UVM Theses and Dissertations
Format:
Print
Author:
Osborne, Brent William
Dept./Program:
Cell and Molecular Biology Program
Year:
2011
Degree:
Ph. D.
Abstract:
The cGMP-dependent protein kinase, PKG, is widely expressed and serves as an integral component of second messenger signaling in a number of biological contexts including vasodilation, motility and memory. The assembly of PKG into homodimers is associated with large conformational changes that are induced by the cooperative binding of cGMP. Despite extensive biochemical characterization, the structure of PKG and the molecular mechanisms associated with protomer communication following cGMP binding remain obscure. This dissertation presents a detailed structural analysis of the regulatory domain of PKG I[alpha] beginning with a 2.5 Å crystal structure of a fragment containing both tandem cGMP binding sites (amino acids 78-355). The overall domain topology of this structure showed a distinct and segregated architecture with the cGMP binding sites separated by an extended central helix with no obvious means of interdomain communication.
The most significant contribution of this structure is the identification of a previously uncharacterized helical domain, termed the switch helix (SW), which promotes the assembly of a critical hydrophobic interface between PKG I[alpha]⁷⁸⁻³⁵⁵ protomers. Disruption of this SW interface in full-length PKG mutants resulted in two significant phenotypes. Both a marked reduction of cGMP-induced activation constants and a loss of cGMP cooperativity were observed. These results suggested a novel trans-mechanistic model of PKG activation whereby the catalytic domain of one protomer is tethered by the cGMP binding domains of the other protomer. The biological integrity of PKG appears to be mediated by the switch helix as it is critical for the maintenance of kinetic fidelity.
These observations inspired a curiosity as to the dynamic nature of the structural changes that follow cGMP association. This dissertation presents the first evidence for a molecular compaction of the regulatory domain of PKG I[alpha] upon cGMP binding. We employed native PAGE analysis and small angle X-ray scattering (SAXS) and demonstrated that the binding of cGMP results in a drastic reduction in molecular shape in PKG⁷⁸⁻³⁵⁵. A more compact, globular fold in the cyclic nucleotide-bound conformation is consistent with the structural rearrangements observed in the regulatory domain of the closely related cAMP-dependent protein kinase (PKA). A better understanding of these dynamic perturbations will allow for the development of novel and improved cyclic nucleotide biosensors, which is also discussed.
This structural work described herein highlights the critical importance of dimer communication in PKG biology and will likely serve as an improved platform for the strategic development of therapeutic agents aimed at treatment and prevention of cGMP-dependent pathologies. Currently, the use of non-cyclic nucleotide modulators of PKG activity is in its infancy. Here, we also performed the first biochemical analysis of two lead compounds, which exhibit the potential to directly target PKG for pharmacological intervention.
The most significant contribution of this structure is the identification of a previously uncharacterized helical domain, termed the switch helix (SW), which promotes the assembly of a critical hydrophobic interface between PKG I[alpha]⁷⁸⁻³⁵⁵ protomers. Disruption of this SW interface in full-length PKG mutants resulted in two significant phenotypes. Both a marked reduction of cGMP-induced activation constants and a loss of cGMP cooperativity were observed. These results suggested a novel trans-mechanistic model of PKG activation whereby the catalytic domain of one protomer is tethered by the cGMP binding domains of the other protomer. The biological integrity of PKG appears to be mediated by the switch helix as it is critical for the maintenance of kinetic fidelity.
These observations inspired a curiosity as to the dynamic nature of the structural changes that follow cGMP association. This dissertation presents the first evidence for a molecular compaction of the regulatory domain of PKG I[alpha] upon cGMP binding. We employed native PAGE analysis and small angle X-ray scattering (SAXS) and demonstrated that the binding of cGMP results in a drastic reduction in molecular shape in PKG⁷⁸⁻³⁵⁵. A more compact, globular fold in the cyclic nucleotide-bound conformation is consistent with the structural rearrangements observed in the regulatory domain of the closely related cAMP-dependent protein kinase (PKA). A better understanding of these dynamic perturbations will allow for the development of novel and improved cyclic nucleotide biosensors, which is also discussed.
This structural work described herein highlights the critical importance of dimer communication in PKG biology and will likely serve as an improved platform for the strategic development of therapeutic agents aimed at treatment and prevention of cGMP-dependent pathologies. Currently, the use of non-cyclic nucleotide modulators of PKG activity is in its infancy. Here, we also performed the first biochemical analysis of two lead compounds, which exhibit the potential to directly target PKG for pharmacological intervention.