Small angle scattering data from bovine lung type I␣ cGMP-dependent protein kinase (PKG) in the absence of cGMP show the protein to have a highly asymmetric structure with a radius of gyration (R g ) of 45 Å and a maximum linear dimension (d max ) of 165 Å. The addition of cGMP induces a marked conformational change in PKG. The R g and d max increase 25-30%, and the protein's mass moves further away from the center of mass; this results in an even more asymmetric structure. Fourier transform infrared spectroscopy data suggest that the conformational change induced by cGMP binding is primarily due to a topographical movement of the structural domains of PKG rather than to secondary structural changes within one or more of the individual domains. Each monomer of the dimeric PKG contains one high and one low affinity cGMP-binding site. A prominent increase in the asymmetry of PKG occurs with binding to high affinity cGMP-binding sites alone, but the full domain movements require the binding to both sets of sites. These conformational changes occurring in PKG with the progressive binding of cGMP to both sets of cGMP-binding sites correlate with past data, which have indicated that cGMP binding to both sets of sites is required for the full activation of the enzyme. These results provide the first quantitative measurement of the overall PKG structure, as well as an assessment of the structural events that accompany the activation of a protein kinase upon binding a small molecular weight ligand.
Chimeric molecules of the cAMP-dependent protein kinase (PKA) holoenzyme (R 2 C 2 ) and of a ⌬ 1-91 RC dimer were reconstituted using deuterated regulatory (R) and protiated catalytic (C) subunits. Small angle scattering with contrast variation has revealed the shapes and dispositions of R and C in the reconstituted complexes, leading to low resolution models for both forms. The crystal structures of C and a truncation mutant of R fit well within the molecular boundaries of the RC dimer model. The area of interaction between R and C is small, seemingly poised for dissociation upon a conformational transition within R induced by cAMP binding. Within the RC dimer, C has a "closed" conformation similar to that seen for C with a bound pseudosubstrate peptide. The model for the PKA holoenzyme has an extended dumbbell shape. The interconnecting bar is formed from the dimerization domains of the R subunits, arranged in an antiparallel configuration, while each lobe contains the cAMP-binding domains of one R interacting with one C. Our studies suggest that the PKA structure may be flexible via a hinge movement of each dumbbell lobe with respect to the dimerization domain. Sequence comparisons suggest that this hinge might be a property of the R II PKA isoforms.Protein phosphorylation is one of the most important mechanisms for the regulation of biochemical function in eukaryotic cells. It is catalyzed by a family of enzymes, the protein kinases, of which several hundred have been identified. The cAMP-dependent protein kinase (PKA) 1 was one of the earliest kinases to be discovered, and it serves as a prototype for understanding kinase structure-function relationships and regulatory mechanisms (1, 2). In the absence of cAMP, PKA is an inactive tetramer (R 2 C 2 ) with two identical regulatory (R) and two identical catalytic (C) subunits. The two R subunits are homodimerized at their amino-terminal ends (3, 4), and, physiologically, the R subunit appears always to exist as a dimer. The R subunit also has two in tandem cAMP-binding sites and a pseudosubstrate autoinhibitory domain that binds to C and inhibits catalysis in the absence of cAMP. Upon binding cAMP, the PKA holoenzyme dissociates into an R 2 homodimer and two active C subunits (5-7). Whether dissociation is absolutely required for activation, however, remains in question (8, 9). Saturation of both cAMP-binding sites on each R is required for activation.There are three isoforms of C (C␣, C, and C␥) and two major isoforms of R (R I and R II ) that are further distinguished into subforms (␣ and ) (10). The physiological importance of these isozyme variations is not fully understood, but anchoring proteins (AKAPs) for R II give it a unique cellular distribution (11,12). R I and R II show sequence homology in their cAMP-binding and pseudosubstrate domains but differ extensively in their dimerization domains as well as in the sequence connecting the dimerization and pseudosubstrate domains.Structural data have been obtained for the individual PKA subunits, but info...
We present here X-ray scattering data that yield new structural information on the multicomponent enzyme methane monooxygenase and its components: a hydroxylase dimer, and two copies each of a reductase and regulatory protein B. Upon formation of the enzyme complex, the hydroxylase undergoes a dramatic conformational change that is observed in the scattering data as a fundamental change in shape of the scattering particle such that one dimension is narrowed (by 25% or 24 A) while the longest dimension increases (by 20% or 25 A). These changes also are reflected in a 13% increase in radius of gyration upon complex formation. Both the reductase and protein B are required for inducing the conformational change. We have modeled the scattering data for the complex by systematically modifying the crystal structure of the hydroxylase and using ellipsoids to represent the reductase and protein B components. Our model indicates that protein B plays a role in optimizing the interaction between the active centers of the reductase and hydroxylase components, thus, facilitating electron transfer between them. In addition, the model suggests reasons why the hydroxylase exists as a dimer and that a possible role for the outlying gamma-subunit may be to stabilize the complex through its interaction with the other components. We further show that proteolysis of protein B to form the inactive B' results in a conformational change and B' does not bind to the hydroxylase. The truncation thus could represent a regulatory mechanism for controlling the enzyme activity.
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