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• W2011105856 abstract "Protein kinase A (PKA) is the main receptor for the universal cAMP second messenger. PKA is a tetramer with two catalytic (C) and two regulatory (R) subunits, each including two tandem cAMP binding domains, i.e. CBD-A and -B. Structural investigations of RIα have revealed that although CBD-A plays a pivotal role in the cAMP-dependent inhibition of C, the main function of CBD-B is to regulate the access of cAMP to site A. To further understand the mechanism underlying the cross-talk between CBD-A and -B, we report here the NMR investigation of a construct of R, RIα-(119–379), which unlike previous fragments characterized by NMR, spans in full both CBDs. Our NMR studies were also extended to two mutants, R209K and the corresponding R333K, which severely reduce the affinity of cAMP for CBD-A and -B, respectively. The comparative NMR analysis of wild-type RIα-(119–379) and of the two domain silencing mutations has led to the definition at an unprecedented level of detail of both intra- and interdomain allosteric networks, revealing several striking differences between the two CBDs. First, the two domains, although homologous in sequence and structure, exhibit remarkably different responses to the R/K mutations especially at the β2-3 allosteric “hot spot.” Second, although the two CBDs are reciprocally coupled at the level of local unfolding of the hinge, the A-to-B and B-to-A pathways are dramatically asymmetrical at the level of global unfolding. Such an asymmetric interdomain cross-talk ensures efficiency and robustness in both the activation and de-activation of PKA. Protein kinase A (PKA) is the main receptor for the universal cAMP second messenger. PKA is a tetramer with two catalytic (C) and two regulatory (R) subunits, each including two tandem cAMP binding domains, i.e. CBD-A and -B. Structural investigations of RIα have revealed that although CBD-A plays a pivotal role in the cAMP-dependent inhibition of C, the main function of CBD-B is to regulate the access of cAMP to site A. To further understand the mechanism underlying the cross-talk between CBD-A and -B, we report here the NMR investigation of a construct of R, RIα-(119–379), which unlike previous fragments characterized by NMR, spans in full both CBDs. Our NMR studies were also extended to two mutants, R209K and the corresponding R333K, which severely reduce the affinity of cAMP for CBD-A and -B, respectively. The comparative NMR analysis of wild-type RIα-(119–379) and of the two domain silencing mutations has led to the definition at an unprecedented level of detail of both intra- and interdomain allosteric networks, revealing several striking differences between the two CBDs. First, the two domains, although homologous in sequence and structure, exhibit remarkably different responses to the R/K mutations especially at the β2-3 allosteric “hot spot.” Second, although the two CBDs are reciprocally coupled at the level of local unfolding of the hinge, the A-to-B and B-to-A pathways are dramatically asymmetrical at the level of global unfolding. Such an asymmetric interdomain cross-talk ensures efficiency and robustness in both the activation and de-activation of PKA. Cyclic adenosine monophosphate (cAMP) is an essential and ubiquitous second messenger that relays extracellular signals and translates them into tightly regulated intracellular responses (1Berman H.M. Ten Eyck L.F. Goodsell D.S. Haste N.M. Kornev A. Taylor S.S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 45-50Crossref PubMed Scopus (168) Google Scholar). In eukaryotes, one of the main cAMP receptors is protein kinase A (PKA), 4The abbreviations used are: PKAcAMP-dependent protein kinase ABBRbase binding regionCBDcAMP binding domainH/Dhydrogen/deuterium exchangeHSQCheteronuclear single-quantum coherencePBCphosphate binding cassettePFprotection factorRregulatory subunit of PKACcatalytic subunit of PKARIαisoform Iα of the R subunit of PKAR2dimeric regulatory subunit of PKATROSYtransverse relaxation optimized spectroscopyNOESYtwo-dimensional nuclear Overhauser effect spectroscopy. which catalyzes Ser/Thr phosphorylation in downstream protein substrates that in turn control a wide range of cellular processes including metabolism and cell death (2Shabb J.B. Chem. Rev. 2001; 101: 2381-2411Crossref PubMed Scopus (285) Google Scholar). PKA exists in two forms: an inactive tetrameric holoenzyme and an active free catalytic subunit (C). In the holoenzyme, two C molecules form a complex with a dimeric regulatory subunit (R2). Upon binding to cAMP synthesized in response to external signals, each R-subunit undergoes a conformational change, releasing the C-subunit in its active state (1Berman H.M. Ten Eyck L.F. Goodsell D.S. Haste N.M. Kornev A. Taylor S.S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 45-50Crossref PubMed Scopus (168) Google Scholar). The cAMP-dependent activation of PKA is reversible, and when phosphodiesterases deplete cAMP levels, the R-subunits revert to their dormant C-bound state in which the kinase function is inhibited (3Dodge K.L. Khouangsathiene S. Kapiloff M.S. Mouton R. Hill E.V. Houslay M.D. Langeberg L.K. Scott J.D. EMBO J. 2001; 20: 1921-1930Crossref PubMed Scopus (402) Google Scholar). The reversibility in the activation of PKA represents a critical aspect of the regulation of its function, as it is required for the cell to revert to resting conditions after cAMP levels have been temporarily raised by transient external stimuli (4Kopperud R. Krakstad C. Selheim F. Døskeland S.O. FEBS Lett. 2003; 546: 121-126Crossref PubMed Scopus (173) Google Scholar). cAMP-dependent protein kinase A base binding region cAMP binding domain hydrogen/deuterium exchange heteronuclear single-quantum coherence phosphate binding cassette protection factor regulatory subunit of PKA catalytic subunit of PKA isoform Iα of the R subunit of PKA dimeric regulatory subunit of PKA transverse relaxation optimized spectroscopy two-dimensional nuclear Overhauser effect spectroscopy. The R-subunit of PKA is a multidomain and highly dynamic protein. All known R-subunit isoforms share a common organization with an N-terminal dimerization/docking domain followed by a flexible linker region that includes the autoinhibitory segment and is C-terminally connected to two tandem CBDs (CBD-A and B, Fig. 1a) (5Taylor S.S. Kim C. Vigil D. Haste N.M. Yang J. Wu J. Anand G.S. Biochim. Biophys. Acta. 2005; 1754: 25-37Crossref PubMed Scopus (198) Google Scholar). Whereas the dimerization/docking domain mediates the subcellular localization of PKA via protein kinase A-anchoring proteins (6Scott J.D. Biochem. Soc. Trans. 2006; 34: 465-467Crossref PubMed Scopus (16) Google Scholar), the interactions with the C-subunit and with cAMP involve the autoinhibitory region and CBDs A and B (7Kim C. Cheng C.Y. Saldanha S.A. Taylor S.S. Cell. 2007; 130: 1032-1043Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Both CBDs share a typical α/β-subdomain architecture, with the β-barrels embedding the two cAMP binding sites and with the non-contiguous α-helical subdomains joined at the interface between the two domains (Fig. 1e) (8Su Y. Dostmann W.R. Herberg F.W. Durick K. Xuong N.H. Ten Eyck L. Taylor S.S. Varughese K.I. Science. 1995; 269: 807-813Crossref PubMed Scopus (346) Google Scholar). The structures of the R-subunit bound to either cAMP or the C-subunit (7Kim C. Cheng C.Y. Saldanha S.A. Taylor S.S. Cell. 2007; 130: 1032-1043Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 8Su Y. Dostmann W.R. Herberg F.W. Durick K. Xuong N.H. Ten Eyck L. Taylor S.S. Varughese K.I. Science. 1995; 269: 807-813Crossref PubMed Scopus (346) Google Scholar, 9Wu J. Jones J.M. Nguyen-Huu X. Ten Eyck L.F. Taylor S.S. Biochemistry. 2004; 43: 6620-6629Crossref PubMed Scopus (68) Google Scholar, 10Wu J. Brown S. Xuong N.H. Taylor S.S. Structure. 2004; 12: 1057-1065Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 11Kim C. Xuong N.H. Taylor S.S. Science. 2005; 307: 690-696Crossref PubMed Scopus (289) Google Scholar) have revealed an ordered sequential mechanism of PKA activation whereby cAMP binds first to CBD-B, making site A accessible to a second molecule of cAMP, which in turn causes the release of the active C-subunit. In other words CBD-B functions as a gatekeeper for CBD-A, whereas the latter acts as the central controlling unit of the PKA system and provides the primary interfaces with the C-subunit (7Kim C. Cheng C.Y. Saldanha S.A. Taylor S.S. Cell. 2007; 130: 1032-1043Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 8Su Y. Dostmann W.R. Herberg F.W. Durick K. Xuong N.H. Ten Eyck L. Taylor S.S. Varughese K.I. Science. 1995; 269: 807-813Crossref PubMed Scopus (346) Google Scholar, 9Wu J. Jones J.M. Nguyen-Huu X. Ten Eyck L.F. Taylor S.S. Biochemistry. 2004; 43: 6620-6629Crossref PubMed Scopus (68) Google Scholar, 10Wu J. Brown S. Xuong N.H. Taylor S.S. Structure. 2004; 12: 1057-1065Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 11Kim C. Xuong N.H. Taylor S.S. Science. 2005; 307: 690-696Crossref PubMed Scopus (289) Google Scholar). The structures of the R-subunit in its active and inhibited states have also demonstrated that although CBD-A and CBD-B play clearly distinct roles in the activation of PKA, they both share similar allosteric features (Fig. 1c). Specifically, in both domains cAMP docks into a conserved phosphate binding cassette (PBC) and a base binding region (BBR), causing a hinge rotation of the helical region C-terminal to the β-barrel (“hinge” or αB/C-helices) and a closure of the hinge region over the adenine base (Fig. 1d). In all CBD domains there is a conserved element called the “lid” that interacts with the adenine ring, but the origin of this lid varies in the different CBD domains (1Berman H.M. Ten Eyck L.F. Goodsell D.S. Haste N.M. Kornev A. Taylor S.S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 45-50Crossref PubMed Scopus (168) Google Scholar). In the CBD-B of RIα, the lid comes from the αC-helix of the same CBD (i.e.“cis-domain lid”), whereas for CBD-A of RIα the lid is provided by the B domain (i.e.“trans-domain lid”) and specifically by the N3A motif (N3A) of CBD-B (Fig. 1c). The N3A constitutes the fourth conserved motif in both CBDs, which is the helical element that lies N-terminal to the β-barrel and is composed of an N-terminal helix, a 310 loop, and the following αA helix (Fig. 1, c–e). The cAMP-dependent rearrangement of the helices C-terminal to the β-subdomain also leads to a concurrent partial re-positioning of the N3A motif that in the case of CDB-B is fused to the αC-helix of CBD-A but is still functionally a part of CBD-B (Fig. 1, c and d) (7Kim C. Cheng C.Y. Saldanha S.A. Taylor S.S. Cell. 2007; 130: 1032-1043Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). This disposition of two CBDs in tandem and fused directly to each other is a distinctive feature of the R-subunits of PKA. However, structural investigations of other CBDs have shown that the four elements outlined above (i.e. PBC, BBR, hinge, N3A, Fig. 1, c and d) are universally conserved among eukaryotic domains that function as cAMP sensors (12Rehmann H. Prakash B. Wolf E. Rueppel A. de Rooij J. Bos J.L. Wittinghofer A. Nat. Struct. Biol. 2003; 10: 26-32Crossref PubMed Scopus (169) Google Scholar, 13Kornev A.P. Taylor S.S. Ten Eyck L.F. PLoS Comput. Biol. 2008; 4: 1-9Crossref Scopus (48) Google Scholar). The crystallographic studies on the structure of CBDs have recently been complemented by NMR analyses of constructs including a single CBD of either PKA or EPAC (exchange protein directly activated by cAMP), which have unveiled an additional conserved allosteric “hot spot” at the loop between strands β2 and β3 (14Das R. Melacini G. J. Biol. Chem. 2007; 282: 581-593Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 15Das R. Esposito V. Abu-Abed M. Anand G.S. Taylor S.S. Melacini G. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 93-98Crossref PubMed Scopus (109) Google Scholar, 16Abu-Abed M. Das R. Wang L. Melacini G. Proteins. 2007; 69: 112-124Crossref PubMed Scopus (36) Google Scholar, 17Mazhab-Jafari M.T. Das R. Fotheringham S.A. SilDas S. Chowdhury S. Melacini G. J. Am. Chem. Soc. 2007; 129: 14482-14492Crossref PubMed Scopus (44) Google Scholar, 18Das R. Mazhab-Jafari M.T. Chowdhury S. SilDas S. Selvaratnam R. Melacini G. J. Biol. Chem. 2008; 283: 19691-19703Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Furthermore, the solution studies have also revealed that cAMP binding controls not only the local environment of the allosteric sites as sensed by chemical shift changes but also the hierarchical distribution of excited states that defines the free energy landscape of these domains, as probed through H/D exchange monitored at residue resolution by NMR. Specifically, it is now clear that cAMP modulates not only global unfolding of the CBDs but also local unfolding at α-subdomain sites well removed from the PBC where cAMP docks (15Das R. Esposito V. Abu-Abed M. Anand G.S. Taylor S.S. Melacini G. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 93-98Crossref PubMed Scopus (109) Google Scholar, 17Mazhab-Jafari M.T. Das R. Fotheringham S.A. SilDas S. Chowdhury S. Melacini G. J. Am. Chem. Soc. 2007; 129: 14482-14492Crossref PubMed Scopus (44) Google Scholar). Whereas the definition of these long range perturbations represents a first step toward mapping intra-CBD signaling pathways and provides initial clues on how cAMP reshapes the free energy landscape of CBDs, the picture emerging from the NMR analysis of single CBD constructs is still somewhat limited by the lack of the adjacent domain. In particular, because of the absence of the “gatekeeper” CBD-B, the previous RIα construct used for NMR purposes (i.e. RIα-(119–244)) did not contain a full integral lid region (15Das R. Esposito V. Abu-Abed M. Anand G.S. Taylor S.S. Melacini G. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 93-98Crossref PubMed Scopus (109) Google Scholar, 18Das R. Mazhab-Jafari M.T. Chowdhury S. SilDas S. Selvaratnam R. Melacini G. J. Biol. Chem. 2008; 283: 19691-19703Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 19Das R. Chowdhury S. Mazhab-Jafari M.T. Sildas S. Selvaratnam R. Melacini G. J. Biol. Chem. 2009; 284: 23682-23696Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and did not provide any insight into the cross-talk between the two cAMP binding domains. Here, we report the investigation by NMR of a two-domain construct of RIα, i.e. RIα-(119–379) (Fig. 1a). This construct not only includes the whole CBD-B, but it provides also an unprecedented representation of eukaryotic CBDs in general because, unlike previous NMR-based investigations (14Das R. Melacini G. J. Biol. Chem. 2007; 282: 581-593Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 15Das R. Esposito V. Abu-Abed M. Anand G.S. Taylor S.S. Melacini G. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 93-98Crossref PubMed Scopus (109) Google Scholar, 17Mazhab-Jafari M.T. Das R. Fotheringham S.A. SilDas S. Chowdhury S. Melacini G. J. Am. Chem. Soc. 2007; 129: 14482-14492Crossref PubMed Scopus (44) Google Scholar, 18Das R. Mazhab-Jafari M.T. Chowdhury S. SilDas S. Selvaratnam R. Melacini G. J. Biol. Chem. 2008; 283: 19691-19703Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 19Das R. Chowdhury S. Mazhab-Jafari M.T. Sildas S. Selvaratnam R. Melacini G. J. Biol. Chem. 2009; 284: 23682-23696Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), it contains the entire lid regions of both CBDs (Fig. 1, a and c). Furthermore, as a first step toward mapping the inter-CBD cross-talk, the NMR investigation of the wild-type two-domain construct RIα-(119–379) was also extended to two single mutants that target two highly conserved Arg residues in the PBCs, i.e. R209K in PBC-A and R333K in PBC-B (Fig. 1, c and d). The guanidiniums of Arg-209 and Arg-333 form salt bridges with the phosphate of cAMP and are an integral part of PBC-A and -B, respectively (8Su Y. Dostmann W.R. Herberg F.W. Durick K. Xuong N.H. Ten Eyck L. Taylor S.S. Varughese K.I. Science. 1995; 269: 807-813Crossref PubMed Scopus (346) Google Scholar, 13Kornev A.P. Taylor S.S. Ten Eyck L.F. PLoS Comput. Biol. 2008; 4: 1-9Crossref Scopus (48) Google Scholar) (Fig. 2, a–e). Indeed, these arginines are highly conserved not only in the PBCs of RIα (Fig. 1, b–d) but also in the majority of other known CBDs (20Kannan N. Wu J. Anand G.S. Yooseph S. Neuwald A.F. Venter J.C. Taylor S.S. Genome Biology. 2007; 8: R264.1-R264.13Crossref Scopus (79) Google Scholar). The mutation of Arg-209 to Lys has been shown to reduce the affinity for cAMP at site A by about 3 orders of magnitude (i.e. KD from 30 nm to the ≥μm range) as well as at the non-mutated site B by about 1 order of magnitude (i.e. KD from 15 to 130 nm) (21Herberg F.W. Taylor S.S. Dostmann W.R.G. Biochemistry. 1996; 35: 2934-2942Crossref PubMed Scopus (107) Google Scholar). A similar reciprocal “domain silencing” effect has been reported also for R333K (21Herberg F.W. Taylor S.S. Dostmann W.R.G. Biochemistry. 1996; 35: 2934-2942Crossref PubMed Scopus (107) Google Scholar). The ability of the R209K and R333K mutations to decrease the cAMP affinity for both PBCs indicates that these point substitutions cause long range perturbations and represent useful tools to start dissecting the pathways that mediate the interdomain communication in RIα. Furthermore, despite the increased dissociation constants for cAMP, the inhibition of the C-subunit by either R209K or R333K RIα is still cAMP-dependent (21Herberg F.W. Taylor S.S. Dostmann W.R.G. Biochemistry. 1996; 35: 2934-2942Crossref PubMed Scopus (107) Google Scholar), indicating that both RIα mutants are functionally active. Overall, the three state (i.e. wild-type, R209K, and R333K RIα-(119–379)) comparative NMR analysis of chemical shifts and solvent protection factors for wild-type, R209K, and R333K RIα-(119–379) (Fig. 1b) has revealed several striking differences between domains A and B. Such differences pertain to intradomain as well as to interdomain cAMP signal propagation and provide new insight not only on the deactivation but also on the activation mechanism of PKA. The two-domain construct RIα-(119–379) of PKA was subcloned into the pRSET-B expression system (Invitrogen), and mutants were prepared by site-directed mutagenesis. The protein was transformed into Escherichia coli BL21(DE3) cells and overexpressed in M9 minimal medium. For 1H,15N HSQC and H/D exchange experiments, a single-labeled 15N sample with >98% 15N incorporation was used. For triple resonance experiments, triple-labeled 13C,2H,15N samples were prepared. Both 15N and 13C were incorporated at levels >98%, whereas 2H was present at levels of 50–98% at the Hα position of the protein after back exchange with purification buffers, depending on what percentage of 2H2O was used in the growth process. Selectively labeled samples of 15N-labeled Gly, Ala, Val, and Leu were also prepared for the wild type to assist in assignment. The Gly-, Ala-, and Val-selective samples were prepared by growing the cells in LB medium and adding 0.5 g of the 15N-labeled amino acid, predissolved in H2O, into the culture 1 h before induction by isopropyl 1-thio-β-d-galactopyranoside, as previously described (22Englander J. Cohen L. Arshava B. Estephan R. Becker J.M. Naider F. Biopolymers. 2006; 84: 508-518Crossref PubMed Scopus (12) Google Scholar). The [15N]Leu sample was prepared in M9 medium with the NH4Cl replaced by 100 mg each of the other unlabeled 19 amino acids and 200 mg of [15N]Leu. The cells were grown and induced under the same conditions as the other isotopically enriched M9 culture. The remaining portion of the sample preparations followed, to a large extent, previously published protocols and the details are available as supplemental material. During the last purification step, i.e. gel filtration, the protein was separated from the cAMP used during the prior elution steps, and the final product was a protein free of excess cAMP. The NMR samples were prepared by concentrating the protein to 0.5 mm, as determined by the Bradford assay (23Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217450) Google Scholar), and adding 2H2O to a final concentration of 5% (v/v). To ensure the long term stability of the NMR samples, 0.4 mm 4-(2-aminoethyl)benzenesulfonyl fluoride and 1 mm Tris(2-carboxyethyl)phosphine were also added. All NMR spectra were recorded at a temperature of 306 K on a Bruker AV 700 spectrometer equipped with a TCI cryo-probe. In all experiments, the 1H and 15N carrier frequencies were set at the water resonance and at the center of the amide region, respectively. The 13C carrier frequency varied depending on the experiment. All one-dimensional data sets were processed and analyzed using Xwinnmr (Bruker Inc.), and all multidimensional data sets were processed using NMRPipe (24Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11626) Google Scholar). Unless otherwise specified, a phase-shifted-squared sine bell window function was employed for both dimensions before zero filling. Processed spectra were analyzed using NMRDraw and Sparky 3.114 (25Goddard T.D. Kneller D.G. SPARKY 3. University of California, San Francisco, CA2006Google Scholar). All of the two-dimensional HSQC spectra acquired were sensitivity- and gradient-enhanced and included water flip-back pulses. After 128 dummy scans, 8 scans were accumulated per t1 point using an inter scan delay of 1 s. Unless otherwise specified, the 15N dimension was digitized with 128 complex points for a spectral width of 31.8 ppm, and the 1H dimension was digitized with 1024 complex points for a spectral width of 15.939 ppm. All TROSY triple resonance three-dimensional experiments (i.e. HNCA, HN(CO)CA, HN(CO)CACB, HNCACB, HN(CA)CO, and HNCO) as well as the HSQC-NOESY-HSQC spectra were acquired on triple-labeled (>98% 15N, >98% 13C, and 50% 2H isotopic enrichment) protein samples with a concentration of 0.5 mm and with an inter-scan delay ranging from 1 to 2 s, depending on the sensitivity of the experiment. Chemical shift predictions were generated with ShiftX (26Neal S. Nip A.M. Zhang H. Wishart D.S. J. Biomol. NMR. 2003; 26: 215-240Crossref PubMed Scopus (466) Google Scholar) using the RIα-(91–379) crystal structure (PBD code 1RGS) (8Su Y. Dostmann W.R. Herberg F.W. Durick K. Xuong N.H. Ten Eyck L. Taylor S.S. Varughese K.I. Science. 1995; 269: 807-813Crossref PubMed Scopus (346) Google Scholar) with corrections to account for deuteration. Secondary structure profiles were calculated with the program PECAN using the NH, HN, Cα, Cβ, and C′ backbone assignments (27Eghbalnia H.R. Wang L. Bahrami A. Assadi A. Markley J.L. J. Biomol. NMR. 2005; 32: 71-81Crossref PubMed Scopus (104) Google Scholar). The compounded changes in chemical shift when comparing the wild-type protein with the mutants were calculated with the equation Δδcompound = ((Δδ1H)2 + (Δδ15N/6.5)2)1/2, where Δδ 1H and Δδ 15N are the ppm differences in nitrogen and hydrogen amide chemical shifts, respectively, between the two states being compared. The samples for the H/D exchange experiments were prepared by first concentrating 7.5 mg of protein to ∼2.5 mm (<100 μl final volume). The sample was prepared as previously described (28Das R. Abu-Abed M. Melacini G. J. Am. Chem. Soc. 2006; 128: 8406-8407Crossref PubMed Scopus (36) Google Scholar) by passing the sample through a Sephadex G10 column with a 3-ml bed volume pre-equilibrated with 98% 2H2O-based gel filtration buffer. A series of HSQC experiments was recorded with preset parameters for a sample with similar buffer and sample height to minimize the dead time (∼25 min) of the H/D exchange experiment. The fast initial decay was monitored through a series of 30 HSQC spectra acquired with four scans (∼10 min per HSQC). The remaining slower decay was monitored through a series of HSQC spectra acquired with eight scans. Data were acquired for a total of 20 h. The sample was kept at 33 °C, and HSQC spectra were acquired once per week to provide additional data points for slowly decaying peaks. For each construct (i.e. wild type and two mutants) H/D exchange data were acquired for both samples with no excess cAMP and 2 mm excess cAMP, resulting in a total of six H/D data sets (Fig. 1b). The HSQC cross-peak heights were quantified by NMRPipe as the sum of the intensities in a 3 × 3 grid centered on the peak maximum (29Tollinger M. Skrynnikov N.R. Mulder F.A. Forman-Kay J.D. Kay L.E. J. Am. Chem. Soc. 2001; 123: 11341-11352Crossref PubMed Scopus (419) Google Scholar). The rates of exchange were measured using the Levenberg-Marqardt nonlinear least-square exponential fitting implemented through the Curvefit software as previously described (15Das R. Esposito V. Abu-Abed M. Anand G.S. Taylor S.S. Melacini G. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 93-98Crossref PubMed Scopus (109) Google Scholar). The error on the intensities was based on the standard deviation of the spectral noise. The H/D decays were normalized relative to the intensities in the first HSQC spectrum acquired after exposure to 2H2O. As a first step toward a high resolution characterization of the tandem CBDs of the PKA R-subunit in solution, the backbone assignment of the cAMP-bound state of RIα-(119–379) was obtained using a combination of TROSY-based triple resonance experiments, HSQC-NOESY-HSQC spectra, selectively labeled amino acid samples (i.e. 15N-labeled Gly, Ala, Val, and Leu), and Cα and Cβ chemical shift calculations. Further details are available in the supplemental material. Overall, our integrated assignment strategy led to a total of 1268 assignments for the NH, HN, Cα, Cβ, and C′ resonances (i.e. ∼97% coverage of the backbone resonances) of the wild-type RIα-(119–379) construct in the presence of a 2 mm excess cAMP. Only two residues were not assigned, the first being Asp-119, which is the N-terminal residue and is typically broadened because of its rapid exchange with the solvent, and the second residue is His-138, which is broadened due to ms-μs internal dynamics of the N3A 310 loop to which it belongs. The NMR assignment of RIα-(119–379)·cAMP2 provides the foundation to further investigate this construct in solution at residue and atomic resolution. The ensemble of locally and globally unfolded states accessible to the RIα-(119–379)·cAMP2 complex was probed at high resolution by NMR-monitored H/D exchange experiments, and the resulting residue-specific protection factors (PFs) are reported in Fig. 3a (black symbols). Based on the magnitude of the PF values shown in Fig. 3a, the residues of each CBD can be subdivided into three major classes. Class I consists of residues that are highly protected (PF > 6.5) and rely mainly on transient global unfolding H/D exchange pathways, i.e. the solvent protection of class I residues is not modulated by local unfolding. Class II encompasses residues with intermediate PF values (4.0 < PF ≤ 6.5). These sites are generally protected from the solvent, but unlike class I residues, they do not strictly require global unfolding to be exchanged with deuterium. Class III includes those amides that exchange rapidly (PF ≤ 4.0), i.e. they are either unprotected from the solvent in the ground state structure or are protected but promptly exposed to the solvent through transient local unfolding events. The partitioning across these three broad H/D exchange classes of the key structural and functional elements of RIα-(119–379) provides an initial basic framework to start comparing the two CBDs of the regulatory subunit of PKA Iα. For both CBD-A and CBD-B of the RIα-(119–379)·cAMP2 complex, class I residues are largely confined to the β-subdomain (Fig. 3a), with the main exception of several residues in the αP helices of both PBCs that are slowly exchanging and display class I protection factors as well. Unlike the PBC sites, almost all residues in the other key functional sites (e.g. the BBR, the N3A motif, and the hinge regions) belong to the intermediate or fast exchange classes (i.e. classes II or III) (Fig. 3a). Whereas this observation applies to both domains, the partitioning of specific loci to class II versus class III is highly domain-specific. For instance, a striking difference between the two domains is the degree of protection observed for the N3A motif and the BBR in each CBD. In domain A, the BBR has several class II residues, and the N3A motif is mostly composed of class III residues (Fig. 3a). On the other hand, a reversed H/D exchange pattern is observed for domain B, with its BBR being highly solvent-exposed (i.e. most BBR-B residues belong to class III) and its N3A motif exhibiting some degree of protection (i.e. several N3A-B residues fall in class II) (Fig. 3a). The variability in the N3A and BBR H/D exchange patterns, therefore, defines a marked difference between CBD-A and -B (Fig. 3a). Further differences between the two domains are revealed by investigating how the H/D protection factors depend on cAMP. Because of the instability of the aggregation-prone apo state of RIα-(119–379) (28Das R. Abu-Abed M. Melacini G. J. Am. Chem. Soc. 2006; 128: 8406-8407Crossref PubMed Scopus (36) Google Scholar), we investigated the cAMP dependence of its H/D PF values through a previously described equilibrium perturbation approach (28Das R. Abu-Abed M. Melacini G. J. Am. Chem. Soc. 2006; 128: 8406-8407Crossref PubMed Scopus (36) Google Scholar). This method effectively circumvents experimental challenges caused by aggregation and/or precipitation of the apo RIα-subunit by requiring only the creation of minor populations of apo state in dynamic" @default.
• W2011105856 created "2016-06-24" @default.
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• W2011105856 title "Communication between Tandem cAMP Binding Domains in the Regulatory Subunit of Protein Kinase A-Iα as Revealed by Domain-silencing Mutations" @default.
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