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Realizing the Allosteric Potential of the Tetrameric Protein Kinase a RIΑ Holoenzyme

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Realizing the Allosteric Potential of the Tetrameric Protein Kinase a RIΑ Holoenzyme Structure Article Realizing the Allosteric Potential of the Tetrameric Protein Kinase A RIa Holoenzyme Angela J. Boettcher,1,6 Jian Wu,1,6 Choel Kim,2 Jie Yang,1 Jessica Bruystens,1 Nikki Cheung,1 Juniper K. Pennypacker,1,3 Donald A. Blumenthal,4 Alexandr P. Kornev,3,5 and Susan S. Taylor1,3,5,* 1Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093, USA 2Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030, USA 3Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093, USA 4Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, UT 84112, USA 5Howard Hughes Medical Institute, University of California at San Diego, La Jolla, CA 92093, USA 6These authors contributed equally to this work *Correspondence: [email protected] DOI 10.1016/j.str.2010.12.005 SUMMARY the active site cleft in the C subunit in the inactive holoenzyme but is disordered in the dissociated free R subunits (Li et al., PKA holoenzymes containing two catalytic (C) 2000). The linker, as summarized in Figure 1, can be divided subunits and a regulatory (R) subunit dimer are acti- into three segments, the consensus inhibitor site (P-3 to P+1), vated cooperatively by cAMP. While cooperativity the N-linker that joins the inhibitor site to the D/D domain, and involves the two tandem cAMP binding domains in the C-linker that becomes ordered in the heterodimeric holoen- each R-subunit, additional cooperativity is associ- zyme complex. While much has been learned from the structures ated with the tetramer. Of critical importance is the of the cAMP binding domains of the RIa and RIIb subunits (Diller et al., 2001; Su et al., 1995), and the C subunit has been crystal- flexible linker in R that contains an inhibitor site (IS). lized in many different conformational states (Akamine et al., While the IS becomes ordered in the R:C hetero- 2003; Knighton et al., 1991a; Madhusudan et al., 2002; Zheng dimer, the overall conformation of the tetramer is et al., 1993), it was not until we solved structures of heterodi- mediated largely by the N-Linker that connects meric holoenzyme complexes that we could appreciate for the the D/D domain to the IS. To understand how the first time how the C subunit was actually inhibited by the R N-Linker contributes to assembly of tetrameric holo- subunits, and how the complex was then activated by cAMP enzymes, we engineered a monomeric RIa that (Brown et al., 2009; Kim et al., 2005, 2007; Wu et al., 2007). contains most of the N-Linker, RIa(73-244), and crys- The challenge now is to understand how the full-length tetra- tallized a holoenzyme complex. Part of the N-linker is meric holoenzymes are assembled as these represent the true now ordered by interactions with a symmetry-related physiological state of PKA. This is essential if we are to appre- dimer. This complex of two symmetry-related dimers ciate the full allosteric potential of this key signaling enzyme. An additional goal is to determine how the N-linker contributes forms a tetramer that reveals novel mechanisms for to the assembly of the tetrameric holoenzyme. allosteric regulation and has many features associ- Mammalian genomes typically code for four separate and ated with full-length holoenzyme. A model of the functionally nonredundant R subunit isoforms (RIa,RIb, RIIa, tetrameric holoenzyme, based on this structure, is and RIIb). Although it has been difficult to crystallize full-length consistent with previous small angle X-ray and R subunits, presumably because the linkers are flexible, struc- neutron scattering data, and is validated with new tures of the CNB domains and the D/D domains have been SAXS data and with an RIa mutation localized to solved (Diller et al., 2001; Kinderman et al., 2006; Sarma et al., a novel interface unique to the tetramer. 2010; Su et al., 1995). To understand how the tetrameric holoen- zymes are spatially organized, we initially used small angle X-ray and neutron scattering (SAXS/SANS). This revealed surprisingly INTRODUCTION that the shapes of the various R subunits and holoenzymes are quite different (Heller et al., 2004; Vigil et al., 2004b, 2006). The The regulatory (R) subunits of cAMP-dependent protein kinase RIa subunit and its corresponding holoenzyme are Y-shaped (PKA) are modular and highly dynamic. In the absence of while the RII subunits are more rod-like and dumb-bell shaped. cAMP, the R subunit dimer is attached to two PKA catalytic (C) The RIIa holoenzyme remains extended while the RIIb holoen- subunits and maintains the enzyme in an inactive tetramer. zyme compacts into a globular protein (Vigil et al., 2006). These Each R subunit contains a dimerization/docking (D/D) domain different architectures are due primarily to differences in the at the N terminus that is joined by a flexible linker to two cyclic N-linker regions (Figure 1) and suggest that the allosteric nucleotide binding domains at the C terminus (CNB-A and signaling in each holoenzyme will be distinct. The inhibitor site CNB-B). The linker contains an inhibitor site (IS) that docks to and the C-linker become organized upon association with the Structure 19, 265–276, February 9, 2011 ª2011 Elsevier Ltd All rights reserved 265 Structure Allostery of the Tetrameric PKA RIa Holoenzyme Figure 1. Extended N-Linker in RIa(73-244):C holoenzyme Complex (A) Domain organization of different RIa constructs. Sequence of the linker between dimerization domain (D/D) and cAMP binding domain A (CNB-A) is shown. Constructs that were crystallized in holoenzyme complexes are marked with asterisks. (B) Sequence alignment of the linkers in different R subunits. Possible sites of phosphorylation are shown as red dots. (C) RIa(73-244):C structure overlaid with the previously solved RIa(91-244):C (dark gray). Catalytic subunit is colored white (N-lobe) and tan (C-lobe). CNB-A is colored teal, the linker is colored red. N-terminal Ca atoms of the RIa constructs are shown as spheres. C subunit; however, the role and ordering of the N-linker, which genesis coupled with fluorescence anisotropy, we explored contains many putative binding motifs, remain unknown. the flexibility of residues that lie in different regions of RIa (Li It is known that cAMP activation of tetrameric holoenzyme is et al., 2000). We found that residues that flank the D/D domain, a cooperative process, that is reflected by the increased Hill Thr6 and Leu66, were quite flexible independent of whether coefficients (Herberg et al., 1994). Other evidence further cAMP or catalytic subunit was bound. Other residues such as supports the importance of the N-linker and the tetrameric Ser99 were flexible in the dissociated R subunit but immobilized configuration of the holoenzyme. Limited proteolysis of free in the holoenzyme. Two residues that lie in the N-linker, Ser75 RIa, for example, cleaves at Arg92 just before the inhibitor and Ser81, were flexible in the free RIa subunit but became site, whereas trypsin cleaves at Arg72 in the holoenzyme sug- much less mobile in the holoenzyme suggesting that they might gesting that at least part of the N-linker is protected in the holo- be interacting with another part of the protein and could enzyme (Cheng et al., 2001). In addition, using cysteine muta- possibly be contributing to forming the tetramer. A final piece 266 Structure 19, 265–276, February 9, 2011 ª2011 Elsevier Ltd All rights reserved Structure Allostery of the Tetrameric PKA RIa Holoenzyme of evidence that supported the importance of the N-linker for RESULTS holoenzyme formation came from mutagenesis of two residues in the C subunit that are predicted to lie on the surface where Overall Structure of RIa(73-244):C Complex the N-linker would bind (Cheng et al., 2001). Mutation of To understand how the RIa N-linker region contributes to the Arg133 selectively interfered with binding of RIIa whereas re- holoenzyme structure, we extended the N terminus of the placing Asp328 with Ala interfered with binding of RIa. These previous construct of RIa(91-244) by 18 residues. We also engi- results led us to predict that the orientation of the N-linker neered the extended form of RIa(91-379). Both proteins were was not only important for formation of the tetrameric holoen- very stable, and both readily formed a high-affinity complex zyme but also that the orientation of the N-linker would be with the C subunit that could be isolated on a gel filtration different in RI and RII (Cheng et al., 2001). column; however, only the RIa(73-244) holoenzyme complex The previous RIa monomers that were crystallized as gave high-resolution crystals. The crystal structure of RIa(73- a complex with the catalytic subunit began with the inhibitor 244) in complex with the C subunit, AMP-PNP and two Mn2+ site and contained no ordered residues from the N-linker. To ions was solved at 3.3 A˚ . Although the overall structure of the further explore the potential role of N-linker residues and, in complex is very similar to that of the previously solved RIa(91- particular, to determine whether they contribute to formation of 244):C complex (Kim et al., 2005)(Figure 1), the crystal packing the tetramer, we engineered two longer constructs of RIa that was different from other previous structures of free or complexed contained 18 additional residues at the N terminus compared R and C subunits. The asymmetric unit contains a single hetero- with the constructs that had been crystallized previously as holo- dimer comprising one R subunit and one C subunit.
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