Background: Activation loop phosphorylation is a conserved mechanism for regulating protein kinases.Results: The unphosphorylated C-subunit structure of protein kinase A shows decoupling of the two lobes of the enzyme.Conclusion: Phosphorylation orients the small and large lobes of the kinase for catalysis.Significance: PKA in its unphosphorylated state shows a great deal of structural disorganization, and this is difficult to predict in advance.

Carbon monoxide oxidation activator protein (CooA) is a dimeric carbon monoxide (CO) binding transcription factor that in the presence of CO promotes the transcription of genes involved in CO oxidation in Rhodospirillum rubrum. The off state (inactive) of Fe(II) CooA has His and Pro as the two axial heme ligands. In contrast, in the on state, which is active in DNA binding, the Pro ligand bond has been replaced by CO. This occurs by the transient loss of the Pro ligand, thus generating a pentacoordinate heme that can bind CO. The active on state of CooA has two major structural differences from the off state, in addition to the displacement of the Pro ligand by CO. There is a repositioning of long C helices at the dimer interface and a concomitant reorientation of the hinge region between the DNA- and effector-binding domains within each monomer [Roberts et al. (2005) J. Inorg. Biochem. 99, 280-292]. To better understand the relationship of these conformational changes, we have removed the DNA-binding domains and compared CO binding to the truncated and full-length protein. The crystal structure of truncated Fe(II) CooA is the same as that of the effector-binding domain of full-length Fe(II) CooA, including the relative orientation of the homodimer and the ligation environment of the heme. Thus, removing the DNA-binding domains has little obvious effect on the structure of CooA in the inactive off state. However, CO binds about 2-fold more tightly and about 10 times more rapidly to truncated CooA. The rate of CO binding is known to be limited by the dissociation of the Pro heme ligand [Puranik et al. (2004) J. Biol. Chem. 279, 21096-21108]. Therefore, the absence of the DNA-binding domain makes it easier for the Pro ligand to dissociate from the heme iron, which also makes it easier for truncated CooA to adopt the on-state structure.

The bacterial CO-sensing heme protein CooA activates expression of genes whose products perform CO-metabolism by binding its target DNA in response to CO binding. The required conformational change has been proposed to result from CO-induced displacement of the heme and of the adjacent C-helix, which connects the sensory and DNA-binding domains. Support for this proposal comes from UV Resonance Raman (UVRR) spectroscopy, which reveals a more hydrophobic environment for the C-helix residue Trp110 when CO binds. In addition, we find a tyrosine UVRR response, which is attributable to weakening of a Tyr55-Glu83 H-bond that anchors the proximal side of the heme. Both Trp and Tyr responses are augmented in the heme domain when the DNA-binding domain has been removed, apparently reflecting loss of the inter-domain restraint. This augmentation is abolished by a Glu83Gln substitution, which weakens the anchoring H-bond. The CO recombination rate following photolysis of the CO adduct is similar for truncated and full-length protein, though truncation does increase the rate of CO association in the absence of photolysis; together these data indicate that truncation causes a faster dissociation of the endogenous Pro2 ligand. These findings are discussed in the light of structural evidence that the N-terminal tail, once released from the heme, selects the proper orientation of the DNA-binding domain, via docking interactions.

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