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...

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...

Ca2؉ -Calmodulin-dependent protein kinase II (CaMKII) is an abundant synaptic protein that was recently shown to regulate the organization of actin filaments leading to structural modifications of synapses. CaMKII is a dodecameric complex with a special architecture that provides it with unique potential for organizing the actin cytoskeleton. We report using biochemical assays that the ␤ isoform of CaMKII binds to and bundles actin filaments, and the disposition of ␤CaMKII within the actin bundles was revealed by cryoelectron tomography. In addition, ␤CaMKII was found to inhibit actin polymerization, suggesting that it either serves as a capping protein or binds monomeric actin, reducing the amount of freely available monomers to nucleate polymer assembly. By means of fluorescent cross-correlation spectroscopy, we determined that ␤CaMKII does indeed bind to monomeric actin, reaching saturation at a stoichiometry of 12:1 actin monomers per ␤CaMKII holoenzyme with a binding constant of 2.4 ؋ 10 5 M ؊1 . In cells, ␤CaMKII has a dual functional role; it can sequester monomeric actin to reduce actin polymerization and can also bundle actin filaments. Together, these effects would impact both the dynamics of actin filament assembly and enhance the rigidity of the filaments once formed, significantly impacting the structure of synapses.Actin and more than 60 different classes of actin-binding proteins form an abundant and highly regulated cytoskeletal network, constituting more than 25% of protein in non-muscle cells and over 60% in muscle cells (1). Actin is estimated to be ϳ4 mg/ml in cells, with half found in the polymerized F-actin state and the remainder in the soluble G-actin state (2). Actin undergoes rounds of polymerization and depolymerization from a soluble (G-actin) pool to a filamentous (F-actin) pool, and it is the kinetics of these forward and backward rates that determines the stability of the actin cytoskeleton. The various actin-binding proteins are, in part, responsible for regulating the kinetics of actin polymerization and depolymerization. Actin is commonly known for its structural and dynamic function in cells, such as its role in muscle contraction and cell locomotion, and has been similarly implicated for its structural role at synapses in the nervous system (3, 4). For example, actin is proposed to serve an important role in coordinating the delivery of synaptic vesicles to the pre-synaptic terminal that appear to underlie certain forms of plasticity mediated by alterations in the probability of stimulus evoked vesicle release (5-7). Additionally, several recent studies have demonstrated the importance of actin dynamics in structural reorganization of the post-synaptic spine compartment of excitatory synapses (8 -10). Several pools of actin have been identified within spines with different turnover rates, and activation of glutamate receptors on the spines regulates the kinetics of actin turnover (9, 10). Spine shape changes have been proposed to underlie forms of synaptic plasticity such as l...

Dihydrolipoamide acyltransferase (E 2 ), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of ␣-keto acids, forms the central core to which the other components attach. We have determined the structures of the truncated 60-mer core dihydrolipoamide acetyltransferase (tE 2 ) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex and complexes of the tE 2 core associated with a truncated binding protein (tBP), intact binding protein (BP), and the BP associated with its dihydrolipoamide dehydrogenase (BP⅐E 3 ). The tE 2 core is a pentagonal dodecahedron consisting of 20 cone-shaped trimers interconnected by 30 bridges. Previous studies have given rise to the generally accepted belief that the other components are bound on the outside of the E 2 scaffold. However, this investigation shows that the 12 large openings in the tE 2 core permit the entrance of tBP, BP, and BP⅐E 3 into a large central cavity where the BP component apparently binds near the tip of the tE 2 trimer. The bone-shaped E 3 molecule is anchored inside the central cavity through its interaction with BP. One end of E 3 has its catalytic site within the surface of the scaffold for interaction with other external catalytic domains. Though tE 2 has 60 potential binding sites, it binds only about 30 copies of tBP, 15 of BP, and 12 of BP⅐E 3 . Thus, E 2 is unusual in that the stoichiometry and arrangement of the tBP, BP, and E 3 ⅐BP components are determined by the geometric constraints of the underlying scaffold.Pyruvate dehydrogenase complexes (PDCs) 1 are among the largest (M r ϳ10 6 -10 7 ) and most complex multienzyme structures known. They consist of a central core that has both functional and structural roles in organizing the complex, the dihydrolipoamide acetyltransferase (E 2 ) subunits associate to form the core complex that also serves as a scaffold to which the other components are attached (1-4). Electron microscopy (5-8) and x-ray crystallography (9 -11) studies have revealed two fundamental morphologies of the E 2 cores. The cubic E 2 core of the Escherichia coli PDC has 24 subunits arranged with octahedral symmetry, whereas the pentagonal dodecahedral E 2 core of the PDC complexes from eukaryotes and some Grampositive bacteria has 60 subunits arranged with icosahedral symmetry. The subunits form cone-shaped trimers at each of the 8 and 20 vertices of the cubic and dodecahedral structures, respectively. These trimers are interconnected by bridges to form a cage-like complex (8 -11).The E 2 subunit is a multidomain structure to which the other constituents of the functional PDC (1-4) bind (see Fig. 1). These include the pyruvate dehydrogenase (E 1 ) and dihydrolipoamide dehydrogenase (E 3 ). E 3 requires a binding protein (BP) to anchor it to the core of the yeast (12) and mammalian PDCs (13-15) though, in E. coli and Bacillus stearothermophilus PDCs, BP is not required (1-4).It is widely held that the constituent proteins are bound to the out...

Three-dimensional electron microscopy reconstructions of the human ␣ 2 -macroglobulin (␣ 2 M) dimer and chymotrypsin-transformed ␣ 2 M reveal the structural arrangement of the two dimers that comprise native and proteinase-transformed molecules. They consist of two side-by-side extended strands that have a clockwise and counterclockwise twist about their major axes in the native and transformed structures, respectively. This and other studies show that there are major contacts between the two strands at both ends of the molecule that evidently sequester the receptor binding domains. Upon proteinase cleavage of the bait domains and subsequent thiol ester cleavages, which occur near the central region of the molecule, the two strands separate by 40 Å at both ends of the structure to expose the receptor binding domains and form the arm-like extensions of the transformed ␣ 2 M. During the transformation of the structure, the strands untwist to expose the ␣ 2 M central cavity to the proteinase. This extraordinary change in the architecture of ␣ 2 M functions to completely engulf two molecules of chymotrypsin within its central cavity and to irreversibly encapsulate them.Human ␣ 2 -macroglobulin (␣ 2 M) 1 is of broad interest to the biological and medical communities as a result of its unique function as a nonspecific proteinase inhibitor. It serves the biological role of a scavenger of most proteinases in the plasma of all vertebrates (1). It also appears to participate in the regulation of proteinase activity in fibrinolysis, coagulation, and complement activation (2). It affects the activity of cytotoxins with which it interacts (3) and has distinct binding sites for ␤-amyloid peptide and therefore may influence the progression of Alzheimer's disease (4).␣ 2 M is a homotetramer in which pairs of subunits are disulfide-linked and form noncovalently associated dimeric protomers (1). Each 1451-residue subunit contains a bait domain with cleavage sequences for almost all known endoproteinases, a receptor binding domain, and a thiol ester linkage between Cys-949 and Glx-952 (1, 5). Cleavage of the four bait domains by an endoproteinase results in a structural change, causing it to irreversibly entrap as many as two molecules of the proteinase, expose the receptor binding domains, and consequently undergo rapid endocytosis by binding to the low density lipoprotein receptor-related protein (1, 6).Three-dimensional electron microscopy (EM) and x-ray crystallography studies have contributed significantly to the understanding of the function of ␣ 2 M. These studies show that the native ␣ 2 M and ␣ 2 M that has reacted with a proteinase have markedly different shapes and that proteinases are entrapped within the body of the complex on its major axis (7-10). A part of the mechanism of proteinase entrapment involves the unusual thiol ester linkage between Cys-949 and Glx-952 in each subunit (1), which directly participates in the conversion of ␣ 2 M from its native architecture to that of the proteinasetransformed molecule. Ind...