents and osmolytes fit into a kinetic framework to describe protein-protein interactions relevant for cellular signaling.

Cytoskeletal-like Filaments of Ca2+-Calmodulin-Dependent Protein Kinase II Are Formed in a Regulated and Zn2+-Dependent Manner

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 micrometers 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 ion-induced filament formation and could disassemble preformed filaments. Interestingly, CaMKII within the filaments retains the capacity to autophosphorylate; however, activity toward 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, in which assembly and activity are further regulated by Ca2+/CaM.

Protein Recognition and Selection through Conformational and Mutually Induced Fit

Protein-protein interactions drive most every biological process but in many instances the domains mediating recognition are disordered. How specificity in binding is attained in the absence of defined structure contrasts with well-established experimental and theoretical work describing ligand binding to protein. The signaling protein calmodulin presents a unique opportunity to investigate mechanisms for target recognition given that it interacts with several hundred different targets. By advancing coarse-grained computer simulations and experimental techniques, novel mechanistic insights were gained in defining the pathways leading to recognition and for how target selectivity can be achieved at the molecular level. A model requiring mutually induced conformational changes in both calmodulin and target proteins was necessary and broadly informs how proteins can achieve both high affinity and high specificity.