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Ca2؉ -calmodulin-dependent protein kinase II (CaMkinase II) is a ubiquitous Ser/Thr-directed protein kinase that is expressed from a family of four genes (␣, ␤, ␥, and ␦) in mammalian cells. We have documented the three-dimensional structures and the biophysical and enzymatic properties of the four gene products. Biophysical analyses showed that each isoform assembles into oligomeric forms and their three-dimensional structures at 21-25 Å revealed that all four isoforms were dodecamers with similar but highly unusual architecture. A gear-shaped core comprising the association domain has the catalytic domains tethered on appendages, six of which extend from both ends of the core. At this level of resolution, we can discern no isoform-dependent differences in ultrastructure of the holoenzymes. Enzymatic analyses showed that the isoforms were similar in their K m for ATP and the peptide substrate syntide, but showed significant differences in their interactions with Ca 2؉ -calmodulin as assessed by binding, substrate phosphorylation, and autophosphorylation. Interestingly, the rank order of CaM binding affinity (␥ > ␤ > ␦ > ␣) does not directly correlate with the rank order of their CaM dependence for autophosphorylation (␤ > ␥ > ␦ > ␣). Simulations utilizing this data revealed that the measured differences in CaM binding affinities play a minor role in the autophosphorylation of the enzyme, which is largely dictated by the rate of autophosphorylation for each isoform.1 is a major downstream effector of Ca 2ϩ signaling in eukaryotic cells. A rise in intracellular Ca 2ϩ concentration leads to binding of Ca 2ϩ ions to calmodulin (CaM), which binds to and activates CaM-kinase II. Upon activation, this enzyme has the ability to autophosphorylate, a process that confers Ca 2ϩ -independent activity upon the kinase (1) and greatly increases its affinity for CaM (2). Once activated, CaM-kinase II phosphorylates numerous target proteins and is involved in many cellular functions, including synaptic plasticity, synaptic vesicle mobilization, regulation of gene expression, regulation of smooth muscle contractility, and modulation of ion channel function (3-7). The fact that CaM-kinase II has so many potential substrates raises the question of the relationship between its activation and a specific response to a particular Ca 2ϩ signal. Possibly, the regulated expression of the multiple isoforms of CaM-kinase II confers these unique properties.CaM-kinase II is expressed from a family of four closely related genes, ␣, ␤, ␥, and ␦, each of which produces mRNA that can be alternatively spliced, giving rise to at least 30 different proteins (8, 9). The overall organization of each of the four kinase isoforms is similar: an N-terminal catalytic domain is followed by a regulatory domain that contains an autoinhibitory region and a CaM-binding site, and a C-terminal association domain, through which the subunits interact to assemble into holoenzymes (10). Between the CaM-binding domain and the association domain is a region termed the...

The three-dimensional reconstruction of the bovine kidney pyruvate dehydrogenase complex (M r Ϸ 7.8 ؋ 10 6 ) comprising about 22 molecules of pyruvate dehydrogenase (E 1) and about 6 molecules of dihydrolipoamide dehydrogenase (E 3) with its binding protein associated with the 60-subunit dihydrolipoamide acetyltransferase (E2) core provides considerable insight into the structural and functional organization of the largest multienzyme complex known. The structure shows that potentially 60 centers for acetylCoA synthesis are organized in sets of three at each of the 20 vertices of the pentagonal dodecahedral core. These centers consist of three E 1 molecules bound to one E2 trimer adjacent to an E3 molecule in each of 12 pentagonal openings. The E1 components are anchored to the E 1-binding domain of the E2 subunits through an Ϸ50-Å-long linker. Three of these linkers emanate from the outside edges of the triangular base of the E 2 trimer and form a cage around its base that may shelter the lipoyl domains and the E 1 and E2 active sites. The docking of the atomic structures of E1 and the E 1 binding and lipoyl domains of E2 in the electron microscopy map gives a good fit and indicates that the E 1 active site is Ϸ95 Å above the base of the trimer. We propose that the lipoyl domains and its tether (swinging arm) rotate about the E 1-binding domain of E 2, which is centrally located 45-50 Å from the E1, E2, and E3 active sites, and that the highly flexible breathing core augments the transfer of intermediates between active sites. T he pyruvate dehydrogenase complex (PDC) serves as the link between glycolysis and the tricarboxylic acid cycle and generally has prominence in the description of these metabolic pathways because they serve as a major source of cellular energy. A central feature of PDCs is a 24-mer (Escherichia coli) or 60-mer (eukaryotes and some Gram-positive bacteria) core with the morphologies of a cube or pentagonal dodecahedron, respectively (1-4). The structures with the latter morphology comprise the largest (M r Ϸ 10 7 ) multienzyme complexes known. Even more remarkable than their exceptional size and morphology, these complexes encompass some of the most unusual features found in structural biology as described below.The E 2 core comprises the dihydrolipoamide acetyltransferase activity and is the only oligomeric enzyme complex known to be organized with the shape of a pentagonal dodecahedron. Moreover, the 250-Å-diameter dodecahedron has a very unusual feature: the tightly bound trimers at each of its 20 vertices seem to be interconnected by 30 flexible bridges enabling the core to ''breathe,'' as evidenced by an extraordinary size variability of 40 Å (17%) at room temperature. The breathing core apparently is a common feature in the phylogeny of the PDCs, suggesting that protein dynamics is an integral component of the function of these multienzyme complexes (5). Moreover, dodecahedral morphology of the core favors a synchronous or harmonious change in the length of the bridges that is relate...

Studies of the structural organization of calcium/ calmodulin-dependent protein kinase II␣ (CaM KII␣) and truncated CaM KII␣ by three-dimensional electron microscopy and protein engineering show that the structures consist of 12 subunits that are organized in two stacked hexameric rings with 622 symmetry. The body of CaM KII␣ is gear-shaped, consisting of six slanted flanges, and has six foot-like processes attached by narrow appendages to both ends of the flanges. Truncated CaM KII␣ that lacks functional domains has a structure that is very similar to the body of CaM KII␣. Thus, the functional domains reside in the foot-like processes, and the association domain comprises the gearshaped core. The ribbon diagram of the bilobate structure of CaM KI fits nicely in the envelope of the foot-like component and indicates that the crevice between the two lobes comprising the functional domains is near the middle portion of the foot. The clustering of the functional domains provides a favorable arrangement for the autophosphorylation reaction, and the unusual arrangement of the catalytic domain on extended tethers appears to be significant for the remarkable functional diversity of CaM KII␣ in cellular regulation.Calcium/calmodulin-dependent protein kinase (CaM K) 1 II phosphorylates Ser and Thr residues in numerous proteins. Its relatively high concentration in brain tissue and its lack of specificity indicate that it has an important role as a kinase in many aspects of neuronal function. Its substrates are involved in neurotransmitter synthesis and release; carbohydrate, lipid, and amino acid metabolism; transcriptional, translational, and cytoskeletal regulation; calcium homeostasis; and receptor and channel function (for reviews, see Refs. 1 and 2).At least four distinct genes that encode isozymes of CaM KII are selectively expressed in different tissues (2). Expression of the ␣ and ␤ isoforms is restricted primarily to the nervous system, and the ␣ isoform is found only in neurons. The abundance of ␣ and ␤ isoforms is both anatomically and developmentally regulated in the nervous system (3, 4), and it has been proposed that the subunit composition of holoenzymes may influence the targeting of the enzyme to distinct subcellular sites, such as the postsynaptic density (5-7). For example, recent investigations have shown that the ␤ isoform is preferentially bound to F-actin in dendritic spines and in the cell cortex and that the ␣ isoform is targeted to these locations when it is co-expressed with the ␤ isoform (8).Studies have implicated the ␣ isoform of CaM KII in neuronal cell function. Electrophysiological and behavioral tests in mice carrying a null mutation for the ␣ isoform showed that this enzyme has a major role in control of neuronal excitability (9) and spatial learning (10). Indeed, this isoform is thought to have a key role in the long lasting synaptic enhancement denoted long term potentiation (11-13).CaM KII is dependent on Ca 2ϩ /calmodulin for activation, and its autophosphorylation has an important r...

Dihydrolipoyl acetyltransferase (E2) is the central component of pyruvate dehydrogenase complex (PDC), which converts pyruvate to acetyl-CoA. Structural comparison by cryo-electron microscopy (cryo-EM) of the human full-length and truncated E2 (tE2) cores revealed flexible linkers emanating from the edges of trimers of the internal catalytic domains. Using the secondary structure constraints revealed in our 8 A cryo-EM reconstruction and the prokaryotic tE2 atomic structure as a template, we derived a pseudo atomic model of human tE2. The active sites are conserved between prokaryotic tE2 and human tE2. However, marked structural differences are apparent in the hairpin domain and in the N-terminal helix connected to the flexible linker. These permutations away from the catalytic center likely impart structures needed to integrate a second component into the inner core and provide a sturdy base for the linker that holds the pyruvate dehydrogenase for access by the E2-bound regulatory kinase/phosphatase components in humans.