Identification of Substrate Binding Site of Cyclin-dependent Kinase 5*

Pushkar Sharma, Peter J. SteinbachDagger , Monica Sharma, Niranjana D. Amin, Joseph J. Barchi Jr.§, and Harish C. Pant

From the Laboratory of Neurochemistry NINDS, the Dagger  Center for Molecular Modeling, Center for Information Technology, and the § Laboratory of Medicinal Chemistry, NCI, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cyclin-dependent kinase 5 (CDK5), unlike other CDKs, is active only in neuronal cells where its neuron-specific activator p35 is present. However, it phosphorylates serines/threonines in S/TPXK/R-type motifs like other CDKs. The tail portion of neurofilament-H contains more than 50 KSP repeats, and CDK5 has been shown to phosphorylate S/T specifically only in KS/TPXK motifs, indicating highly specific interactions in substrate recognition. CDKs have been shown to have a high preference for a basic residue (lysine or arginine) as the n+3 residue, n being the location in the primary sequence of a phosphoacceptor serine or threonine. Because of the lack of a crystal structure of a CDK-substrate complex, the structural basis for this specific interaction is unknown. We have used site-directed mutagenesis ("charged to alanine") and molecular modeling techniques to probe the recognition interactions for substrate peptide (PKTPKKAKKL) derived from histone H1 docked in the active site of CDK5. The experimental data and computer simulations suggest that Asp86 and Asp91 are key residues that interact with the lysines at positions n+2 and/or n+3 of the substrates.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cyclin-dependent kinases (CDKs)1 phosphorylate proline-directed serine and threonine residues in peptide/protein substrates. These kinases are active only when bound to their regulatory partners, cyclins. However, maximal kinase activation is achieved only after phosphorylation of these kinases by CDK-activating kinase (1 and references therein). CDK5 is slightly different from other CDKs; it is active only in neuronal cells because of the presence of its cyclin-like neuronal activator p35 and does not seem to require phosphorylation for its catalytic activity (1-3). However, like other CDKs, it phosphorylates S/T in S/TPXK/R motifs unique to the CDK family of kinases (4, 5). The specificity of this interaction is demonstrated by the phosphorylation of the tail portion of high molecular weight neurofilament-H (NF-H) by CDK5. The tail portion of this molecule contains 52 KSP repeats that can be classified as either KSPXK or KSPXXX. CDK5 specifically phosphorylates only KSPXK motifs and does not phosphorylate any other KSP site (4, 5). Histone H1 is phosphorylated in vitro at a KTPKK motif (6). These studies indicate that the CDKs have high affinity for a basic residue like lysine or arginine at the n+3 position of the sequence, where n is the position of the phosphoacceptor serine or threonine. The mechanism of activation of CDK2 by cyclin A was revealed by the crystal structures of CDK2 (7) and its complex with cyclin (8). A recently published structure of a cyclin A-CDK2 complex phosphorylated by CDK-activating kinase demonstrated the structural basis for further activation by phosphorylation (9). However, because of the lack of a crystal structure of a CDK-peptide substrate complex, the highly specific interactions addressed above are not well understood.

Alignment of more than 60 protein kinases (PKs) revealed the minimal catalytic domain to be ~ 260 amino acids; this group exhibited 20-60% sequence homology (10). The crystal structures of the PKs solved to date reveal that the sequence homology and secondary structural elements are conserved at the functionally important sites, such as those involved in ATP binding and catalysis. When these amino acids are used to align the PKs, the major differences occur in the regions coding for loops (10). Despite their homologous sequences and similar global structures, PKs differ surprisingly in the substrate sequences they recognize, indicating highly specific interactions between the active sites of the enzyme and the substrates.

cAMP-dependent protein kinase (PKA) was the first kinase to be crystallized without (11) and with (12) a RRXS/A pseudo substrate peptide (PKI). Consequently, the crystal structure of the PKA·PKI complex has been used as a model kinase-substrate system. The recently published structure of phosphorylase kinase with a peptide substrate further confirms the specificity of these interactions (13). These structures reveal a highly specific network of interactions between the charged residues in the substrate with their oppositely charged partners in the enzyme. The differences in amino acid composition in the catalytic clefts of PKs account for differences in substrate specificity for these kinases (14, 15).

To understand the key interactions involved in this highly specific enzyme-substrate interaction, we have used site-directed mutagenesis, reaction kinetics, and molecular modeling. We constructed a homology model of CDK5 using the CDK2 coordinates from the cyclin-CDK2 crystal structure (9) and docked a peptide derived from histone H1, which is an in vitro substrate for CDK5 and other CDKs (6). Site-directed mutagenesis was used to confirm the interactions suggested by the computer modeling. Further refinement of the model included inter-residue restraints suggested by the enzyme kinetics measurements of the designed CDK5 mutants.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

All fine chemicals were purchased from Sigma. The peptide substrates were synthesized by either Research Genetics or QCB Chemicals. [gamma -32P]ATP was purchased from NEN Life Science Products.

Expression and Purification of Proteins-- GST-CDK5 in pGEX 2TK was constructed by putting the BamHI fragment of His-tag CDK5 (3) into BamHI-cut pGEX 2TK. GST-p25 in pGEX 4T-2 was constructed by a polymerase chain reaction method using the oligonucleotides 5'-GTCCGGATCCGCCCAGCCCCCGCCG-3' as a forward primer, 5'-GTGATGAATTCTGGATCACCGATC-3' as a reverse primer, and DNA from the His-tag p35 construct as a template (3). The polymerase chain reaction products were digested with BamHI and EcoRI, and the resulting fragments were cloned into BamHI-EcoRI-cut pGEX 4T-2. The proteins were expressed as described earlier (4, 5). Purification of GST fusion proteins was carried out using glutathione-agarose chromatography by standard procedures (Amersham Pharmacia Biotech) and as described earlier (5). The purity of the proteins was assessed by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining of the gels. The protein concentration was estimated by densitometric analysis of these gels.

Site-directed Mutagenesis-- The "charged to alanine" mutations were made using a commercial kit (Quick ChangeTM, Stratagene). In brief, a pair of complementary primers of 30-40 bases was designed with desired mutations placed in the middle of the sequence. Parental cDNA inserted in pGEX 2TK was amplified using Pfu DNA polymerase with these primers for 15 cycles in a DNA thermal cycler (Perkin-Elmer). After digestion of the parental DNA with DpnI, the mutants were transformed into Escherichia coli (DH5alpha strain, Life Technologies, Inc.). The mutations were confirmed by DNA sequencing. The GST fusion mutant proteins were expressed and purified as described above.

Phosphorylation Assay and Enzyme Kinetics-- Standard assay mixtures contained 50 mM Tris, pH 7.4, 2.5 mM MgCl2, 1 mM EGTA, 150-165 ng of wild-type CDK5 or different mutants complexed with a molar excess of p25 (preincubated enzyme preparation), 100 µM [gamma -32P]ATP, and 0.1-10 mM peptide substrate in a total volume of 30 µl. Reactions were initiated by adding [gamma -32P]ATP and were carried out at 30 °C for 60-80 min. Under these conditions, the rate of phosphorylation was constant for more than 2 h and proportional to the amount of enzyme. Reactions were terminated by adsorption of the assay mixture on phosphocellulose paper P81(Whatman). Phosphopeptide formation was measured by counting the radiolabeled 32P incorporation after several washings of the phosphocellulose paper with 75 mM phosphoric acid. Kinetic constants were derived by fitting the Michaelis-Menten equation to the data using the Kaleidagraph program. Each experiment was done at least twice. Representative kinetic constants are shown in Tables I and II, and the variation in these constants from one experiment to another was typically 10-15%. The apparent peptide affinities are compared on the basis of the Km and Vmax/Km values.

Molecular Modeling-- Using the program LOOK (16), CDK5 was aligned to CDK2 (9), and a model of CDK5 was built exploiting its high homology to CDK2 (62% identity). Atoms of CDK5 were assigned coordinates using the SegMod algorithm (16). An ATP molecule and magnesium were docked to CDK5 by copying the corresponding coordinates from the CDK2 complex.

The CDK5 model was then superimposed on the crystal structure of PKA (pdb file 2CPK) by best-fitting only the backbone (N, Calpha , C) atoms in the 13-residue "catalytic loops." Of particular interest was the close proximity (1.0 Å separating Calpha atoms) of Asp86 in CDK5 to Glu127 of PKA. The Glu127 of PKA forms a salt bridge with Arg18 of PKI (2cpk), and Arg18 of PKI corresponds to Lys6 of the H1 peptide in the antiparallel alignment (Fig. 1B). This structural alignment and subsequent modeling of CDK5 complexed with the H1 peptide were performed with the program CHARMM (17) and an all-atom parameter set (18, 19) running on Hewlett-Packard workstations.


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Fig. 1.   Panel A, sequence alignment and comparison of CDK2, CDK5, and ERK2. The residues mutated in CDK5 are underlined. Panel B, alignment of H1 peptide in parallel and antiparallel orientations. Thr3 of H1 and Ala21 of PKI are the phosphorylation sites. Basic residues that are aligned are boxed.

In modeling the CDK5·H1·peptide complex, several approximations were employed. A distance-dependent dielectric (epsilon  = r) coefficient was used to screen electrostatic interactions with a potential energy "shifted" to zero at 12 Å. The shifted potential monotonically damps the electrostatic forces for this dielectric model (20). The distance-dependent dielectric is a rather crude approximation to an implicit solvent, but it maintains reasonable hydrogen bond distances.

Entropic effects were ignored, and a limited (non-ergodic) search of conformations was performed. In other words, we did not attempt to solve the ab initio docking problem, which would require a more accurate energy function and more exhaustive conformational sampling. Rather, the goal of the modeling was to aid in the choice and interpretation of experiments. Although our electrostatic approximations overly stabilize salt bridges, our intent was to enforce these interactions, not to predict them from first principles. The details of the peptide backbone conformation and side chain packing should not be overinterpreted, but our model can be used to test "lower resolution" hypotheses. For example, we can predict whether the H1 peptide is long enough and flexible enough to interact simultaneously with the ATP and a specific subset of CDK5 residues. Thus, prospective interactions between CDK5 and the peptides identified in early stages of the modeling led us to perform specific mutation experiments. Similarly, the measured enzyme kinetics were incorporated into later stages of modeling through the addition of inter-residue distance restraints favoring specific salt bridges between CDK5 and the H1 peptide.

Multiple families of short Monte Carlo (MC) simulations were run in succession, differing in the moves attempted and the restraints employed. In the latter stages of the modeling, protein residues near the peptide were free to move, but in each MC simulation the CDK5 molecule was held fixed while random rigid body translations and rotations of the entire H1 peptide were explored as well as random rotations of the peptide dihedral angles. Configurations were energy minimized somewhat to relieve bad contacts before applying the Metropolis criterion (21) for accepting conformations at a temperature of 600 K. A subset of torsions was included in the MC move set, but all peptide degrees of freedom were allowed to relax upon minimization. Positional restraints were used to guide the docking of the H1 peptide in a configuration antiparallel to that of PKI binding to PKA. Again, this configuration was investigated because it allows interaction between Asp86 of CDK5 and Lys6 of the peptide. In each MC simulation, a restraint was used to keep the gamma oxygen of the peptide Thr3 close to the third phosphate group of the ATP molecule.

H1 Peptide Docked in the Antiparallel Orientation-- The goal of the first family of MC simulations was to dock only the first several residues of the H1 peptide. 100 runs were started with the peptide in an extended conformation near the ATP molecule. Inter-residue distance restraints were used to favor energetically four interactions between a CDK5 residue and a peptide residue: Asp86 and Lys5, Asp86 and Lys6, Asp125 and Lys2, and Asp143 and Lys2. Only 11 phi  and psi  torsions (excluding proline phi  angles) of the first seven residues were eligible for rotation during MC moves.

The lowest energy conformation obtained from these simulations was used to initiate the second family of 50 MC runs. The goal here was to guide the peptide toward an orientation like that of PKI bound to PKA. Two additional inter-residue restraints were added: Asp91 and Lys8, Glu193 and Lys9. Only the 11 phi  and psi  angles not involving the first four residues of the peptide were moved by the MC procedure.

Next, MC simulations were performed with both backbone and side chain dihedral angles included in the move set, but the fixed CDK5 molecule resulted in the acceptance of very few moves. Therefore, the protein was made increasingly flexible in the neighborhood of the peptide as follows. First, simulated annealing was employed during which the peptide and all atoms within 5 Å of it were free to move. The system was heated to 600 K in 20 ps and cooled to 100 K over 230 ps and energy minimized. Next, 100 ps of molecular dynamics at 300 K was simulated during which all residues with any atom within 5 Å of the peptide were in motion. The 100 structures saved (1/ps) were energy minimized to a root mean square gradient of 0.01 kcal/mol/Å. Finally, all residues in the lowest energy structure with any atom within 10 Å of the peptide were freed of constraints and energy minimized to a root mean square gradient of 0.001 kcal/mol/Å. The resulting structure is depicted in Fig. 2.


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Fig. 2.   Model of CDK5 after H1 peptide (not shown) was docked as described under "Experimental Procedures." The ATP (red) and magnesium ion (blue) are shown. Side chains of acidic residues potentially involved with the peptide binding in the antiparallel (cyan) and parallel (yellow) configuration are represented as balls and sticks. The PSSALRE helix (Pro45-Leu55) and the loop (Phe150-Asp170) analogous to the T-loop of CDK2 are colored purple. Glycines 11, 13, and 16 of the glycine-rich motif are colored green. This figure was produced using the programs Molscript (30) and Raster 3D (29).

H1 Peptide Docked in the Parallel Orientation-- Docking of the H1 peptide in an orientation parallel to that of PKI relative to PKA was also investigated (Fig. 1B). Analogous to the first family of MC runs described above, 40 simulations were performed during which inter-residue restraints were applied between Asp86 and Lys2, Asp143 and Lys5, and the ATP and Thr3. Again, 11 phi  and psi  torsions of the first seven residues were rotated during MC moves. The lowest energy structure was inspected visually. Aside from Asp143 and Asp125, the only acidic residues within reach of the terminus of the peptide were observed to be in two clusters. The first includes Asp38, Asp39, Asp40, Asp41, and Glu42. Based on the cyclin A-CDK2 structure, it seems likely that these residues are buried in the CDK5·p35 complex. The other cluster includes Glu160, Asp206, Asp208, Asp209, and Asp234 (9).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Homology Model of CDK5-- It is well known that CDKs are not active on their own; they require binding to regulatory cyclins to be activated (1). However, maximal activation is achieved after phosphorylation at Thr/Ser in the T-loop of most CDKs. In the case of CDK2, it is Thr160 in the cyclin A-CDK2 complex. This complex has been crystallized in both phosphorylated (9) and unphosphorylated (8) forms. These crystal structures help elucidate the mechanism of activation of CDKs. Briefly, the binding of cyclin A to CDK2 causes the T-loop (146-166) to move 21 Å from its position in the CDK2 structure, thereby opening the catalytic cleft, allowing access to the substrate, and orienting ATP in a position that favors phosphotransfer (8, 9). The main regulator of CDK5 is a neuron-specific protein p35 or its truncated form p25 (2, 3), accounting for the activity of this enzyme mainly in neurons. p35 does not have any sequence similarity to cyclins. Sequence homology among cyclins is rare; even two structurally similar cyclin motifs typically have very different sequences (22, 23). Recent studies by two groups have suggested that p35 consists of a cyclin-like fold (24, 25); however, unlike the regulators of other CDKs, p35 seems to achieve maximal activation of CDK5. Other CDKs seem to be catalytically active only upon phosphorylation in the T-loop subsequent to cyclin binding (1, 2). A recent crystal structure of cyclin A-CDK2 in this phosphorylated state has revealed that the T-loop moves further away from the catalytic cleft upon phosphorylation at Thr160 in the T-loop (9). There seems to be no difference in substrate specificity caused by this effect; it only increases the rate of phosphorylation. Therefore, we assume that the active conformation of CDK5 is similar to that of CDK2 in the cyclin A-CDK2 (phosphorylated) complex (9). Thus, we modeled CDK5 in its active form using coordinates from the cyclin A-CDK2 (1jst.pdb) complex using the program LOOK (as described under "Experimental Procedures") taking advantage of the 62% sequence identity and a high sequence homology between these two kinases (Fig. 1A). The CDK5 model exhibits a bilobal structure (Fig. 2), a conformation representative of protein kinases. The small lobe is rich in beta -sheets and contains the glycine-rich motif important for ATP binding. The ATP binding site is common to the entire PK family.

The N-lobe of CDK5 also contains the PSSALRE helix, which corresponds to the PSTAIRE helix in CDK2. This helix is another feature found in all CDKs and is important for interaction with cyclins. The PSTAIRE helix rotates by several Å into the catalytic cleft (7- 9) upon cyclin A binding to CDK2. Residues 150-170 in the model of CDK5 are analogous to the T-loop in CDK2. Three conserved residues, Lys33, Glu51, and Asp143, form a catalytic triad that helps orient ATP and facilitate catalysis. These interactions are similar to those found for PKA and other PKs (10). Mutation of Lys33 to Ala in case of CDK5 abolishes the kinase activity, implicating the importance of this residue in catalysis since the salt bridge between Lys33 and Glu51 is broken (3). The bigger C-lobe is predominantly helical but contains loops that are important for interaction with suc family proteins (26) and also the T-loop, which is important for the kinase activity as described above. The catalytic site of the enzyme lies between the two lobes, enabling the ATP to transfer phosphate to Ser/Thr residues of the peptide substrate. The residues important for substrate binding have not been identified; but based on the PKA·PKI structure, some residues in the C-lobe have been suggested in substrate binding (9).

Peptides Bound to the Active Site of CDK5-- Both of the peptides studied here (H1 and NF-H, Table I) contain at least one S/TPXK motif, which is a consensus sequence for CDK family kinases. The H1 peptide used for docking was derived from histone H1 and contains the TPKK motif, which is the in vitro site phosphorylated by CDK5 in histone H1 (6). It is possible that this peptide has a higher affinity because of the presence of 5 lysines (out of 10 residues) that interact with acidic residues in the catalytic cleft and with the ATP. We also used an NF-H-derived peptide for our enzyme kinetics studies with different mutants because NF-H is phosphorylated at similar SPXK motifs by CDKs. We showed recently that CDK5 has a higher affinity for the first KSPXK repeat (X = A) than for the second repeat (X = E) (5), consistent with the work of Beaudette et al. (6), who showed that CDK5 prefers X = K or R over X = neutral amino acid. When X =D or E, the peptides make very poor substrates.

                              
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Table I
Kinetic parameters of CDK5 mutants for peptide substrates
Phosphorylation assays and calculation of kinetic constants were performed as described under "Experimental Procedures." H1, histone peptide (PKTPKKAKKL); NF, neurofilament peptide (VKSPAKEKAKSPEK).

It is likely that the substrate in CDK5 or other CDKs binds to a site analogous to that observed in the PKA·PKI complex (12) because CDKs and PKA have similar overall structures and almost superimposable catalytic clefts that include the glycine-rich ATP binding domains and the catalytic base, Lys33. When the peptide substrate sequences are aligned with the PKI sequence in a parallel (conventional) fashion with the Ser/Thr matching the corresponding residue in PKI, none of the lysines of the CDK5 substrate peptides corresponds to any arginine of PKI (Fig. 1B). However, if the substrate sequences are aligned with PKI in an antiparallel fashion, the n+3 lysine of either NF or H1 peptide aligns with the n-3 arginine in the RRXS motif of PKI. For the H1 peptide, two other lysines align with PKI arginines in the antiparallel alignment (Fig. 1B).

Effect of Site-directed Mutagenesis of Residues Implicated in Substrate Recognition-- Charged to alanine scanning mutagenesis of CDK5, combined with kinetic analyses of the mutant enzymes, was used to investigate interactions involving acidic residues near the ATP binding pocket in our homology model of CDK5. As described above, Asp86 and Asp91 were implicated in binding to the peptide substrates. Independent mutation of these residues to alanine caused a 2-fold decrease in apparent affinity for the NF peptide (Table I). However, there was no significant difference measured in the Km for ATP binding to the D86A (Km = 140 µM) and D91A (Km = 210 µM) mutants compared with CDK5 (Km = 160 µM).

For the higher affinity H1 substrate, the decrease in apparent affinity was 5-fold for both CDK5 mutants (Table I). Assuming that the Asp to Ala mutations do not greatly affect the structure of CDK5, it would appear that both Asp86 and Asp91 form stabilizing interactions with both peptides, arguably salt bridges to one or more lysines. To identify the alleged salt bridge interactions between CDK5 and the H1 substrate, the D86A and D91A mutants were subjected to kinetic analyses with Ala-substituted H1 peptides in which either the n+2 or n+3 residue (Lys5 or Lys6) was replaced by alanine (Table II). We report Vmax/Km in Table II to represent the catalytic efficiency of the enzyme. Because, in general Vmax/Km decreases as Km increases, we interpret our results in terms of Km, which reflects apparent binding affinity.

                              
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Table II
Kinetic studies of CDK5 mutants with Ala-substituted H1 peptide
Phosphorylation assays and calculation of kinetic constants were performed as described under "Experimental Procedures." Alanine substitutions in H1 peptide are shown in bold. n represents threonine, the phosphoacceptor residue.

In H1 peptide binding to wild-type CDK5, the n+2 alanine substitution increased Km by a factor of 5, whereas the n+3 alanine substitution increased Km by a factor of 7 (Table II). These results are in agreement with reports that the presence of non-basic residues in the n+2 or n+3 position makes for a very poor substrate for CDKs (6, 14).

The Km values for H1 peptide binding to CDK5 and to the D86A mutant differ by a factor of 5. For the n+2 alanine peptide, these Km values differ by a factor of only 1.5. Thus, the n+2 alanine peptide is considerably less sensitive to changes in Asp86 than is the H1 peptide, suggesting that Lys5 of H1 probably interacts with Asp86 of CDK5. The weakening of the Lys5-Asp86 interaction was essentially independent of whether it was Lys5 or Asp86 (factor of 5) that was replaced by Ala (Table II).

For the n+3 alanine peptide, the Km values for binding to CDK5 and to D86A differ by a factor of 2.5 compared with the factor of 5 for the H1 peptide. The effects observed for Lys6 (n+3) are less dramatic than those discussed above for Lys5 (n+2). Still, the data suggest that Lys6 also interacts with Asp86. The proximity of Lys6 to Lys5 certainly makes this conclusion plausible.

When Asp91 was mutated to Ala, the reduction in apparent H1 affinity was comparable to that resulting from the D86A mutation, implicating Asp91 as another residue that interacts favorably with H1. D91A binds both the n+2 alanine and n+3 alanine peptides very poorly compared with wild type, suggesting that a second interaction has been weakened, i.e. that Asp91 interacts with an H1 residue other than Lys5 or Lys6. The data in Table II suggest that disruption of any one of the interactions between Asp86 or Asp91 and the peptide substrate results in a reduced apparent affinity but not a complete loss of binding. Multiple interactions stabilize the binding of the substrates to CDK5.

The simultaneous disruption of Asp86 and Asp91 interactions with the peptide was studied using a CDK5 double mutant (D86A/D91A). Relative to wild-type CDK5, this mutant showed an ~18 fold decrease in apparent affinity (Km = 0.7 mM) for the H1 peptide, and relative to either D86A or D91A, it showed a 3.5-fold decrease. These results are further evidence that these residues are important for peptide binding.

The modeled complex (Fig. 3) highlights the interactions implicated by the measured enzyme kinetics while suggesting additional interactions that may stabilize the complex. Our model suggests that Asp91 may interact with Lys8 and/or Lys9 of the substrate, which could account for the large Km values for the binding of the n+2 alanine and n+3 alanine peptides to D91A. To identify any interactions between Asp91 and Lys8 or Lys9, we measured enzyme kinetics with two additional Ala-substituted H1 peptides, n+5 alanine and n+6 alanine. The results indicate no increase in Km for the binding of these peptides to D91A (Table II). In comparison, the binding to wild-type CDK5 and D86A showed a 3-5-fold and 2-fold increase in Km, respectively. Taken together, the kinetic data for the n+5 alanine and n+6 alanine peptides support the possibility of interactions between Lys8 and Lys9 of the H1 peptide and Asp91 of CDK5, as was suggested originally by the molecular modeling.


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Fig. 3.   Panel A, modeled binding site of CDK5 with H1 peptide docked. The CDK5 surface is colored according to the electrostatic potential calculated with the program GRASP (31). Regions of negative potential are red; positive regions are blue. Selected residues of CDK5 visible at the surface are labeled in white; selected peptide residues are labeled in green. Panel B, stereo view of the H1 peptide residues from Lys5 to Lys9, as docked to CDK5. Asp86 (top) and Asp91 (bottom) are also shown. Each of the four Nzeta -Odelta distances shown (Lys5- Asp86, Lys6-Asp86, Lys8-Asp91, Lys9-Asp91) is between 2.5 and 2.6 Å.

In the model of the H1 peptide bound to CDK5 (Fig. 3), each of the peptide's five lysines interacts with one or more negatively charged groups. Additional salt bridges identified in the model involve the ATP, Asp143, and Lys88 (Table III).

                              
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Table III
Salt bridges involving H1 peptide in model of peptide docked to CDK5
terminal atoms are OT1 and OT2.

Effect of Mutation of Residues Implicated in Substrate Binding in the Parallel Orientation-- The crystal structure of the PKA·PKI complex can serve as a template for substrate binding. We modeled the H1 substrate peptide in an orientation comparable to that of PKI bound to PKA (Fig. 1B) with a distance restraint that kept the phosphoacceptor threonine within hydrogen bonding distance of the ATP. It appeared from the modeling (not shown) that the only likely salt bridge partners for Lys5 and Lys6 were Asp143 and Asp125. Other acidic residues in the vicinity of the substrate included Asp206, Asp208, Asp209, Asp234, and Glu160 (Fig. 2). We mutated each of these six residues (except Asp208) to Ala. The D143A and E160A mutants were not active. None of the other three Asp right-arrow Ala mutations resulted in a significantly altered Km value for H1 peptide (Table I). These data strongly support the conclusions based on the D86A and D91A data, namely that the peptide substrate binds in the antiparallel orientation.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

There is no available crystal structure of a CDK-substrate complex. Thus, the mechanism of this interaction is unknown. Because of the very high specificity of CDKs for the S/TPXK motifs, it is important to probe the mode of binding of these substrates. Although unambiguous interpretation of mutagenesis experiments is difficult, we assume that non-local conformational changes induced by mutations and many-body interactions can be neglected to a first approximation. We therefore interpret our mutagenesis data in terms of pairwise interactions, specifically salt bridges between CDK5 and the peptide substrates.

Based on the crystal structure of the PKA·PKI complex, it has been speculated that some of the acidic residues (e.g. Glu160, Glu206, Glu209) in the C-lobe of CDKs might interact with the essential lysines of the peptide substrate (9). The homology model and mutagenesis of CDK5 have implicated several acidic residues in the binding of peptide substrates: Asp86, Asp91, Asp125, Asp143, and Glu193 (Fig. 2A). The consensus sequence for PKA is RRXS/A. Therefore, Lys5 and Lys6 of the CDK5 substrates align well with the n-2 and n-3 arginines in an antiparallel alignment (Fig. 1B). Furthermore, Lys9 of the H1 peptide corresponds to the n-6 arginine.

The parallel alignment of H1 peptide to PKI is not as compelling because no lysines line up with the arginines of PKI (Fig. 1B). Preliminary docking of the peptide in this parallel orientation suggested the possibility of salt bridges involving residues Asp143, Asp206, Asp208, Asp209, Asp234, and Glu160. These acidic residues have been considered important in substrate binding by other reports (9). We mutated each of them (except Asp208) to Ala. There was no significant change in Km values for peptide interaction with these mutants except for D143A and E160A. For these mutants, there was no kinase activity, and determining the apparent peptide affinity was not possible. Along with Lys33, Asp143 orients the ATP in a conformation that facilitates phosphotransfer (7-9). It is thus not surprising that the D143A mutant is inactive. Glu160 is part of the flexible T-loop, and it may be that the conformation of CDK5 is especially sensitive to mutations in this region (Fig. 2).

The pairwise combinations of the D86A and D91A mutants (each defective in substrate binding) with Ala-substituted peptides suggest that n+2 and n+3 lysines interact with Asp86 in the KTPKK peptide substrate. Presumably, when binding to the D86A mutant, Lys5 and Lys6 interact with the ATP or Asp91. The first family of MC simulations suggested that Asp91 is within reach of Lys8 and Lys9 when the N2-terminal half of the peptide is docked to the ATP. Although these lysines are not part of the TPKK consensus CDK motif, the D91A mutant data suggest that a stabilizing interaction outside the TPKK region is present in the native system. Interestingly, in the case of other major proline-directed kinases, extracellular signal-regulated kinases (ERKs), the residue corresponding to Asp91 is a neutral residue (Fig. 1A). These kinases do not have any known preferences for charged residues following the proline (15). Recently, it was shown that the NF peptide and other related peptides containing n+3 as basic residues were very poorly recognized by ERKs (27) compared with the peptides with n+3 as nonbasic residues. A proline at the n+1 position is a common essential feature in substrates for both CDKs and ERKs. These kinases do not phosphorylate substrates without proline. It is possible that orientation of the serine hydroxyls for hydrogen bonding with ATP requires proline at the n+1 position. CDKs seem to require additional interactions with the substrate lysines at the n+2 and n+3 positions to stabilize the substrate-enzyme complex. Replacement of Lys5 or Lys6 with alanine decreases the apparent affinity for the peptide substrate (Table II). However, when both of these lysines are replaced by a neutral or acidic residue, the peptide is not phosphorylated by CDK5 (27). Our data and model also suggest additional stabilization of the complex by salt bridges involving Lys8 and Lys9 of the substrate and Asp91 of CDK5.

PKA and phosphorylase kinase have been the only two serine/threonine kinases to be crystallized as ternary complexes with ATP and their peptide substrates (12, 13). These two kinases show a high degree of structural similarity in their ternary complexes. Because the cyclin A-CDK2 (phosphorylated) crystal structure differs from PKA and phosphorylase kinase in several ways, it has been speculated that CDKs might have a different catalytic mechanism (10). A recently reported crystal structure of CDK2 complexed with staurosporine, an inhibitor of CDKs and other protein kinases, which binds at the ATP binding pocket, revealed that staurosporine interacts with Asp86 (28). This interaction suggested that the peptide substrate binding site of CDKs is very close to the ATP pocket, consistent with our observation that mutation of Asp86 results in a decreased apparent affinity for the peptide. Also, recent work in our laboratory has shown that a pseudo substrate analogue of the NF peptide was surprisingly competitive with ATP.2 Given the close proximity of the ATP and peptide binding sites, peptide binding at or near Asp86 could affect ATP binding. Interestingly, in the same study we showed that peptide binding promoted ATP binding.2 Taken together, these data suggest that a conformational change resulting from peptide binding could trigger events important for catalysis.

Because of the lack of a crystal structure of a CDK-substrate complex, the peptide substrate binding site has not been identified. Therefore, design of specific inhibitors for CDK family kinases has not been possible. Most of the available inhibitors bind in the ATP pocket, which makes them less specific since the ATP pockets of most PKs are structurally very similar. But in the case of CDKs, the interactions with the substrate are very specific; CDKs specifically target SPXK-type motifs. The enzyme kinetics and molecular modeling reported here indicate that the H1 peptide most likely binds to CDK5 in an orientation that is antiparallel to that of PKI bound to PKA. The binding site implicated by this study should help guide the design of specific inhibitors for CDKs.

    ACKNOWLEDGEMENTS

We thank Jim Neagle for assistance with DNA sequencing, Robert A. Pearlstein and R. W. Albers for thoughtful discussions, and Bernard R. Brooks for computational resources.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Laboratory of Neurochemistry, NINDS, National Institutes of Health, Bldg. 36, Rm. 4D20, Bethesda, MD 20892. Tel.: 301-402-2124; Fax: 301-496-1339; E-mail: hcp{at}codon.nih.gov.

2 P. Sharma, N. D. Amir, R. W. Albers, and H. C. Pant, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: CDK(s), cyclin-dependent kinase(s); NF, neurofilament(s); NF-H, high molecular weight NF protein; PK, protein kinase; PKA, cAMP-dependent protein kinase; PKI, cAMP-dependent protein kinase inhibitor; GST, glutathione S-transferase; MC, Monte Carlo; ERK, extracellular signal-regulated kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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