Allosteric Communication between cAMP Binding Sites in the RI Subunit of Protein Kinase A Revealed by NMR*

The activation of protein kinase A involves the synergistic binding of cAMP to two cAMP binding sites on the inhibitory R subunit, causing release of the C subunit, which subsequently can carry out catalysis. We used NMR to structurally characterize in solution the RIα-(98–381) subunit, a construct comprising both cyclic nucleotide binding (CNB) domains, in the presence and absence of cAMP, and map the effects of cAMP binding at single residue resolution. Several conformationally disordered regions in free RIα become structured upon cAMP binding, including the interdomain αC:A and αC′:A helices that connect CNB domains A and B and are primary recognition sites for the C subunit. NMR titration experiments with cAMP, B site-selective 2-Cl-8-hexylamino-cAMP, and A site-selective N6-monobutyryl-cAMP revealed that cyclic nucleotide binding to either the B or A site affected the interdomain helices. The NMR resonances of this interdomain region exhibited chemical shift changes upon ligand binding to a single site, either site B or A, with additional changes occurring upon binding to both sites. Such distinct, stepwise conformational changes in this region reflect the synergistic interplay between the two sites and may underlie the positive cooperativity of cAMP activation of the kinase. Furthermore, nucleotide binding to the A site also affected residues within the B domain. The present NMR study provides the first structural evidence of unidirectional allosteric communication between the sites. Trp262, which lines the CNB A site but resides in the sequence of domain B, is an important structural determinant for intersite communication.

absence of cAMP, the enzyme is an inactive, tetrameric holoenzyme complex, composed of two regulatory (R) and two catalytic (C) subunits (R 2 C 2 ). The catalytic site of the C subunit is occluded by a short inhibitory sequence in the R subunit (residues 94 -99 in bovine RI␣) that connects the N-terminal dimerization domain to the two cyclic nucleotide binding (CNB) domains. Multiple contacts exist between the CNB domains and the C subunit. The enzyme is allosterically activated by cAMP (3,4), whose binding to the R subunits causes dissociation of the C subunits from the holoenzyme complex, thereby rendering C catalytically active (5). Two CNB domains (A and B) are present in all four isoforms (RI␣, RI␤, RII␣, and RII␤) of mammalian PKA, and both need to be occupied by cAMP to achieve PKA dissociation under physiologically relevant conditions (for reviews, see Refs. 2 and 6). Newly transcribed R subunit (apoR) in the cell can complex with either cAMP or the C subunit of PKA. Binding of cAMP leads to a dramatically decreased affinity for the C subunit, whereas binding of the C subunit lowers the cAMP affinity by about 3 orders of magnitude (7), allowing the holoenzyme to respond to fluctuations in physiological cAMP concentrations (8,9).
Comparing the crystal structures of RI␣-(103-376) (numbering for bovine RI␣) with cAMP bound in both the A and B domains (10) with the structure of RI␣-(91-379) (R333K) complexed with the C subunit (4) revealed pronounced differences in the two CNB domains, in particular with respect to their relative positioning (Fig. 1). However, little is known about the structure of the ligand-free (apo) state of the R subunits. A truncated RI␣ (residues 119 -244), comprising most of the A domain, has been investigated by NMR (11)(12)(13). Note, however, that this truncated form lacks not only the B domain but also the C-terminal end of the A domain, in particular the ␣C:A and ␣CЈ:A helices. These helices are at the junction between domains A and B and are important elements for interaction with the C subunit (4). Moreover, this region is conserved in all CAP-related eukaryotic cAMP binding proteins, as recently pointed out by Taylor et al. (14). For structural assessment of both the apo-state and the cAMP-bound state of RI␣, and in order to reveal any allosteric communication between the domains, it is therefore necessary to investigate a protein with two intact CNB domains.
Here, we present an NMR study of RI␣-(98 -381), a construct that includes both CNB domains and the N-terminal region between the inhibitory sequence and the A domain, in the absence (apo-form) and presence of cAMP. We show that cAMP binding induces conformational stabilization of residues close to the binding sites as well as of regions that contact the C subunit of PKA. The conformational change of cAMP-ligated RI␣ in the area that interacts with the C subunit may explain why C binds to apoRI␣ with several orders of magnitude higher affinity than to cAMP-saturated RI␣ (15,16). Titration of apoRI␣ with site-selective cAMP analogs permitted us to map the effects caused by single site occupancy at the amino acid level. Conformational changes observed for the ␣C:A helix, as well as the regions immediately preceding and following this helix, provided direct evidence for communication between the two binding sites and for additional structural effects that ensue only when both sites are occupied by cyclic nucleotide.

Mutagenesis, Expression, and Purification of RI␣ Proteins-
Recombinant human wild-type full-length RI␣ (wt-RI␣) and the C subunit of bovine PKA were obtained as described previously (17,18). The DNA sequences corresponding to RI␣-(92-381) and RI␣-(98 -381) were amplified by PCR from genomic DNA of human RI␣ (18) and cloned into pGEX-2T vectors (Amersham Biosciences). RI␣-(98 -381) was expressed as a Factor Xa-cleavable, N-terminal maltose-binding proteintagged fusion protein. Escherichia coli BL21 (DE3) codon plus cells were used for protein production, induced at A 600 0.6 -0.7 with 1 mM isopropyl 1-thio-␤-D-galactopyranoside, and grown for an additional 7-10 h at 28°C. Cells were harvested by centrifugation, resuspended in 20 mM sodium phosphate buffer (pH 7.3) containing 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 5 mM benzamidine, 1 mM DTT, 1 mM phenylmethylsul-fonyl fluoride, and 2 g/l leupeptin and lyzed by passage through a French press. The fusion protein was purified by affinity chromatography on amylose resin (New England Bio-Labs), using 10 mM maltose in 20 mM Hepes buffer (pH 7.5) containing 0.2 M NaCl and 1 mM DTT for elution. Cleavage was carried out overnight at 4°C with a Factor Xa/protein ratio of 1:240 (w/w). The cleaved protein was fractionated by gel filtration chromatography on a HiLoad Superdex 200 HR (1.6 ϫ 60-cm) column (Amersham Biosciences) in 20 mM Hepes buffer (pH 7.0) containing 0.2 M NaCl and 1 mM DTT, followed by a final purification step using a prepacked RESOURCE TM Q column (Amersham Biosciences) and a linear NaCl gradient (0 -0.5 M) in 20 mM Hepes buffer (pH 6.35) at a flow rate of 0.5 ml/min.
Removal of cAMP from the RI␣ Subunits-Purified recombinant RI␣ subunits (full-length and truncated forms) contain bound cAMP to various degrees, and fully saturated R subunits (R-cAMP 2 ) can be easily prepared by adding cAMP (60 mM) prior to the gel filtration step. In order to prepare cAMP-free proteins (apo-forms), an unfolding/refolding procedure was devised and optimized. Briefly, RI␣-(98 -381) and RI␣-(92-381) proteins were incubated in 5 M urea in 10 mM potassium phosphate buffer (pH 7.4) containing 50 mM KCl, 1 mM EGTA for 5 h at 4°C for partial unfolding. Complete removal of cAMP from the proteins was achieved by passage over a prepacked PD-10 column (GE Healthcare) and a couple of buffer exchanges using Amicon concentrators (Millipore, Billerica, MA). Refolding of the proteins was carried out by extensive dialysis against the same buffer without urea. Additional purification involved gel filtration on a HiLoad Superdex 200 HR (1.6 ϫ 60-cm) column (Amersham Biosciences) for removal of soluble aggregates due to incomplete refolding. The absence of cAMP in the protein was verified by measuring the cAMP occupancy with [ 3 H]cAMP (Amersham Biosciences) as described previously (19).
Binding of cAMP Analogs to CNB Sites A and B of RI␣-(98 -381) and Full-length RI␣-cAMP and cAMP analogs of 98% or better purity were obtained from Dr. G.-G. Genieser (BioLog Life Science Institute, Bremen, Germany). The relative affinity of each cAMP analog for the RI␣ subunit was estimated by competition experiments using protein-bound [ 3 H]cAMP by the validated ammonium sulfate precipitation method (17,20).  ation of whether labeled cAMP is bound to site A or site B (19). Kinase activity was measured by the phosphotransferase assay. The phosphorylation of heptapeptide substrate by PKA was determined essentially as described previously (21). The incubations were at 25°C for 18 h with a 3.7 pM concentration of the catalytic subunit of PKA and various concentrations of the inhibitory full-length RI␣ and the noninhibitory RI␣-(98 -381), in assay buffer (18 mM Hepes, pH 7.2, containing 0.1 mM heptapeptide substrate (Leu-Arg-Arg-Ala-Ser-Leu-Gly; Kemptide), 0.1 mM [␥-32 P]ATP, 2 mM Mg(CH 3 COO) 2 , 1 mM NaHPO 4 , 0.4 mM EGTA, 0.1 mM EDTA, 130 mM KCl, 0.5 mM dithioerythritol, and 0.2 mg/ml each of bovine serum albumin and soybean trypsin inhibitor (0.2 mg/ml)).

Exchange of Bound cAMP at CNB Sites in Domains
Dynamic Light Scattering (DLS)-Solutions of RI␣ proteins in 10 mM potassium phosphate buffer, pH 7.4, 50 mM KCl (buffer A) were prepared, starting at ϳ0.74 mM and diluting 4-, 8-, and 32-fold, in the absence or the presence of cAMP. Samples were passed through a 0.22-m Millipore filter, and 12 l were loaded into a quartz cuvette and placed in the DynaPro LSR (Protein Solutions Inc.) DLS instrument with a temperature-controlled microsampler. 25 data acquisitions were collected for each solution at 22°C. The data were analyzed using Dynamics version 6 (Protein Solutions) software to extract hydrodynamic radii, which are reported as the mean value for the dominant peak.
Preparation of U-2 H/ 13 C/ 15 N-Labeled Samples for NMR Studies-5 ml of starter cultures of E. coli expressing human RI␣-(98 -381) were grown in LB medium up to A 600 ϳ0.4 and then centrifuged at 3000 rpm for 5 min. The cell pellet was added to 150 ml of 2 H/ 13 C/ 15 N-modified M9 medium (prepared with 13 C-perdeuterated glucose and 15 N-ammonium chloride in D 2 O). Cells were grown at 37°C up to A 600 ϳ0.4, harvested by centrifugation, resuspended in 850 ml of 2 H/ 13 C/ 15 N-M9-D 2 O-based medium, and grown again up to A 600 ϳ0.4 at 37°C for induction by 1 mM isopropyl 1-thio-␤-D-galactopyranoside. Growth was continued at 25°C for 14 h. Protein purification was carried out as described above for unlabeled RI␣-(98 -381).

RI␣-(98 -381) Monomer Binds cAMP and cAMP Analogs
Similar to wt-RI␣-Due to its large molecular mass, dimeric full-length wt-RI␣ is not easily amenable to high resolution NMR studies. Monomeric RI␣-(92-381) that lacks the dimerization/docking domain (residues 12-61) has been used extensively to mimic full-length RI␣ (2, 10). Our initial tests with human RI␣-(92-381), however, revealed that the protein selfassociates at concentrations above 0.1 mM, providing little prospect of obtaining high quality NMR data. A similar construct from bovine RI also formed homodimers through the N-terminal linker region (27). Fortunately, the removal of six additional residues rendered the protein, RI␣-(98 -381), monomeric at concentrations of 0.1 mM and above. Size exclusion chromatography of the cAMP-saturated-RI␣-(98 -381) protein revealed a single peak, and DLS measurements at 22°C yielded an apparent hydrodynamic radius of 3.2-3.3 nm (supplemental Table S1), in agreement with dimensions extracted from the x-ray structure of the cAMP-bound RI␣-(113-376) monomer (Protein Data Bank code 1RGS) (10). In contrast, apoRI␣-(98 -381) exhibited slight polydispersity, with an apparent radius of 3.8 nm. The hydrodynamic radius and polydispersity reverted readily back to the values for the cAMP-liganded protein after the nucleotide addition (supplemental Table S1), indicating that aggregation of apoRI is reversible upon cAMP binding.
RI␣-(98 -381) exhibited binding affinities similar to those of wt-RI␣ for cAMP and its analogs (Table 1) and the dissociation rate of bound cAMP is essentially identical between RI␣-(98 -381) and RI␣-(92-381) and full-length RI␣ (supplemental Fig. S1A). The dissociation rate from site B of RI␣ is slowed 7-fold by site A occupancy (3,19). The fact that a similar dissociation rate is found from site B in RI␣-(98 -381) and full-length RI␣ (supplemental Fig. S1A) suggests that the unidirectional effect of site A occupancy on the dissociation from site B (19) is unimpaired in the truncated RI␣-(98 -381). RI␣-(98 -381) also retained the ability to bind to the C subunit, as evidenced by its ability to competitively displace full-length RI␣ from its inhibitory complex with the C subunit (supplemental Fig. S1B). Therefore, we reasoned that RI␣-(98 -381) was a good candi-  Fig. 2A) is well dispersed (10.3-6.2 ppm for 1 H and 132-106 ppm for 15 N frequencies), indicative of a well folded protein. However, apoRI␣-(98 -381) had a tendency to slowly aggregate, resulting in about 50% NMR signal intensity loss after 1-2 weeks at 30°C, in agreement with the aggregation behavior observed by DLS (supplemental Table S1). Similar instability/aggregation behavior has been reported for bovine RI␣-(119 -244) containing only one CNB domain (12). Fortunately, the addition of a trace amount of cAMP (about 15% of the cAMP-binding capacity of the protein) remarkably improved the long term stability of RI␣-(98 -381). NMR samples prepared in this manner were stable without detectable signal loss for more than 1 month, the time required for acquiring the full set of three-dimensional NMR experiments for complete backbone assignments, and exhibited essentially the same TROSY-HSQC spectrum as a completely cAMP-free sample, except for the presence of additional low intensity resonances from the cAMP-bound RI␣-(98 -381) fraction (supplemental Fig. S2). However, even with this sample, it was impossible to obtain complete assignments for the apoprotein because many of the resonances are weak and broad and reside in the severely crowded central spectral region exhibiting significant overlap (dashed box in Fig. 2A). Nevertheless, ϳ70% of all resonances were assigned, using perdeuter-ated protein samples, high sensitivity cryoprobes, and TROSY-type pulse sequences with 2 H decoupling (24).
NMR assignments of the backbone resonances of cAMP-bound RI␣-(98 -381) were carried out on a cAMP-saturated sample. Its 1 H-15 N TROSY-HSQC spectrum exhibited narrower line widths and better spectral dispersion than the spectrum of the apoprotein (Fig. 2B), permitting backbone assignments for 92% of the residues. The superior quality of the cAMP-saturated RI␣-(98 -381) spectrum is clearly apparent in the most crowded region (dashed box) and by the presence of resonances for residues such as Glu 202 , Leu 205 , and Ala 212 in domain A, and Glu 326 , Leu 329 , and Ala 336 in domain B that are absent in the apoprotein spectrum (circled in Fig. 2).
Structural Differences between cAMP-free and cAMP-saturated RI␣-(98 -381)-The secondary structures of apoRI␣-(98 -381) and cAMP-bound RI␣-(98 -381) were deduced from secondary chemical shifts of all assigned RI␣ residues (Fig. 3). As is well established (28), particular patterns of positive and negative secondary chemical shifts are reflective of helical and sheet conformations, respectively, and the observed shifts for cAMP-saturated RI␣-(98 -381) (Fig. 3B) agree with the ␣-helical and ␤-sheet regions in the x-ray structure of the bovine RI␣-(92-379)-cAMP complex (Fig. 1A) (10). This implies that the overall structure in solution is similar to that observed in the crystal. On the other hand, several distinct differences were noted for the apoprotein. First, residues Glu 202 -Tyr 207 , Thr 209 -Ala 212 , and Glu 326 -Ala 336 , which belong to the phosphate-binding cassettes (PBCs), could only be assigned for the cAMP-bound protein (Figs. 2 and 3). Residues 202-207 and 326 -331 constitute the ␣BЈ:A and ␣BЈ:B helices, respectively (Fig. 3), that stabilize cAMP binding by an N-terminal capping mechanism with the phosphate group (10,12). Second, helices ␣C and ␣CЈ at the C terminus of the A domain (␣C:A and ␣CЈ:A) and helices ␣B and ␣C in the B domain (␣B:B and ␣C:B) are also only present in the cAMP-bound protein (Fig. 3, green shaded regions). Note that the ␣C:A and ␣CЈ:A helices connect the A and B CNB domains, whereas helix ␣C:B is located at the C terminus of the entire RI␣ polypeptide chain and caps domain B upon cAMP binding (10,29). Because a large proportion of resonances are broad and overlap in the "random coil" region of the 1 H-15 N TROSY-HSQC spectrum of the apoRI sample but not in the cAMPbound sample (dashed boxes in Fig. 2, A and B), it is likely that the associated RI␣ segments comprising PBC residues and the ␣C helices are unstructured or ill structured in the apo-state, exhibiting conformational averaging on an intermediate chemical shift (micro-to millisecond) time scale. This is in agreement with previous NMR studies of the apo form of the isolated A domain that reported a destabilization of the PBC region, evidenced by increased solvent exposure and dynamics upon the removal of cAMP (11,12). Interestingly, all regions associated with high conformational flexibility in the apo-form are localized within the primary recognition area for the C subunit (4).
In addition to identifying the areas that are structured upon cAMP binding, the extent of any structural changes caused by ligand binding can be derived by analyzing the combined 1 H, 15 N chemical shift changes for all assignable 1 H-15 N TROSY-HSQC resonances of apoRI and cAMP-bound RI (ϳ60% of all residues; Fig. 4A). Binding of cAMP induced noticeable (Ͼ0.05 ppm) chemical shift changes for most resonances, with an average value of 0.15 ppm. In particular, resonances from residues in the loop between ␤2 and ␤3 in domain A showed substantial changes (Ͼ0.31; average change (0.15 ppm) plus one S.D. (0.16 ppm)) (Fig. 4A). This loop has recently been recognized as an important conserved area for the allosteric modulation of the CNB domains (14). It contains negatively charged residues (e.g. Asp 172 in domain A) that position the conserved Arg in the PBC (14,30). Other regions associated with substantial chemical shift changes are strands ␤6:A and ␤7:A and helices ␣B:A and ␣A:B. Note that these strands and helices, respectively, flank the PBC in domain A and the ␣C:A and ␣CЈ:A helices (Fig. 4A), whose regions are not assignable (visible) in the 1 H-15 N TROSY-HSQC spectrum of the apo-state protein (see above and Fig. 3A). Moreover, Trp 262 , the residue for which cAMP binding induced the largest chemical shift change (1.2 ppm), is located in the ␣A:B helix (Fig. 4A). Finally, a stretch of residues (positions 103-118) in the N-terminal region of RI␣-(98 -381) that in full-length RI resides between the inhibitory sequence and the A CNB domain also exhibited noticeable (up to 0.3 ppm) chemical shift differences between the cAMP-free and cAMPsaturated protein. All chemical shift changes associated with cAMP binding are mapped onto the structure of RI␣ complexed with either cAMP (Fig. 4B) or the C subunit (Fig. 4C), with the position of Trp 262 highlighted.
Binding of Site-selective Cyclic Nucleotide Analogs-The 2-Cl-8-AH-cAMP and N 6 -MB-cAMP analogs show preferential binding for the B-and A-sites of CNB, respectively, of RI␣-(98 -381) ( Table 1; see also Ref. 31). These analogs were used to study the effects of single site binding to RI␣-(98 -381). We first ascertained that the bound RI␣-(98 -381) chemical shifts (red resonances in the Tyr 185 (Y185) panels) were similar for the protein fully saturated with cAMP (Fig. 5A), with the B analog (Fig. 5C), or with the A analog (Fig. 5E). This showed that these analogs induced very similar structural changes to cAMP.
Titration of RI␣-(98 -381) with the B site preferring analog 2-Cl-8-AH-cAMP up to a molar ratio of 1:1 revealed no spectral changes for residues located in the A domain (e.g. Val 164 , Tyr 185 , and Ala 191 ). In contrast, for B domain residues, such as Ala 301 , Arg 306 , and Gly 319 , new resonances (red) appeared (Fig.  5, C and D), clearly confirming that this analog binds preferentially to the B site (Table 1). It is interesting to point out that resonances associated with residues in the PBC, the ␣B and ␣C helices in the B domain as well as the interdomain ␣C:A and ␣CЈ:A helices that were unassigned/undetected in the apoRI spectrum (Figs. 2 and 3), became readily visible upon the addition (1:0.5 ratio) of this B site analog (e.g. see Val 253 (V253) in Fig. 5C). This indicates that nucleotide binding to site B alone is sufficient to induce structure in these regions. In particular, a distinct and interesting pattern was observed for residues located in helices ␣C:A and ␣CЈ:A. 2-Cl-8-AH-cAMP binding  (46) for the residues whose chemical shifts were assigned unambiguously. ␣-Helices and ␤-strands present in the x-ray crystal structure of cAMP-bound bovine RI␣-(92-379) (10) are depicted by rectangles and arrows, respectively. resulted in two types of bound resonances for Val 253 (shown in Fig. 5C), Ser 251 , and Leu 259 . A new Val 253 resonance (purple in Fig. 5C) becomes visible at the first addition of the nucleotide (1:0.5 ratio) and shows maximum intensity at a molar ratio of 1:1, when only the B site is occupied, whereas a second new resonance (red in Fig. 5C) gradually replaces the purple resonance at higher analog concentrations, when also the A site becomes occupied. Therefore, two distinct conformations of the ␣C:A and ␣CЈ:A helices can exist, one for the protein with cyclic nucleotide bound only in the B site and the other with ligand bound in both A-and B sites. Saturation of the A site induced no further changes of the bound resonances of the B domain residues (Fig. 5C). Therefore, binding of 2-Cl-8-AH-cAMP to site B is communicated to the ␣C:A and ␣CЈ:A helices, at the junction between the A and B domains, but not to domain A itself.
Titration with the A site preferring N 6 -MB-cAMP up to molar ratio 1:0.6 resulted in essentially monosaturation of site A (Fig. 5, E  and F). Interestingly, the titration data showed that binding to the A site alone caused widespread effects throughout the protein (Fig. 5, E and F). First and not surprisingly, spectral changes occurred for A domain residues like Gly 180 , Tyr 185 , and Gly 198 (Fig. 5E), with gradual replacement of the free protein resonances (blue) by new ligand-bound resonances (red). Second, new (purple) resonances were also observed for B domain residues (Gly 286 , Gly 319 , and Ala 341 in Fig. 5E). Moreover, the undetectable interdomain  resonances exhibited their maximum intensities at a molar ratio of ϳ1:0.9, where other new resonances (red) started to appear, most likely due to actual ligand binding at the B site. The red resonances completely replaced the purple resonances at a molar ratio of 1:1.7. Therefore, they indeed are caused by ligand binding to the B domain, whereas the purple resonances belong to protein with ligand bound only in the A site.
In summary, our titration data demonstrate that ligand binding to the A site alone affects the conformation of the interdomain ␣C:A helix region as well as the B domain, providing unequivocal evidence for allosteric communication from the A domain to the B domain.
Titration of RI␣-(98 -382) with cAMP, the natural ligand (Fig. 5, A and B), revealed a slight B site preference, in accord with previous knowledge (7,20). At a molar ratio of 1:0.2, spectral changes were only seen for B domain residues, such as Gly 286 , Gly 319 , and Ala 341 (red in Fig. 5A), reflecting binding to the B site only. Importantly, amino acids that are located in the domain-connecting helices, such as Ser 251 , Val 253 , and Leu 259 (shown in purple in Fig. 5A) also exhibited new resonances, similar to those seen with the B analog 2-Cl-8-AH-cAMP. For increasing molar ratios of cAMP/RI␣-(98 -382), new resonances (red) were also observed for A domain residues, and the Leu 259 resonance of the B site saturated form (purple) is replaced by one for fully saturated (red) RI␣-(98 -382) (Fig. 5A).
In conclusion, residues in the ␣C:A and ␣CЈ:A helices that connect domains A and B and are critically involved in commu-nicating with the catalytic subunit clearly experience an allosteric conformational change imparted by binding of either the natural ligand cAMP or cAMP analogs to either of the two sites and followed by complete conformational change upon full saturation to both sites. The observed distinct stepwise conformational change induced by single to double site binding may be the structural correlate of the positive cooperativity observed in the PKA holoenzyme by cAMP binding (4,7).

Conformational Flexibility of ApoRI␣, a Crucial Feature for Understanding PKA Regulation-
The structural investigation of the tandem CNB domains of the R subunit of PKA in its apo-state (cAMPand C-free) has been hampered by conformational dynamics and instability of the protein. Here, we investigated RI␣-(98 -381), which comprises both cAMP binding domains structurally by NMR. The critical feature for our ability to study the apo-form of RI␣-(98 -381) is the presence of trace amounts of cAMP, rendering the sample stable for extended NMR data collection, necessary for resonance assignment. However, titrations with cyclic nucleotides were performed starting from the nucleotide-free apo-state, allowing us to dissect and define the role of each binding site. It therefore was possible to detect nucleotide-specific effects for each binding site throughout the regulatory subunit, including the interdomain regions. The cAMP binding sites of domains A and B were intact in RI␣-(98 -381) because they behaved like those in wt-RI␣ (Table 1 and supplemental Fig. S1). The ability of the cAMP binding domains to interact with the C subunit of PKA appeared also to be intact, the slightly lower affinity for the C subunit of RI␣-(98 -381) compared with wt-RI␣ (supplemental Fig. S1B) being easily explained by the loss of the pseudosubstrate motif (residues 92-96), whose binding to C is controlled by peptide substrates rather than by cAMP (21).
ApoRI␣ binds to the C subunit of PKA several orders of magnitude tighter than cAMP-saturated RI␣ (15,16). Comparison of conformational changes upon cAMP binding to RI␣ is therefore likely to provide important clues about the cAMP-mediated regulatory effects on the interaction with the catalytic domain. Crystal structures, NMR, small angle x-ray scattering analysis, and MD simulations of truncated forms of RI␣ either bound to cAMP or to the C subunit have already revealed that the R subunit is modular and dynamic and that a large conformational reorganization of RI␣ occurs upon binding to its partners (4, 10, 11, 32, 33) (see also Fig. 1). Our NMR studies con-