2H Exchange Reveals Communication Between the cAMP and Catalytic Subunit-binding Sites in the RIα Subunit of Protein Kinase A

doi:10.1016/S0022-2836(02)00919-1 available online at http://www.idealibrary.com on

w B

J. Mol. Biol. (2002) 323, 377–386

Amide H/2H Exchange Reveals Communication Between the cAMP and Catalytic Subunit-binding Sites in the RIa Subunit of Protein Kinase A Ganesh S. Anand1, Carrie A. Hughes2, John M. Jones2 Susan S. Taylor1,2 and Elizabeth A. Komives2* 1

Howard Hughes Medical Institute, University of California, San Diego 9500 Gilman Drive, La Jolla, CA 92093-0359, USA 2

Department of Chemistry and Biochemistry, University of California, San Diego 9500 Gilman Drive, La Jolla, CA 92093-0359, USA

The changes in backbone hydrogen/deuterium (H/2H) exchange in the regulatory subunit (RIa(94 – 244)) of cyclic AMP-dependent protein kinase A (PKA) were probed by MALDI-TOF mass spectrometry. The three naturally occurring states of the regulatory subunit were studied: (1) free RIa(94 –244), which likely represents newly synthesized protein, (2) RIa(94 – 244) bound to the catalytic (C) subunit, or holoenzyme, and (3) RIa(94 –244) bound to cAMP. Protection from amide exchange upon C-subunit binding was observed for the helical subdomain, including the A-helix and B-helix, pointing to regions adjacent to those shown to be important by mutagenesis. In addition, C-subunit binding caused changes in observed amide exchange in the distal cAMP-binding pocket. Conversely, cAMP binding caused protection in the cAMP-binding pocket and increased exchange in the helical subdomain. These results suggest that the mutually exclusive binding of either cAMP or C-subunit is controlled by binding at one site transmitting long distance changes to the other site. q 2002 Elsevier Science Ltd. All rights reserved

*Corresponding author

Keywords: MALDI-TOF; H/2H exchange; protein kinase; signal transduction; cAMP

Introduction Protein phosphorylation and dephosphorylation play a central role in the regulation of cellular functions in response to changes in external stimuli.1 Phosphorylation is mediated by diverse kinases that are ubiquitous in cells. One of the first recognized and most extensively characterized protein kinases is cAMP-dependent protein kinase (PKA). PKA is among the first downstream targets activated by increases in the second messenger, cAMP, in response to binding of hormones and other ligands to receptors on the extracellular surface.2 PKA is a tightly regulated enzyme that is inhibited by an intracellular inhibitor, PKI, and two classes of regulatory subunits. In the absence of cAMP, PKA exists as an inactive, tetrameric holoenzyme that is composed of two regulatory (R) and two catalytic (C) subunits. cAMP binding Abbreviations used: PKA, protein kinase A; MALDITOF, matrix-assisted laser desorption ionization – timeof-flight. E-mail address of the corresponding author: [email protected]

mediates cooperative dissociation of the R and Csubunits, thereby unleashing the catalytic activity of the C-subunit. The R-subunit is thus a locus for binding cAMP that controls the activity of PKA in a cAMP-dependent manner. There are two classes of R-subunits, type I and II, with distinct structural and functional differences. Nevertheless, they share a similar domain organization with an Nterminal docking/dimerization domain, a pseudosubstrate region and two tandem, homologous cAMP-binding domains A and B (Figure 1(a)).3 There are two principle loci on the R-subunit that maintain interactions with the C-subunit; one is the pseudosubstrate sequence (micromolar KD) and the other is within the A-domain.4 The two binding sites together result in very high affinity binding for the holoenzyme complex (KD 0.2 nM).5 In the PKA holoenzyme, cAMP binds the Bdomain first, triggering conformational changes that are propagated to the A-domain allowing a second molecule of cAMP to then access the binding site in the A-domain, which then mediates dissociation of the holoenzyme complex.6 Conversely, the opposite holds true for the reverse reaction with C-subunit binding to the cAMP-bound

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

378

RIa Subunit of Protein Kinase A

Figure 1. (a) A schematic showing the domain organization of intact RIa and RIa(94 – 244). The dimerization/docking domain (10 – 61) is hatched, the pseudo substrate region (94 –97) is indicated in red, the linker region (98– 140) connecting the pseudo substrate to the cAMP-binding domain in cyan, the cAMP-binding domains A and B in blue with the consensus sequences for cAMP binding in the A (199 – 210) and B-domains (323 –334) in gray. (b) The structure of RIa(113 – 244) bound to cAMP.14 The Adomain is in blue, the linker region is in cyan and cAMP is represented in gold. The cAMP-binding cassette residues and the segment of the Chelix predicted to be necessary for high affinity binding to the C-subunit are indicated in gray.11 Sidechains of residues identified to be important for mediating interactions with the C-subunit and cAMP are indicated in green.4,11 (c) A schematic representation of the phosphate-binding cassette of the A-domain shows the residues involved in interactions with cAMP. Main-chain nitrogen atoms of residues 199, 202 and 210 and the side-chain atoms of 200 and 209 that are in close proximity to cAMP in the structure of RIa(113 – 379)14 are highlighted. This cassette is the unique motif that is evolutionarily conserved in cAMP-binding proteins.29

R-subunit promoting cAMP release from the Adomain first followed by release of cAMP from the B-domain.7 While the A and the B-domains maintain the same fold, homologous to the cAMPbinding domain of the catabolite gene activator protein (CAP) in Escherichia coli,8 they differ in binding affinity for cAMP analogs and the Adomain has a faster off-rate than the B-domain for cAMP.9 The A-domain shows a slightly greater affinity for cAMP relative to the B-domain in the type I PKA holoenzyme.10 Deletion mapping studies of RIa have been used to probe the interactions between the R and C-subunits in the holoenzyme. These studies have identified that in addition to the pseudosubstrate region, the A-domain (RIa(94 – 244)) provides the primary determinants for interactions with the C-subunit.11 An understanding of one of the two interactions between the R and the C-subunits, that of the pseudosubstrate region with C-subunit, has been obtained from the structure of the ternary complex of the C-subunit with ATP bound to an inhibitor peptide.12 A detailed structural understanding of the interactions between the A-domain and the Csubunit has not yet been possible. Deletion map-

ping studies pointed to residues 236– 244 in maintaining high binding affinity to the C-subunit.11 Chemical footprinting and site-directed mutagenesis to alanine have identified two residues in the A-domain of RIa, Asp140 and Glu143, that contribute to holoenzyme formation (Figure 1(b)).4,13 The structures of cAMP-bound RIa(D1 – 91)14 and RIIb(D1 –112)15 provide an understanding of the modular organization of the A and B-domains in the two classes of the R-subunit. Within the eightstranded b-barrel lies the cAMP-binding cassette that uniquely defines this motif (Figure 1(c)). Taken together, it appears as though distinct subdomains are involved in recognition of the C-subunit (a-helical subdomain) and binding to cAMP (b-barrel subdomain). The truncated A-domain includes the minimum determinants for C-subunit interaction and shows the same affinity for cAMP as the intact RIa subunit.16 Thus, by itself, RIa(94 – 244) is suitable for understanding the interaction of the R and C-subunits and cAMP binding. Amide H/2H exchange measured by mass spectrometry is an elegant method that has been used to analyze conformational changes in proteins, protein folding and in mapping protein

379

RIa Subunit of Protein Kinase A

Results Measurement of solvent accessibility changes in PKA

Figure 2. (a) MALDI-TOF mass spectrum of the peptic digest from RIa(94– 244). The inset is an expansion of the spectrum showing the mass envelope of the m/z ¼ 1782.99 peak corresponding to residues 111 – 125. The different peaks in the envelope are the isotopic peaks. (b) MALDI-TOF mass spectrum of the peptic digest from the RIa(94– 244) – C holoenzyme with the m/z ¼ 1782.99 peak shown in the inset.

conformational changes.17,18 Analysis of the fast exchanging amides in the uncomplexed and complexed states has been used to map the interfaces of protein– protein interactions.19,20 Kinetic analysis combined with structural information can be used to decouple interface changes from conformational changes.21 Here, we have used amide hydrogen exchange coupled to MALDI-TOF mass spectrometry to map the interactions of RIa(94 –244), which includes the A-domain and pseudosubstrate region, with the C-subunit. We also probed the role of cAMP association in mediating this interaction. Our results have enabled identification of residues important for holoenzyme formation. The results further indicate that interactions with the C-subunit cause distal changes within the cAMP-binding region of the protein. A model is proposed in which C-subunit binding primes the R-subunit for cAMP release and conversely cAMP binding primes the R-subunit for C-subunit release.

Amide H/2H exchange measurement by MALDI-TOF mass spectrometry provides a suitable technique to map the interaction surfaces between the regulatory and catalytic subunits in the holoenzyme of PKA.19,22 Previous studies using this approach have mapped the binding sites of ATP and an inhibitor peptide to the C-subunit,19 and characterized of changes within the C-subunit upon nucleotide binding.23 Here, we have used amide H/2H exchange to probe the interactions between the A-domain of the RIa subunit, RIa(94 – 244), and the C-subunit in the type I PKA holoenzyme as well as to characterize the effects of cAMP binding to the R-subunit. While cAMP is bound to the free R-subunit in solution, the R-subunit when bound to the catalytic subunit is free of cAMP. In order to understand how the R-subunit changes upon formation of the holoenzyme, it is necessary to separate the effects of removal of cAMP on the R-subunit from binding of the C-subunit. Three states of the R-subunit have, therefore, been examined here: the free Rsubunit, R-subunit in complex with the C-subunit, and the R-subunit bound to cAMP. Deuterium incorporation into the rapidly exchanging backbone amides in the free RIa(94 – 244) with and without cAMP, and the RIa(94 – 244) – C-subunit complex was carried out by incubation of all protein samples in Mops buffer in 2 H2O (pH 7.0) at 25 8C. The H/2H exchange reaction was quenched by tenfold dilution into cold 0.1% (v/v) TFA to rapidly lower the temperature to 0 8C and pH to 2.5. This enabled quantification of backbone amide protons that exchanged with deuterium under native-like conditions. In order to localize the observed changes in solvent accessibility in the R-subunit upon complexation with the C-subunit or cAMP, the protein was cleaved by pepsin under quench conditions. A total of 16 peptides from RIa(94 – 244) were analyzed from each MALDI mass spectrum of the A-domain (Figure 2) and these covered 70% of the sequence of the A-domain of the R-subunit. MALDI-TOF conditions were optimized to minimize loss of coverage when the complex with the C-subunit was studied, so that the same peptides were used for all three states of RIa(94 – 244) (Figure 3). Table 1 summarizes the extent of deuteration for all the peptides from which quantitative data were obtained. The data are reported as the average number of deuterons incorporated after ten minutes for three independent determinations. For those segments where changes were observed, plots of the time-course of deuteration are also shown and either method of data presentation results in the same conclusions.20 Extending the time-course of the experiment did not result in observation of any further differences. Peptides

380

RIa Subunit of Protein Kinase A

The a-Xn helix, which is not defined as part of the A-domain, but was present in the crystal structure, was represented by two peptides, m/z ¼ 1783.00, residues 111– 125, m/z ¼ 1619.93, residues 122 –136. These segments also showed decreased solvent accessibility when the C-subunit was bound. This region lies directly behind the A and B-helices that are also protected upon C-subunit binding (see below).

Figure 3. Sequence of RIa(94– 244) showing the peptides that were observed in the mass spectrum of the peptic digest. Thick lines are peptides for which quantifiable data were obtained and thin lines are peptides for which quantifiable data could not be obtained from the holoenzyme complex.

have been grouped on the basis of their location on the X-ray crystal structure of RIa(113– 379).14 The pseudosubstrate region and the a-Xn helix (residues 112 – 136) showed decreased solvent accessibility upon binding C-subunit A peptide (m/z ¼ 946.51, residues 94 – 101) from the N terminus of RIa(94 –244), was highly deuterated, and was protected when the C-subunit was bound. These results are consistent with the fact that residues 94 –97 correspond to the pseudosubstrate site. This site is also protected from limited proteolysis in the holoenzyme complex.24

The B-helix (residues 222 – 229) and the C-helix (residues 230 – 244) show decreased solvent accessibility upon binding C-subunit Three peptides, m/z ¼ 1011.46, residues 222 – 229, m/z ¼ 1046.61, residues 230 –238, and m/z ¼ 881.51, residues 239– 244, which cover the B and C-helices in RIa(94 – 244), had the same solvent accessibility whether the R-subunit was free or bound to cAMP (Table 1). Residues 222 – 229 were protected from deuteration when the C-subunit was bound (Figure 4(a) and (b)). Similar results were obtained for residues 230– 238 (data not shown). No significant differences in deuteration were observed for the end of the C-helix, residues 239 –244 (Figure 4(c)). Although this is the C terminus of our truncated A-domain, it is interesting to note that the segment incorporates deuterium into only 2.5 of five possible amide positions, suggesting that it is tucked against the phosphate-binding cassette. This is consistent with cross-linking results showing that Tyr244 is modified by azido-cAMP only in the truncated A-domain.25 Overall, it appears that the B-helix is one site of interaction with the C-subunit.

Table 1. Summary of H/2H exchange data for PKA regulatory subunit Deuteration after ten minutes Number of amides

Free RIa

Holoenzyme complex

cAMP-bound RIa

Pseudosubstrate 94–101 (946.51)

8

6.68 ^ 0.07

4.77 ^ 0.08

5.53 ^ 0.11

a-Xn 111–125 (1783.00) 122–136 (1619.93)

13 14

11.49 ^ 0.39 12.17 ^ 0.24

10.73 ^ 0.18 9.75 ^ 0.12

12.29 ^ 0.24 10.06 ^ 0.08

C-helix 222–229 (1011.46) 230–238 (1046.61) 239–244 (881.51)

7 8 5

1.56 ^ 0.19 4.23 ^ 0.24 2.48 ^ 0.25

0.91 ^ 0.03 3.86 ^ 0.17 2.6 ^ 0.06

1.55 ^ 0.05 4.40 ^ 0.13 2.77 ^ 0.09

A-helix 136–143 (976.40) 140–148 (1110.47) 136–148 (1594.73) 148–170 (2473.12)

7 8 12 21

3.06 ^ 0.17 2.87 ^ 0.13 5.31 ^ 0.41 8.16 ^ 0.36

2.60 ^ 0.09 2.45 ^ 0.08 3.88 ^ 0.14 6.23 ^ 0.36

2.87 ^ 0.07 3.70 ^ 0.05 5.81 ^ 0.17 8.34 ^ 0.12

Phosphate-binding cassette 202–221 (2115.27) 204–221 (1931.15)

18 16

7.55 ^ 0.18 4.92 ^ 0.18

6.72 ^ 0.03 5.83 ^ 0.09

4.28 ^ 0.05 2.11 ^ 0.02

Region of PKA regulatory subunit

381

RIa Subunit of Protein Kinase A

Figure 4. (a) MALDI-TOF mass spectra of one of the 16 peptides from the analysis of RIa(94 –244) that experienced slowed exchange in the RIa(94 – 244) –C holoenzyme complex. The spectra are expanded so as to show the isotopic distribution for the peptide of interest (m/z ¼ 1011.46). (i) The undeuterated sample. The higher mass peaks in the envelope are caused by naturally occurring isotopes. (ii) The isotopic envelope for the same peptide from free RIa(94 – 244) (-cAMP) after ten minutes of deuteration. (iii) The isotopic envelope for the same peptide from RIa(94– 244) – C holoenzyme after ten minutes of deuteration. (iv) The isotopic envelope for the same peptide from cAMP-bound RIa(94– 244) after ten minutes of deuteration. (b) Plot of deuterium incorporation into the amide positions of the region of RIa(94– 244) for the peptide, m/z ¼ 1011.46 corresponding to the residues (222 – 229) for free RIa(94– 244) (-cAMP) (O), RIa(94 –244) – C-subunit holoenzyme (B) and cAMP-bound RIa(94 – 244) (X). (c) The structure of RIa(113 –244) showing the protection from deuterium exchange for two regions, 222– 229 and 230– 238, that comprise part of the B and C-helices. Both regions were protected in the C-subunit bound state compared to the cAMP-bound RIa(94– 244) and are shown in red. No difference in deuterium exchange was observed for a third region, residues 239– 244, comprising the C terminus of the C-helix (green).

Figure 5. (a) Comparative analysis of the amount of deuteration after ten minutes for the observable amides from three overlapping peptides (continuous lines), m/z ¼ 1594.73, residues 136– 148; m/z ¼ 976.40, residues 136– 143; m/z ¼ 1110.47, residues 140– 148 from cAMPbound RIa(94 – 244), free RIa(94– 244) (-cAMP) and RIa(94 – 244)– C holoenzyme. The broken lines and the parenthetic values listed above them represent the difference in deuteration between the full-length peptide (residues 136–148) and the two overlapping fragments. (b) The structure of RIa(113 – 244) showing all of the segments that were protected upon C-subunit binding including peptide corresponding to residues 136–143, 140– 148, 136– 148 and 148– 170 (red) that also contained two amides that were protected.

A-helix of the R-subunit (residues 141 –150) show decreased solvent accessibility upon binding C-subunit Residues preceding (Asp140) and within the Ahelix (Glu143) were shown to disrupt C-subunit binding upon mutation to alanine.4 Furthermore, the mutants RI (E143A) and C (K213A) were found to compensate each other, suggesting that the Glu143 from RI and Lys213 from the C-subunit form a salt bridge in the holoenzyme complex.26

382

RIa Subunit of Protein Kinase A

Figure 6. (a) MALDI-TOF mass spectra of one of the 16 peptides from the analysis of RIa(94 – 244) that experienced higher exchange in the RIa(94–244)–C-subunit holoenzyme complex. (i) The undeuterated peptide (m/z ¼ 2115.27). (ii)– (iv) are the same as already described in the legend to Figure 4. (b) Plot of deuterium incorporation into the amide positions of residues 202–221 of RIa(94–244) (m/z ¼ 2115.27). The symbols correspond to the same samples as already described in the legend to Figure 4. (c) Comparative analysis of the amount of deuterium exchange after ten minutes for two overlapping peptides, m/z ¼ 2115.27, residues 202– 221 and m/z ¼ 1931.15, residues 204– 221 from cAMP-bound RIa(94 – 244), free RIa(94– 244) (-cAMP) and RIa(94 – 244) –C-subunit holoenzyme. (d) The structure of RIa(113 – 244) showing the results from deuterium exchange experiments. The phosphate-binding cassette (residues 205– 221, yellow) became more deuterated upon cAMP removal and even more so upon C-subunit binding. Residues 203 and 204 (green) were highly deuterated in all three states of the R-subunit.

Four peptides, m/z ¼ 976.40, residues 136 –143, m/z ¼ 1594.73, residues 136– 148, m/z ¼ 1110.47, residues 140– 148 and m/z ¼ 2473.12, residues 148 –170 covered this region. All four peptides showed protection of backbone amide hydrogen atoms in the holoenzyme complex compared to the cAMP-bound R-subunit. A more thorough understanding of this region was obtained by analyzing the peptides that overlap the 137 –148 region (Figure 5). Protection upon C-subunit binding extended across the entire region from residues 137 –148. Comparison of the data from free RI with that from the cAMP-bound form showed differences, with the entire region becoming more deuterated in the cAMP-bound form. This increased deuteration could be largely localized to the 144– 148 region.

less when the R-subunit was bound to cAMP (Figure 6(a) and (b), results from residues 204 –221 not shown). Residues 204– 221 showed a higher level of deuteration when the R-subunit was complexed to the C-subunit as compared to when it was free, perhaps, indicating an increase in backbone dynamics of this region in the holoenzyme complex. However, under all conditions and even after 24 hours, this region was highly shielded from solvent, indicating clearly that solvent does not rush into this pocket when cAMP is released. In contrast, residues 203 and 204 are highly deuterated, but become less so when the C-subunit binds (Figure 6(c)). Thus C-subunit binding in the helical subdomain appears to subtly tighten residues 203 and 204 while allowing a loosening of the rest of the cAMP-binding pocket (Figure 6(d)).

Phosphate-binding cassette (residues 203 – 221) showed changes in solvent accessibility upon cAMP and C-subunit binding

Discussion

Two peptides, m/z ¼ 2115.27, 202– 221 and m/z ¼ 1931.15, 204– 221 spanned a large part of the phosphate-binding cassette, composed of b-strand 6, a short P-helix, a loop, and b-strand 7. Consistent with the fact that this is the cAMP-binding site, whose primary function is to shield the phosphate from solvent and access to phosphodiesterases, the amount of deuteration was much

The C-subunit-binding site in RIa(94– 244) In addition to the pseudosubstrate region, previous work to identify the C-subunit-binding sites in RIa(94 –244) had pointed to the end of the Chelix and residues 140 – 143. Protection from deuterium exchange in the R-subunit was indeed observed, as was expected for the pseudosubstrate region. In addition, a single contiguous surface

383

RIa Subunit of Protein Kinase A

was protected by C-subunit binding that is adjacent to but not identical with the previously identified regions. The surface is formed from the C-terminal end of the A-helix and the B-helix. No differences in amide deuteration were observed for residues 239 –244. Although deletion mutagenesis had shown that residues 236 –244 were essential for C-subunit binding, our results suggest that this may have been due to loss of structure of the C-helix rather than direct loss of the C-subunit-binding interface.11 Similarly, greater protection was also seen for residues 144 –148 in the A-helix than for residues 137 – 143. Behind this surface is the aXn-helix, which also shows protection, probably due to the C-subunit pressing on the two helices adjacent to it. Alternatively, the aXn region may become more ordered when the pseudo substrate and helical-binding surface regions simultaneously dock to the C-subunit. It is striking that such a small patch on the surface of the R-subunit is all of the A-domain that is involved in direct contact with the C-subunit. This may be because the A-domain-binding site contributes only part of the binding affinity, the rest being provided for by the pseudosubstrate region, residues 94 –101, that were also highly protected upon C-subunit binding. If both bind independently, each with micromolar affinity, the two together would account for the nanomolar-binding affinity. It is interesting to speculate that cAMP binding to the holoenzyme may dissociate the A-domain, but not the pseudo substrate site, leaving the complex partly associated. Amide protection upon cAMP binding The cAMP-binding pocket (residues 202 – 221) was, as expected, protected from exchange when cAMP bound. This is consistent with the region being hydrogen bonded to cAMP as seen in the structures of both isoforms of the R-subunit.14,15 In these structures, three backbone amide nitrogen atoms from residues 199, 202 and 210 were found to be within H-bonding distance of the cyclic nucleotide and phosphate moiety of cAMP. We did not obtain peptides that covered residues 199– 202, but some observations can be made regarding the remainder of the cAMP-binding site. This region is not highly deuterated, indicating that it is not very solvent accessible even when cAMP is not bound. Comparison of the free and cAMP-bound states indicates that cAMP binding protects residues 205– 221 from amide exchange. C-subunit binding-mediated conformational changes are propagated to the cAMP-binding pocket of the R-subunit Residues 204 –221 in the cAMP-binding pocket showed differences in amide exchange for all three states of the R-subunit. Beyond the expected

increase seen for removal of cAMP, C-subunit binding caused a further increase in amide deuterium exchange at this site. This region contains R209, which when mutated to K results in an Adomain that can no longer bind cAMP with high affinity, but can still bind the C-subunit.10 The increased amide exchange may be due to increased backbone dynamics in the cAMP-binding pocket induced by C-subunit binding. This could partly explain why binding of cAMP and C-subunit are mutually exclusive. In fact, it appears that C-subunit binding primes the R-subunit for cAMP release.

cAMP binding propagates changes to the C terminus of the A-helix As with the cAMP-binding pocket, the A-helix showed differences in amide exchange for all three states of the R-subunit. As already discussed, the A-helix becomes protected upon C-subunit binding. In addition, this region becomes more deuterated when cAMP is bound as compared with free RI. Analysis of all of the overlapping peptides showed that the increased deuteration could be localized to the 144 – 148 region, which was exactly where the C-subunit binding was also localized. If the increased deuteration is interpreted as increased dynamics in the ground state, we can conclude that not only does C-subunit binding shake the cAMP loose, but also cAMP binding shakes the C-subunit loose.

A model for the yin and yang of the R-subunit A closer look at the structure of the RIa(113 – 244) reveals that the A-domain really consists of two subdomains, a non-contiguous helical subdomain that binds the C-subunit, and a contiguous b-barrel subdomain that binds cAMP. The amide H/2H exchange results presented here show that these two subdomains “talk” to each other by inducing dynamics at the opposing binding site. Across the subdomain interface, hydrophobic side-chains from Y229, M234 and I204 are packed against each other and may provide the connection between the two subdomains (Figure 7). Thus, binding of the C-subunit at Y229 appears to transmit dynamic or conformational changes to residues 204 –221. Conversely, binding of cAMP appears to alter the conformation of I204, causing increased dynamics in the C-subunit-binding site. Both of these could occur via subtle alterations of packing at this hydrophobic hinge point. It is interesting to note that this hinge point was also identified as a region where RIa differs structurally from RIIb when the structures of the A-domains from the two isoforms were superimposed.13 This hypothesis, which remains to be tested, suggests an intriguing mechanism for exclusivity of binding.

384

RIa Subunit of Protein Kinase A

Removal of cAMP from R-subunits To obtain cAMP-free RIa(94– 244) the polyhistidinetagged protein was unfolded with 8 M urea and dialyzed and purified as described by Buechler et al.28 Refolding of the protein was carried out by step-wise dialysis against 50 mM Mops (pH 7.0), 1 mM b-mercaptoethanol (bME) buffer containing decreasing concentrations of urea (6, 4, 2, 1, 0.5 M) for at least four hours. The protein was then dialyzed overnight at 4 8C against three 1 l changes of 50 mM Mops (pH 7.0), 1 mM bME. There was no visible precipitation at any of the unfolding and refolding steps. The refolded protein was tested for its ability to inhibit the catalytic activity of the C-subunit in an in vitro assay. Free RIa(94– 244) was concentrated to 133 mM prior to deuterium exchange experiments. Figure 7. Summary of amide exchange data shown on the structure of RIa(113 – 244) showing the proposed residues that mediate inter-subdomain communication. The residues previously shown to be important for C-subunit binding are D140, E143, R239 and R241 and for cAMP binding are E200 and R209. The three additional residues proposed to be involved in inter-subdomain interactions are I204, Y229, and M234. The contiguous surface predicted to be the C-subunit-binding site from analysis of all of the H/2H exchange data is shown in red. Regions showing increased deuteration upon C-subunit binding are shown in yellow. Regions that showed little change in deuteration in the three states of RIa(94– 244) are colored blue. Other regions of the protein from which no peptides were observed are colored gray.

Materials and Methods Materials ATP, cAMP, Mops and cAMP immobilized on 6% (w/v) agarose were obtained from Sigma. Nickel-NTA resin was obtained from Qiagen. Deuterium oxide, 2H2O (99.9% deuterium) was obtained from Cambridge Isotopes. Pepsin immobilized on 6% agarose was obtained from Pierce. Trifluoroacetic acid and acetonitrile were obtained from Fisher and were of peptide synthesis grade and optima grade, respectively. The matrix compound, a-cyano-4-hydroxycinnamic acid, was obtained from Aldrich and recrystallized once from ethanol. PD10 columns for buffer exchange were obtained from Amersham Pharmacia Biotech. All other materials were reagent grade from standard commercial sources.

Preparation of RIa(94–244) and C-subunits RIa(94 –244) was expressed as a polyhistidine-tagged protein and purified by passing through a Nickel-NTA column and eluted with sodium phosphate buffer (pH 7.0), containing 100 mM imidazole as described.11 The buffer was changed to 50 mM Mops (pH 7.0), with 10 mM cAMP on a PD10 column, and the protein was concentrated to 133 mM. The protein was stored at 4 8C. The Ca-subunit of murine PKA was expressed in E. coli and purified as described.27 Isozyme II, the second isoform eluting from the cation exchange column, was used for all experiments.

Formation of holoenzyme The C-subunit and 1.2 molar excess of cAMP-bound RIa(94 – 244) were combined in holoenzyme buffer (10 mM Mops (pH 7.0), 50 mM NaCl, 5 mM b-Me) with 0.2 mM ATP, 2 mM MgCl2 and dialyzed against two 1 l changes of holoenzyme buffer overnight. The dialysate was then passed through a Sephacryl S-200 (2.5 cm £ 20 cm) column equilibrated with holoenzyme buffer. Fractions containing holoenzyme were characterized by SDS-PAGE, pooled and concentrated. The buffer was changed to 50 mM Mops (pH 7.0), 5 mM b-ME on a PD10 column, and the protein was concentrated to 172 mM prior to deuterium exchange. Measurement of amide H/2H exchange by MALDITOF mass spectrometry All of the data for each protein analysis was obtained from a single mass spectrum of the peptic digest of the cAMP-bound and free states of RIa(94 – 244) and holoenzyme of PKA (Figure 2). A total of 16 and 66 peptides were observed in the MALDI mass spectra of 2.8 pmol of the mixture of peptic fragments of RIa(94 – 244) and the holoenzyme complex, respectively. The 16 RIa(94 – 244) cleavage peptides were identified by a combination of sequence searching for accurate masses, post-source decay sequencing, and C-terminal ladder sequencing as described.22 Deuterium exchange experiments The exchange mixtures for the RIa(94 – 244) contained 50 mM Mops for analysis of the free RIa(94– 244) and included 1 mM cAMP for analysis of the cAMP-bound state of RIa(94 – 244). The exchange mixture for the holoenzyme contained 50 mM Mops. The final pH was 7.0. The deuterium exchange was initiated by combining the protein solution in H2O (10 ml) with Mops buffer in 2 H2O (90 ml) at 25 8C. After 0, 0.5, one, two, five, or ten minutes, the deuterium exchange reaction was quenched by adding to ice-cold 0.9 ml 0.1% TFA. Deuterium exchange at time t ¼ 0, was determined by adding the protein solution in H2O (10 ml) to a mixture of ice-cold 0.9 ml 0.1% TFA and Mops buffer in 2H2O (90 ml). A mock experiment was performed to determine the amount of 2% TFA required so that upon quenching, the pH would be 2.5. A portion of the quenched reaction (0.1 ml) was mixed with 50 ml of pepsin bead slurry (previously washed two times in 1 ml of cold 0.1% TFA). The mixture was incubated on ice with occasional mixing for

385

RIa Subunit of Protein Kinase A

five minutes to facilitate complete pepsin proteolysis of the protein samples. The mixture was then centrifuged for 15 seconds at 12,000g at 4 8C to remove the pepsin beads, and the solution was divided in aliquots and frozen in liquid N2. The samples were stored at 2 80 8C until MALDI-TOF analysis. Frozen samples were quickly defrosted to 0 8C, mixed with matrix (5 mg/ml a-cyano-4-hydroxycinnamic acid in 1:1:1 acetonitrile, ethanol, 0.1% TFA, final pH 2.5, 0 8C), and 1 ml was spotted on a chilled MALDI target. The target was immediately placed in a dessicator under a moderate vacuum to dry the spots in one to two minutes. The chilled plate was then transferred quickly to the mass spectrometer. The back exchange that occurred during the analysis was 46% as determined by carrying out control experiments where each of the protein samples was deuterated for 24 hours at 25 8C.

3.

4.

5.

6. Data analysis Mass spectra were calibrated using the theoretical mass of two prominent peptides (theoretical m/ z ¼ 1011.4609 and 1594.7138). For some deuterated samples the monoisotopic peak was not present, so higher mass peaks of the same envelope were used; these peaks were identified based upon the internal calibration of the mass spectrometer. The average mass of a peptide was calculated by determining the centroid of its isotopic envelope as described.22 The difference between the average masses of the deuterated and nondeuterated peptide gave the number of deuterons incorporated. Side-chain deuteration, which requires correction in MALDI data analysis because some residual deuterium remains in the dried spot, was carried out prior to back exchange correction. The number of exchangeable side-chains was multiplied by the residual deuterium content (2.2%) and this value was subtracted from each number of deuterons incorporated. Finally, a correction was applied to account for the 46% back exchange. Kinetic plots of deuteration fit best to a single exponential model accounting for deuterons exchanging at a rapid rate (mainly solvent-accessible amides). The fit was implemented in KaleidaGraph 3.0 (Synergy Software, Inc.). Errors in the fits were approximately 10%, and similar errors were obtained from individual timepoints measured in triplicate.

Acknowledgements We thank Celina Juliano for assistance with protein purification. This work was supported in part by Howard Hughes Medical Institute, NIH grant GM34921 to S.S.T. and by NSF grant MCB9808286 to E.A.K. C.A.H. was supported by the Molecular Biophysics Training Program.

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Edited by P. E. Wright (Received 31 May 2002; received in revised form 16 August 2002; accepted 20 August 2002)