Abstract
Chimeric molecules of the cAMP-dependent protein kinase (PKA) holoenzyme (R2C2) and of a Delta1-91RC 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 RII PKA isoforms.
Highlights
Inactive tetramer (R2C2) with two identical regulatory (R) and two identical catalytic (C) subunits
Small angle scattering studies (16) revealed that in the absence of the pseudosubstrate peptide the catalytic cleft is in a more “open” configuration resulting from a hinge movement at residue Gly125 located in the sequence segment joining the two lobes
We report here small angle x-ray scattering and neutron contrast variation measurements on the protein kinase (PKA) holoenzyme (R2C2) and a ⌬1–91RC heterodimer, each reconstituted with partially deuterated R
Summary
Vol 273, No 46, Issue of November 13, pp. 30448 –30459, 1998 Printed in U.S.A. Quaternary Structures of a Catalytic Subunit-Regulatory Subunit Dimeric Complex and the Holoenzyme of the cAMP-dependent Protein Kinase by Neutron Contrast Variation*. Small angle scattering studies (16) revealed that in the absence of the pseudosubstrate peptide the catalytic cleft is in a more “open” configuration resulting from a hinge movement at residue Gly125 located in the sequence segment joining the two lobes. This glycine is closely conserved throughout most of the kinase phylogenetic family (16). The different neutron scattering properties of hydrogen (protium) and deuterium enable us to extract the structural parameters for the C and R subunits within the complexes From these measurements, we have determined the molecular boundaries of the RC dimer, the PKA holoenzyme, and the subunits within each complex.
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