Mechanisms associated with cGMP binding and activation of cGMP-dependent protein kinase

Abstract

Using small-angle x-ray scattering, we have observed the cGMP-induced elongation of an active, cGMP-dependent, monomeric deletion mutant of cGMP-dependent protein kinase (Delta(1-52)PKG-I beta). On saturation with cGMP, the radius of gyration of Delta(1-52)PKG-I beta increases from 29.4 +/- 0.1 A to 40.1 +/- 0.7 A, and the maximum linear dimension increases from 90 A +/- 10% to 130 A +/- 10%. The elongation is due to a change in the interaction between structured regulatory (R) and catalytic (C) domains. A model of cGMP binding to Delta(1-52)PKG-I beta indicates that elongation of Delta(1-52)PKG-I beta requires binding of cGMP to the low-affinity binding site of the R domain. A comparison with cAMP-dependent protein kinase suggests that both elongation and activation require cGMP binding to both sites; cGMP binding to the low-affinity site therefore seems to be a necessary, but not sufficient, condition for both elongation and activation of Delta(1-52)PKG-I beta. We also predict that there is little or no cooperativity in cGMP binding to the two sites of Delta(1-52)PKG-I beta under the conditions used here. Results obtained by using the Delta(1-52)PKG-I beta monomer indicate that a previously observed elongation of PKG-I alpha is consistent with a pure change in the interaction between the R domain and the C domain, without alteration of the dimerization interaction. This study has revealed important features of molecular mechanisms in the biochemical network describing PKG-I beta activation by cGMP, yielding new insight into ligand activation of cyclic nucleotide-dependent protein kinases, a class of regulatory proteins that is key to many cellular processes.

Figure 1

P(R) distributions from all experiments. The apparent degree of elongation of the molecule increases with increasing amounts of cGMP, until saturation (see Table 1). The apparent isosbestic point in the P(R) distributions suggests a mixture of just two molecular species in solution.

Figure 2

I(Q) from Δ^1–52PKG-Iβ without cGMP. Scattering data from Δ^1–52PKG-Iβ (points) are similar to simulated scattering calculated from a model of the PKA R/C interaction (line); the fit has χ²/n = 1.07. crysol (39) was used to calculate the model scattering.

Figure 3

Two-state model fit overlayed with 1.5:1 cGMP:PKG titration data. All titration point fits resulted in χ²/n ≤ 1.0.

Figure 4

Modeling the cGMP-dependent elongation of PKG. (Left) Model of cGMP binding to monomeric PKG that is used to generate binding curves. States are annotated with respect to size and activity. (Right) Extended state occupancy vs. cGMP:PKG ratio for monomeric PKG. Predicted binding site occupancy curves for cGMP binding to site A, site B, and sites A and B combined are shown. In our experiments, the PKG:B state and the PKG:AB state cannot be distinguished.

Figure 5

Singular value decomposition analysis of cGMP titration data. (Left) Normalized singular values are shown for SVD of the titration data. The first two components account for 96% of the signal. (Right) The first three SVD components of the scattering signal for the titration data are shown offset, sorted top-to-bottom. The third component and higher are dominated by noise. Combined, these plots provide evidence that the titration data are explained by two underlying signals, consistent with a two-state model of the elongation.