Siv Garrod

Isoform-specific Differences between the Type Iα and IIα Cyclic AMP-dependent Protein Kinase Anchoring Domains Revealed by Solution NMR

Cyclic AMP dependent protein kinase (PKA) is controlled, in part, by the subcellular localization of the enzyme (1). Discovery of dual specificityanchoring proteins(d-AKAPs) indicates that not only is the type II, but also the type I, enzyme localized (2). It appears that the type I enzyme is localized in a novel, dynamic fashion as opposed to the apparent static localization of the type II enzyme. Recently, the structure of the dimerization/docking (D/D) domain from the type II enzyme was solved (3). This work revealed an X-type four-helix bundle motif with a hydrophobic patch that modulates AKAP interactions. To understand the dynamic versus static localization of PKA, multidimensional NMR techniques were used to investigate the structural features of the type I D/D domain. Our results indicate a conserved helix-turn-helix motif in the type I and type II D/D domains. However, important differences between the two domains are evident in the extreme NH2 terminus: this region is extended in the type II domain, whereas it is helical in the type I protein. The NH2-terminal residues in RIIα contain determinants for anchoring, and the orientation and packing of this helical element in the RIα structure may have profound consequences in the recognition surface presented to the AKAPs.

Synergistic Binding of Nucleotides and Inhibitors to cAMP-dependent Protein Kinase Examined by Acrylodan Fluorescence Spectroscopy

We have engineered an acrylodan-modified derivative of the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK) whose fluorescence emission signal has allowed the synergistic binding between nucleotides and physiological inhibitors of cAPK to be examined (Whitehouse, S., and Walsh, D. A. (1983) J. Biol. Chem. 258, 3682-3692). In the presence of the regulatory subunit, RI, the affinity of cAPK for adenosine, ADP, AMPPNP (adenosine 5′-(β,γ-imino)triphosphate), or ATP was 5-, 50-, 120-, and 15,000-fold enhanced, while in the presence of the heat-stable inhibitor protein of cAPK (PKI), there was a 3-, 20-, 33-, and 2000-fold enhancement in the binding of these nucleotides, respectively. A short inhibitor peptide, PKI-(14-22), enhanced the binding of ADP to the same degree as did full-length PKI (20-fold) but, in contrast, did not significantly enhance the binding of ATP or AMPPNP. The full binding synergism between PKI and either ATP (2000-fold) or AMPPNP (33-fold) to cAPK could, however, be mimicked by a longer peptide, PKI-(5-24), suggesting that the PKI NH2 terminus (residues 5-13) is most likely critical. Since this region is remote from the ATP γ-phosphate, the binding synergism must arise through an extended network communication mechanism between the PKI NH2 terminus and the ATP binding site.

Isoform-Specific Differences Between the Type Iα and IIα PKA Anchoring Domains Revealed by Solution NMR

Cyclic AMP dependent protein kinase (PKA) is controlled, in part, by the subcellular localization of the enzyme (1). Discovery of dual specificityanchoring proteins(d-AKAPs) indicates that not only is the type II, but also the type I, enzyme localized (2). It appears that the type I enzyme is localized in a novel, dynamic fashion as opposed to the apparent static localization of the type II enzyme. Recently, the structure of the dimerization/docking (D/D) domain from the type II enzyme was solved (3). This work revealed an X-type four-helix bundle motif with a hydrophobic patch that modulates AKAP interactions. To understand the dynamic versus static localization of PKA, multidimensional NMR techniques were used to investigate the structural features of the type I D/D domain. Our results indicate a conserved helix-turn-helix motif in the type I and type II D/D domains. However, important differences between the two domains are evident in the extreme NH2 terminus: this region is extended in the type II domain, whereas it is helical in the type I protein. The NH2-terminal residues in RIIα contain determinants for anchoring, and the orientation and packing of this helical element in the RIα structure may have profound consequences in the recognition surface presented to the AKAPs.

Dissecting interdomain communication within cAPK regulatory subunit type IIβ using enhanced amide hydrogen/deuterium exchange mass spectrometry (DXMS)

cAMP‐dependent protein kinase (cAPK) is a heterotetramer containing a regulatory (R) subunit dimer bound to two catalytic (C) subunits and is involved in numerous cell signaling pathways. The C‐subunit is activated allosterically when two cAMP molecules bind sequentially to the cAMP‐binding domains, designated A and B (cAB‐A and cAB‐B, respectively). Each cAMP‐binding domain contains a conserved Arg residue that is critical for high‐affinity cAMP binding. Replacement of this Arg with Lys affects cAMP affinity, the structural integrity of the cAMP‐binding domains, and cAPK activation. To better understand the local and long‐range effects that the Arg‐to‐Lys mutation has on the dynamic properties of the R‐subunit, the amide hydrogen/deuterium exchange in the RIIβ subunit was probed by electrospray mass spectrometry. Mutant proteins containing the Arg‐to‐Lys substitution in either cAMP‐binding domain were deuterated for various times and then, prior to mass spectrometry analysis, subjected to pepsin digestion to localize the deuterium incorporation. Mutation of this Arg in cAB‐A (Arg230) causes an increase in amide hydrogen exchange throughout the mutated domain that is beyond the modest and localized effects of cAMP removal and is indicative of the importance of this Arg in domain organization. Mutation of Arg359 (cAB‐B) leads to increased exchange in the adjacent cAB‐A domain, particularly in the cAB‐A domain C‐helix that lies on top of the cAB‐B domain and is believed to be functionally linked to the cAB‐B domain. This interdomain communication appears to be a unidirectional pathway, as mutation of Arg230 in cAB‐A does not effect dynamics of the cAB‐B domain.