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1 Department of Chemistry and Biochemistry, 2 Howard Hughes Medical Institute, and 3 Department of Biology and Physics, University of California, San Diego, La Jolla, California 92093, USA
4 San Diego Supercomputer Center, San Diego, California 92186, USA
Reprint requests to: Susan S. Taylor, Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, Leichtag 415, La Jolla, CA 92093, USA; e-mail: staylor{at}ucsd.edu; fax: (858) 534-8193.
(RECEIVED July 20, 2005; FINAL REVISION July 20, 2005; ACCEPTED August 23, 2005)
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Abstract |
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 TOP Abstract IntroductionResultsDiscussionMaterials and methodsReferences |
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-helix conformation. Finally, based on temperature factors, this mutant structure is more stable than the wild-type C-subunit in the apo state. Keywords: E230Q mutant; crystal structure; apoenzyme; extended N-terminal helix; electrostatic surface
Abbreviations: PKA, cAMP-dependent protein kinase • C, catalytic subunit • IP20, the inhibitor peptide
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051715205.
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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Glu230 is a surface residue that contributes to the recognition of the P-2 arginine in the peptide substrate (second residue N-terminal to the P-site phosphoacceptor Ser or Thr) (Fig. 1
). This Glu is actually highly conserved in many protein kinases and also contributes significantly to the overall electrostatic properties of the enzyme. The conservative replacement of Glu230 with Gln (E230Q) demonstrated the kinetic importance of this electrostatic contribution (Tsigelny et al. 1996). The mutant protein not only is defective in its recognition of substrates, but also is deficient in phosphoryl transfer (Grant et al. 1996). Specifically, it exhibited a ~200-fold increase in Km for substrate peptide, Kemptide (LRRASLG), and a ~20-fold increase in Ki for the inhibitory Ala-Kemptide (LRRAALG). The mutation did not affect the rate-limiting product release step but caused a 25-fold reduction in phosphoryl transfer rate, and thus resulted in only a twofold decrease in the over-all kcat. It thus appeared that the electrostatic contribution of Glu230 is important not only for peptide affinity, but also for catalysis, most likely due to perturbation of the electrostatic field at the surface and at the active site of the enzyme (Grant et al. 1996; Tsigelny et al. 1996).
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, it is linked to the P+1 loop through its interaction with Tyr204 and to the catalytic loop mediated through interaction of the P-2 Arg with Glu170. The circuitry that connects the two loops is only completed fully when the peptide with its P-2 arginine binds between Glu170 and Glu230. In the absence of peptides, the two carboxyl groups are linked more loosely through Arg133 in the D helix (Akamine et al. 2003). Although replacement of Glu230 with Ala resulted in an insoluble protein, replacement with Gln does not have a major effect on the overall stability of the protein. In trying to understand the altered kinetic properties of the mutant from the structural point of view, we have solved its crystal structure at 2.8 Å. It appears that the mutant prefers an open conformation with a preserved overall structure of the wild-type protein. Interesting features of the structure also include the observation of the full N terminus in an extended helical conformation and partial occupancy of the substrate-binding groove by the N-terminal helix from a neighboring molecule, presumably due to crystal packing. |
Results |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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The mutant crystallized in the P42 space group under crystallization conditions that typically yielded P212121 crystals for the wild-type C-subunit in binary or ternary complexes (Knighton et al. 1991, 1993; Zheng et al. 1993a; Madhusudan et al. 1994, 2002; Narayana et al. 1997a, b, 1999). The only previous exceptions were the apo-form of C-subunit that crystallized in the monoclinic space group P21 (Akamine et al. 2003) and the binary complex of the myristylated mammalian C-subunit with IP20 that crystallized in the P4132 space group (Zheng et al. 1993b). For the E230Q structure, each asymmetric unit contained two molecules that had very similar overall conformations as indicated by a small root mean square deviation (RMSD) of 0.11 Å for the -carbon atoms. The R-factor and Rfree of the E230Q mutant structure were 0.202 and 0.249, respectively, with good stereochemistry statistics (Table 1). The Ramachandran plot showed that 86.8% of all of the nonglycine residues were in the most favored regions, and the rest fell into the allowed regions.
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levels, two well-ordered phosphate moieties covalently bound to Thr197 and Ser338 were built into the model. This is consistent with the mass-spectrometry analysis showing that the protein had two phosphates. Unexpectedly, no electron densities could be found for either MgATP or IP20 in either molecule, although these ligands were included in the crystallization buffer. The E230Q mutant structure thus exhibited an open conformation most similar to that of the apo-form of the wild-type enzyme (Fig. 2A). When the two structural models were superimposed, the RMSD of -carbon atoms for the small lobe alone, large lobe alone, and the overall structure were 1.3, 0.5, and 1.1 Å, respectively. Conformational differences lay mostly in the small lobe.
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). However, residues that form the ATP-binding pocket were mostly well ordered and adopt a very similar conformation as that of the wild-type protein. This again supports the concept of a largely preformed ATP-binding pocket proposed earlier (Akamine et al. 2003).
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). In the wild-type structure, the average B-factor for the small lobe (79.8 Å2) is significantly higher than that of the large lobe (55.3 Å2), and many of the side chains in the small lobe were disordered (Akamine et al. 2003). In contrast, in the E230Q mutant structure the average B-factor for the small lobe (31.3 Å2) is similar to that of the large lobe (27.6 Å2). In addition, many residues had well-defined electron densities for both main chains and side chains, including the residues in the ATP-binding pocket. Notably, although the "gate" region in the C-terminal tail (Phe318–Tyr330) is disordered in the wild-type apoenzyme, it was reliably traced in one of the molecules (A) in the E230Q crystal, even though the B-factors were high (Fig. 2). This reduced dynamics of the small lobe in the E230Q structure may partly result from its crystal contacts (see below), however, it is possible that the surface electrostatics also introduce internal dynamics into the protein.
Substrate-binding site
Glu230 is positioned in the center of the surface acidic cluster that is critical for substrate recognition (Grant et al. 1996; Tsigelny et al. 1996; Fig. 1). In the E230Q mutant structure, the inhibitor peptide could not be traced due to the void of the electron density (Fig. 6B, below). The substrate-binding sites, however, retained a very similar overall conformation compared with those structures where substrate or inhibitory peptide was bound. Comparison of the substrate-binding site in the E230Q structure with the closed ternary structure (PDB ID 1L3R
[PDB]
) and the wild type apoenzyme structure (PDB ID 1J3H
[PDB]
) indicates that most of the residues that are involved in direct interaction with substrate, such as Asp166, Glu170, Glu203, and Tyr204 adopt a stable conformation (Fig. 4). The side-chain conformation of the mutated glutamine itself is quite stable, presumably due to retention of hydrogen-bonding interactions with Tyr204 in the P+1-binding pocket. Interestingly, the side chain of Arg133 in the D-helix, another key binding partner of Glu230, is highly dynamic and could not be modeled in the mutant structure (Fig. 4). In fact, most of the D-helix segment (residues 125–136) was found to have high-temperature factors relative to the ternary and apoenzyme structure. Since residues within the D-helix are involved in direct interactions with the peptide inhibitor (Fig. 5), it is likely that internal dynamics induced within this region may influence the surface electrostatics and subsequently substrate binding as well as the closure of the active site cleft.
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Crystal packing
One of the striking features of the E230Q mutant structure was that in one of the two molecules in the asymmetric unit, molecule A, the entire N terminus (Gly1–Thr32) assumed an extended -helical conformation with very well-defined electron density (Fig. 3B). This is in contrast to what has been observed in all of our previous recombinant C-subunit structures where the first 10–15 residues were disordered and the N-terminal A-helix begins at residue 10 or 15. The only exception is the mammalian binary complex with IP20. In this myristylated protein, the N terminus, including the acyl group, was folded into a hydrophobic pocket on the surface of the large lobe.
The N-terminal segment in molecule B was similar to previous structures, with the helix starting at residue 13. In molecule A, however, residues Gly1, Asn2, Ser10, Glu13, and Lys17 made tight contacts with a neighboring symmetry-related molecule. Ala3 and Lys16 also had weak hydrogen-bond interactions with a symmetry-related molecule. These intermolecular contacts may account for the formation and stabilization of the long N-terminal helix. Table 2 lists all of these intermolecular hydrogen bonds between the N terminus of molecule A and its symmetry-related molecule in the E230Q mutant structure. However, the packing environment for molecule B was different. None of these intermolecular interactions mentioned above occurred in molecule B or the previous C-subunit structures.
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, part of the A-helix (13–21) is bound at the hydrophobic groove where the N-terminal helix of the IP20 peptide would have bound. The interactions made by the A-helix (13–21) involved both hydrophobic contacts and hydrogen-bonds, but were different from those found by the N-terminal helix of IP20 (Fig. 6). |
Discussion |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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A comparison of these two residues, Glu230 and Tyr204, which converge at the same site, provides novel insights into the mechanisms by which the structure and function of this enzyme are integrated and potentially provide some new ways of thinking about the larger enzyme family. Glu230 and Tyr204 come together at the site that recognizes the P-2 Arg in substrate and inhibitor peptides (Fig. 1). Each residue also integrates distant parts of the protein with the active site in novel ways. Tyr204 in the P+1 loop is part of the hydrophobic core. It not only reaches over to the P-2 Arg site through its hydrogen bonding on the surface with Glu230, but also helps to anchor the catalytic loop through hydrophobic interactions with the backbone of Lys168 and with the side chain of Leu169 (Fig. 1). Glu230, in contrast, is part of the surface continuum of negative charges that extend from the activation loop (P-Thr197), through the active site (Asp166 and Asp184), and to the peptide binding site (Glu170, Glu127, and Glu203) as well as the highly dynamic C-terminal tail with its six acidic residues surrounding Tyr330 (Fig. 5). Its location at the center of this electrostatic surface allows Glu230 to play a key role in orchestrating catalysis, as was predicted computationally (Tsigelny et al. 1996). Both residues are also highly conserved in many protein kinases, even when there is not always a requirement for a P-2 Arg or P-3 Arg in the peptide substrate. Mutation has allowed us to appreciate the different contributions that these two residues make. Replacement of Glu230 with Gln retains the geometry of the site but alters the electrostatic properties, while replacement of Tyr204 with Ala not only abolishes hydrogen bonding but also removes the hydrophobic contributions. The general catalytic mechanism for PKA indicates that ATP and substrate bind to the enzyme in a random fashion with a preference for ATP binding preceding peptide binding (Cook et al. 1982; Whitehouse et al. 1983; Adams and Taylor 1992). Much evidence supports the notion that the C-subunit exhibits an ensemble of open and closed conformations in solution and binding of ATP and peptide synergistically promote a closed conformation, thereby creating the right conformational state for phosphoryl transfer (Taylor et al. 2004). Based on thermostability of the apoenzymes, the Tyr204 mutation shows a 10° reduction in Tm, while in its apo-form E230Q is comparable to the wild-type C-subunit (Yang et al. 2005). However, one of the characteristic features of the C-subunit is the synergistic binding of ATP and the inhibitor proteins, PKI or RI. Each binds with high affinity in a highly synergistic manner and this is reflected in the increase in thermostability (48.9° for free C-subunit vs. 61.2° for the C:PKI:MgATP complex) (Yang et al. 2005). The Y204A mutant shows this same synergy even though the stability is less (42.3° vs. 51.6°). However, the E230Q mutant showed little enhanced stability. This is consistent with the structure that we observed. Even when MgATP and IP20 were present in millimolar concentrations, the mutant crystallized in its apo form. The biophysical differences that result from these two mutations are also reflected in their hydrogen/deuterium exchange properties (Yang et al. 2005). While apo E230Q is similar to wild-type C-subunit, consistent with its surface location, Y204A alters the long-range allosteric network that permeates the large lobe by causing enhanced deuterium exchange in the catalytic loop, in the G helix, and in the loop that joins the H and I helices (Yang et al. 2005). The Glu to Gln mutation retains the geometry of the mutant, but not the electrostatic surface.
Electrostatic force is the only strong long-range interaction involved in protein-substrate recognition. Electrostatic contributions can direct a substrate toward the active site by "precollision electrostatic guidance" (Cudd and Fridovich 1982; Getzoff et al. 1983). Along the substrate-binding groove of the C-subunit, the cluster of acidic residues includes Asp127, Glu170, Glu203, and Glu230. Their roles in guiding the positively charged substrate to the enzyme and stabilizing the interactions are clear. Molecular dynamic calculation predict that the charge-to-neutral mutation of one of these residues, such as Glu230, not only affects the substrate binding, but also affects the entire electrostatic environment of the active site, so that this long-distance change could affect the pKa of the P-site Serin the substrate peptide (Tsigelny et al. 1996). Although mutation of Glu230 to Gln did not cause major perturbation in the overall structure of the enzyme (Yang et al. 2005), it does appear to have a major effect on how this molecule is seen from the outside and on its ability to interact with other proteins or peptides. This is caused by the change in the electrostatic potential and is also reflected in the different crystal packing for the E230Q mutant.
The other unusual feature of this structure is the extension of the A helix. This extended helix emphasizes again that this N-terminal motif is quite dynamic and likely has different binding partners. The N-terminal 39 residues that precede the core are conserved in all mammalian C-subunits, but there are many splice variants for the first exon, which include residues 1–14. At the time this structure was first solved, work by Breitenlechner et al. (2004) was published. They generated a four-site mutation of the C-subunit that crystallized as a ternary complex in the same P212121 space group seen previously with different crystal contacts compared with the previous crystal structure of the wild-type protein. Most of those mutations were of hydrophobic residues within the ATP-binding pocket. The investigators concluded that lack of phosphorylation at Ser10 is critical for the observation of the entire N-terminal helix and proposed the biological relevance of the helical conformation. In the current E230Q structure, for the two molecules in the asymmetric unit, molecule A has an ordered N terminus, but molecule B’s N terminus is disordered. In our previous 1.26 Å structure of Tyr204 to Ala mutant, even though Ser10 is not phosphorylated, the N-terminal residues 6–14 were disordered (Yang et al. 2004). Furthermore, in the previously solved myristoylated mammalian C-subunit structure, the N terminus was disordered (Fig. 7). Thus, in the absence of any mutations, we can only conclude that the whole N terminus indeed has the propensity to assume a helical conformation, although the absence of phosphorylation of Ser10 is not sufficient to promote helix formation of the N terminus. Crystal packing and other effects introduced by mutations may all contribute to the observation of an extended N-terminal helix.
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B, a transcription factor, led to the prediction that PKA plays a role in regulating NF-B activity through a cAMP-independent mechanism (Zhong et al. 1997). The recent identification of a novel nuclear A kinase interacting protein (AKIP) that binds the A helix of the C-subunit, introduces the possibility that this region can also contribute to targeting or localization (Sastri et al. 2005). The wild-type and several mutants of the PKA catalytic subunit, which all lack the phosphorylation at Ser10, still show variable conformation in the N terminus (Fig. 7). Furthermore, several crystal contacts are made by the N-terminal residues when the N terminus forms a completely ordered helix. Thus, the extension of the Helix A to the N terminus is most likely due to crystal packing. From the available data one cannot conclude that the absence of phosphorylation at Ser10 alone will induce an extended helix. Certainly these studies all support the conclusion that the N terminus is highly dynamic and an extended helical structure can be switched from a disordered conformation or from the previous structure of the myristoylated protein where the N-terminal segment is folded into a hydrophobic pocket (Zheng et al. 1993b). This potential feature could lead to PKA adopting its extended helical conformation when associated with membranes or bound to the other binding partners.
So far we have seen three conformations for the first 15 residues. The mammalian enzyme has no phosphate on Ser10, is myristoylated at Gly1, and has an Asn at position 2. This structure shows the N terminus folded into a highly conserved hydrophobic pocket (Fig. 7). The same enzyme purified from mammalian tissue with deamidation at Asn2 showed a disordered N-terminal. Deamidation of Asn2 appears to disrupt the hydrophobic capping. All of the recombinant proteins that lack myristoylation show residues 1–12 to be disordered, with the exception of these two mutants discussed here, which show an extended -helix (Fig. 7). Fluorescence anisotropy studies predicted a highly dynamic myristoylation switch and predicted, furthermore, that the dynamic behavior of the N terminus is enhanced in an isoform specific manner by binding to RII
but not RI. This also promotes association with membranes. Thus, many lines of evidences point to a highly dynamic N terminus that likely has a significant biological function.
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Materials and methods |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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-ME at 4°C overnight and concentrated to 8 mg/mL. The protein concentration was measured by a Bradford assay using bovine serum albumin as standard. The inhibitor peptide IP20 was synthesized at the Peptide and Oligonucleotide Core Facility at University of California at San Diego (UCSD) and purified by HPLC.
Crystallization and data collection
To obtain ternary complex crystals, a mixture of E230Q mutant with ATP, MgCl2, and IP20 was prepared in molar ratios of 1:5:10:10, then crystallized using the hanging drop vapor-diffusion method. Crystals were harvested into the reservoir solution (13% MPD, 11% MeOH, 0.1 M Bicine at pH 8.0) at 4°C. The crystallization condition is similar to that used for the wild-type C-subunit (Madhusudan et al. 2002). Crystals were flash-frozen in the liquid nitrogen stream mounted by nylon loops (purchased from Hampton) after dipping them in cryo-protectant solution (15% MPD, 15% glycerol, 0.1 M Bicine at pH 8.0).
Diffraction data were collected up to 2.8 Å resolution using one single crystal on a MarResearch Imaging Plate-345 detector system mounted on a Rigaku RU-200 rotating anode X-ray generator operated at 100 mA and 50 KV. This crystal form, different from all of the earlier crystal forms of C-subunit, belongs to ternary space group P42 with the unit cell dimensions of a = b = 120.3 Å, and c = 58.7 Å (Table 1). There are two molecules in an asymmetric unit (Vm = 2.7 Å3/Da). Data were processed using DENZO and scaled with SCALEPACK (Otwinowski and Minor 1997).
Structure solution and refinement
The phasing was conducted using the molecular replacement method by applying the program Amore of CCP4 suite (CCP4 1994). Crystal structures of the C-subunit in ternary complex form containing PKI (5–24) and Mn:ATP (PDB ID 1ATP
[PDB]
; Zheng et al. 1993a) and in binary form complexed with the inhibitor peptide (PDB ID 1CTP
[PDB]
; Zheng et al. 1993b) were used as the search models, respectively. The R-factor was significantly lower for a solution when using 1CTP (42.4%) than 1ATP (46.3%). The overall electron density of small lobe, especially the glycine-rich loop, is also much better when using 1CTP as the starting model. The temperature factors for all of the atoms were set to 25 Å2, and the occupancy values were set to 1.0.
The model was refined with CNS protocols using a maximum-likelihood target function (Brünger et al. 1998). The graphics software TURBO-FRODO (A. Roussel and C. Cambillau, Silicon Graphics Inc.) was used for model building. Five percent of the reflection data were randomly selected as a test set used for cross-validation. Rigid body refinement was carried out, leading to an R-factor of 0.273 and Rfree of 0.329 at 2.8 Å resolution. Structure was then refined for both simulated annealing from 5000°C and individual B-factor for a number of rounds. A noncrystallographic symmetry (NCS) restraint between molecules A and B in the asymmetric unit was performed for the whole model initially, and then those residues in different orientation, either main chain or side chain, were released from the NCS restraint. The final structural model gave an R-factor of 0.202 and Rfree of 0.249. The refinement statistics are summarized in Table 1. Ramachandran plot (Ramachandran and Sasisekharan 1968) using PROCHECK (Laskowski et al. 1993) revealed that all of the nonglycine residues are located in the allowed regions, with 86.8% in the most favored regions.
Accession number
The coordinate and structure factor are deposited with the Protein Data Bank (PDB ID 1SYK
[PDB]
).
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Acknowledgments |
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References |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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