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Published online before print January 4, 2005, 10.1110/ps.04959705
Protein Science (2005), 14:445-451. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
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The mechanism of inhibition of the cyclin-dependent kinase-2 as revealed by the molecular dynamics study on the complex CDK2 with the peptide substrate HHASPRK

Iveta Bártová1, Michal Otyepka2,3, Zdenek KrÍz1 and Jaroslav Koca1

1 National Centre for Biomolecular Research, Faculty of Science, Masaryk University, 611 37 Brno, Czech Republic
2 Department of Physical Chemistry, Palacky University, 771 46 Olomouc, Czech Republic
3 Laboratory of Growth Regulators, Palacky University and Institute of Experimental Botany AS CR, 783 71 Olomouc, Czech Republic

Reprint requests to: Michal Otyepka, Department of Physical Chemistry, Palacky University, tr. Svobody 26, 771 46 Olomouc, Czech Republic; e-mail: otyepka{at}aix.upol.cz; fax: +420 585634425; or Jaroslav Koc a, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic; e-mail: jkoca{at}chemi.muni.cz; fax +420 549492556.

(RECEIVED June 29, 2004; FINAL REVISION October 13, 2004; ACCEPTED October 18, 2004)


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
Molecular dynamics (MD) simulations were used to explain structural details of cyclin-dependent kinase-2 (CDK2) inhibition by phosphorylation at T14 and/or Y15 located in the glycine-rich loop (G-loop). Ten-nanosecond-long simulations of fully active CDK2 in a complex with a short peptide (HHASPRK) substrate and of CDK2 inhibited by phosphorylation of T14 and/or Y15 were produced. The inhibitory phosphorylations at T14 and/or Y15 show namely an ATP misalignment and a G-loop shift (~5 Å) causing the opening of the substrate binding box. The biological functions of the G-loop and GxGxxG motif evolutionary conservation in protein kinases are discussed. The position of the ATP {gamma}-phosphate relative to the phosphorylation site (S/T) of the peptide substrate in the active CDK2 is described and compared with inhibited forms of CDK2. The MD results clearly provide an explanation previously not known as to why a basic residue (R/K) is preferred at the P2 position in phosphorylated S/T peptide substrates.

Keywords: cell cycle; CDK inhibition; phosphorylated tyrosine and threonine; glycine-rich loop; GxGxxG motif

Abbreviations: p denotes phosphorylation, i.e., pT160 is phosphothreonine 160 • G-loop, glycine-rich loop (CDK2 residues 11–) • JST, pT160-CDK2/Cyclin A/ATP • QMZ, pT160-CDK2/Cyclin A/HHASPRK/ATP • pT14-QMZ, pT14,pT160-CDK2/Cyclin A/HHASPRK/ATP • pY15-QMZ, pY15,pT160-CDK2/Cyclin A/HHASPRK/ATP • pT14,pY15-QMZ, pT14, pY15,pT160-CDK2/Cyclin A/HHASPRK/ATP

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04959705.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 References
 
Cyclin-dependent kinase 2 (CDK2) is a member of the eukaryotic S/T protein kinase family and its function is to catalyze the phosphoryl transfer of ATP {gamma}-phosphate to serine or threonine hydroxyl (denoted as S0/T0) in a protein substrate. CDK2 participates in eukaryotic cell cycle regulation at the G1/S boundary. CDK2 deregulation has been proved to occur in tumor cells, evoking a strong interest in artificial (Otyepka et al. 2000, 2002; Fischer et al. 2003; Meijer and Raymond 2003) and native (Davies et al. 2002) inhibitors. CDK2 activity is tightly regulated by a complex mechanism, including a positive regulatory subunit binding, and phosphorylations at positive and/or negative regulatory sites (Morgan 1997). For activation it requires a binding to Cyclin A or Cyclin E and phosphorylation of the T160 residue in the activation segment (T-loop) (Jeffrey et al. 1995).

The structure of CDK2 exhibits the classical bi-lobal kinase fold (Morgan 1997), where the N-terminal domain is composed mainly of the {beta}-sheet, containing five anti-parallel {beta}-strands, and one {alpha}-helix (the C-helix). The larger C-terminal domain is predominantly {alpha}-helical, and it is linked to the N-terminal domain by a flexible hinge. The catalytic cleft that natively binds ATP is located between both domains (Fig. 1Go). The ATP phosphate binding pocket is partly formed by the G-loop and it is believed that the primary function of the G-loop is to help in correct alignment of the ATP phosphate moiety for reaction. The G-loop motif (GxGxxG) is conserved in many kinases (Hanks and Quinn 1991). In the CDK2/substrate complexes, the substrate S/T is directly hydrogen bonded to the catalytic aspartate, D127, and to the conserved lysine, K129 (Brown et al. 1999; Cook et al. 2002). The first and the most important step of the ATP {gamma}-phosphate transfer to the substrate is the correct alignment of both molecules, i.e., the peptide and ATP-Mg2+, to favor an in-line mechanism for phosphoryl transfer.



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  		Figure 1. The picture of pT160-CDK2/Cyclin A/HHASPRK/ATP (QMZ) (coordinates taken from the PDB database: 1QMZ [PDB] code) represents the CDK2/Cyclin secondary structure. The T160 (shown in black colored licorice representation) activation site is located in the T-loop. The G-loop includes two possible inhibitory sites, T14 and Y15 (black colored licorice representation). Peptide substrate (HHASPRK), and ATP-Mg2+ are shown in licorice representation.

 
The peptide libraries’ analyses for CDK2 substrate preference have detected the sequence X-1(S/T)0P1X*2(K/R)3 to be an optimal substrate. Here, S/T are phosphorylation residues (serine or threonine), X is any amino acid, but K/R are favored at the X* position. Subscripts denote amino acid positions in the substrate numbered from the phosphorylation residue with increasing numbers toward the C-terminus (Songyang et al. 1994; Holmes and Solomon 1996). The published structure of pT160-CDK2/Cyclin A/ATP (JST) in complex with the substrate peptide comprising the optimal motif (HHASPRK; PDB code 1QMZ [PDB] ; Brown et al. 1999) explains the basis of the peptide binding to CDK2. The substrate peptide binds in an extended conformation across the catalytic site and its binding site is located close to the active site on the C-terminal lobe surface. Some parts of the binding site are formed by the G-loop and T-loop (Fig. 1Go). The T-loop forms a suitably shaped pocket to accept the substrate proline P1 residue, next to the phosphorylation residue (S0). The arginine R2 makes no contact with the protein and is directed to the solvent. The lysine K3 is H-bonded to the pT160 phosphate and to the Cyclin A I270 main chain oxygen. Interaction of the pT160 phosphate with lysine K3 explains the specificity for basic residues at this position. Histidine H–2 makes one H-bond from N{varepsilon} to the main chain of G205 (Brown et al. 1999), the glycine of the conserved CDK motif GDSEID of the CMGC protein kinase group (Hanks and Quinn 1991).

CDK2 can be deactivated by phosphorylation of Y15 and T14 residues in the glycine-rich loop (G-loop). This process is known from kinetics experiments (Coulonval et al. 2003) but the structural aspects of inhibition remain unclear. Endicott and Johnson deduced from a not yet published crystal structure of the pY15,pT160-CDK2/Cyclin A/ATP (pY15-JST) complex that the phosphorylation of the Y15 residue may perturb the protein substrate binding at the catalytic site through a steric hindrance (Endicott et al. 1999; Johnson and Lewis 2001). The structural aspects of the inhibitory phosphorylation of the pT160-CDK2/Cyclin A/ATP (JST) complex were recently studied by molecular dynamics simulations (Bártová et al. 2004), concluding that the inhibitory sites utilize a different mechanism of action. Phosphorylation of either T14 residue or both inhibitory sites’ T14 and Y15 residues together causes an ATP misalignment for phosphorylation and a G-loop conformational change, which leads to the opening of the CDK2 substrate binding box. On the other hand, the Y15 residue phosphorylation can lead to an incorrect ATP terminal phospho-group alignment for transfer to the CDK2 substrate. Consequently, we have proposed, similar to Endicott and Johnson, that the phosphorylated Y15 residue can negatively affect substrate binding (Endicott et al. 1999; Johnson and Lewis 2001).

The objective of this work is to study the dynamics of the QMZ system, i.e., fully active CDK2 in complex with HHASPRK (an optimal peptide substrate), namely interactions of CDK2 with peptide substrate and the dynamics of the G-loop. These findings are also compared with the behavior of QMZ systems inhibited by phosphorylation in the G-loop.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 References
 
The pT160-CDK2/Cyclin A/HHASPRK/ATP (QMZ) system
During the early stage (the first nanosecond) of QMZ simulation a minor conformational change of the ATP phosphate moiety is observed in comparison with its conformation in the crystal structure (Fig. 2Go). This reconformation causes a change in the position of the ATP terminal phospho-group, but this change does not influence its orientation to the peptide substrate S0 hydroxyl group. Thereafter, the conformation of the ATP phosphate moiety is stable during the remaining part of the molecular dynamics (MD) simulation. The Mg2+ ion is hexacoordinated by oxygen atoms from the ATP phosphate moiety (O{alpha}2, O{beta}1, and O{gamma}2 atoms), N132 (O{delta}1 atom), D145 (O{delta}2 atom), and by one water molecule during the whole production part (Table 1Go).



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  		Figure 2. Comparison (stereoview) of the MD averaged structure of QMZ (black) with the X-ray structure (gray; PDB code 1QMZ [PDB] ). The ATP and SPRK peptide substrate residues are shown in licorice representation and G-loop in tube representation.

 

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  		Table 1. Coordination of the Mg2+ ion was monitored during the entire simulation period of all trajectories; the results show that the magnesium ion was hexacoordinated during all simulations; also, changes of the ligands coordinating the Mg2+ ion are shown
 
The average distance S0-O{gamma}...P{gamma}-ATP between the ATP terminal phospho-group (P{gamma}) and the phosphorylation serine hydroxyl group (terminal O{gamma} atom) of the peptide substrate is 3.75 ± 0.70 Å, and this is close to the X-ray crystal structure value (3.7 Å). This distance sharply increases twice to 8.5 Å (1420–1870 psec, 10,750–11,200 psec) due to the movement of the N-terminal substrate peptide moiety (HHASPRK) away from the substrate binding box to the solvent. When the two above periods are excluded from the calculation, then the mean distance S0-O{gamma}...P{gamma}-ATP is equal to 3.63 ± 0.27 Å. Table 2Go collects the lengths of occurrences of the distance S0-O{gamma}...P{gamma}-ATP below the value of 3.90 Å (obtained as + s, i.e., 3.63 + 0.27 Å) during the simulation. The distance remains lower than the threshold during 87.7% of simulation time. The V164 residue exhibits a left-handed conformation ({phi}= 60 ± 9, {Psi}= 130 ± 11) during the whole simulation that agrees well with the X-ray study (Brown et al. 1999). This unfavorable conformation is stabilized by two H-bonds to R169 and R126. The K3 residue is strongly H-bonded to the pT160 phosphate group during the simulation. The R2 side chain moves toward the ATP phosphate moiety at 2.8 nsec of the MD simulation (Fig. 2Go). The H–2 makes a strong H-bond to the D206 side chain of the conserved CDK GDSEID motif.


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  		Table 2. The occurrence of distance S0-O{gamma}. . . P{gamma}-ATP below the value of 3.90 Å, and the mean value during the simulation
 
The G-loop, bearing residues crucial for appropriate ATP alignment for phosphorylation (Hanks and Quinn 1991), moves periodically during the QMZ simulation, causing opening and closing of the substrate binding box (Fig. 3Go). The G-loop shift is remarkable on five occasions during the simulation. Each shift away-and-back lasts ~1.2–1.8 nsec and occurs every 2–4 nsec (Fig. 4Go).



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  		Figure 3. Stereoview of superimposed snapshots taken every 1 nsec from MD simulations of QMZ (A) and inhibited systems pT14-QMZ (B), pY15-QMZ (C), and pT14,pY15-QMZ (D).

 


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  		Figure 4. The time dependence of distance between the G-loop (represented as the mass center of residues E12, G13, and T14) and the mass center of active site residues (D127, N132, and D145) for the QMZ (A) and phosphorylated systems pT14-QMZ (B), pY15-QMZ (C), and pT14,pY15-QMZ (D) simulations. Distances are calculated during equilibration and the production part of the molecular dynamics simulations.

 
The pT14-QMZ, pY15-QMZ, and pT14,pY15-QMZ systems
The phosphorylation at inhibitory sites in the G-loop affects the ATP phosphate moiety orientation. It causes its misalignment due to the ATP phospho-group conformational changes (the pT14-QMZ and pY15-QMZ complexes) or due to the shift of the ATP terminal phospho-group toward the ATP adenine base (the pT14,pY15-QMZ complex) (Fig. 5Go). These changes lead to an increase in the S0-O{gamma}...P{gamma}-ATP distance and a significant decrease of the optimal distance occurrences (Table 2Go).



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  		Figure 5. Stereoview of the superimposition of QMZ (black) and complexes phosphorylated in the G-loop (gray); pT14-QMZ (A), pY15-QMZ (B), and pT14,pY15-QMZ (C). The ATP, phosphorylated residues (T14 and/or Y15), and SPRK peptide substrate residues are shown in licorice representation and the G-loop is shown in tube representation.

 
Inhibitory phosphorylations affect also the Mg2+ ion coordination sphere (Table 1Go), resulting in the loss of Mg2+ ion coordination by N132 (O{delta}1). This ligand is replaced by D145 (O{delta}1) in pT14-QMZ during the whole simulation. In pY15-QMZ the replacement takes 6 nsec, then D145 (O{delta}1) is replaced by a water molecule. The phosphorylations cause N132 side chain reconformation, leading to a change of the H-bonds network within the CDK2 active site. The N132 (O{delta}1) creates a new H-bond to the conserved K129 (N{xi}) residue and the N132 (N{delta}2) makes a new H-bond to the catalytic aspartate D127 (O{delta}1 ({delta}2)) residue. The Mg2+ ion co-ordination by N132 (O{delta}1) is replaced by an oxygen atom from the water molecule (during the whole simulation) in the pT14,pY15-QMZ system (Table 2Go). The Mg2+ ion coordination by water is replaced by the ATP (O{gamma}3) at 2.1 nsec of the MD simulation in the pT14,pY15-QMZ in relation to a shift of the ATP terminal phospho-group toward the ATP adenine base.

The interaction of the K3 residue with pT160 phosphate is not significantly affected by inhibitory phosphorylations in the G-loop. However, the R2 residue in pT14-QMZ moves toward the ATP phosphate moiety in the same way as in the QMZ system and its positively charged terminal group exhibits electrostatic interaction with the negatively charged pT14 terminal phospho-group. The Y15 phosphorylation causes the R2 residue to interact strongly with the pY15 phospho-group, blocking the R2 motion toward the ATP (Fig. 5Go). In the pT14,pY15-QMZ system, the R2 residue forms two H-bonds to phospho-groups of both pT14 and pY15. The inhibitory phosphorylations decrease interaction between H–2 (N{delta}1) and the D206 terminal carboxyl group. The phosphorylations cause a remarkable shift of the G-loop during the first 2 nsec of the MD simulations in all studied systems (pT14-, pY15-, and pT14,pY15-QMZ; Figs. 4Go, 5Go). The distances between mean G-loop positions (calculated for snapshots between 200 psec and the end of MD simulations) in the QMZ structure and in the pT14-, pY15-, and pT14,pY15-QMZ systems were equal to 5.3, 5.5, and 6.2 Å, respectively.


   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 References
 
The pT160-CDK2/Cyclin A/HHASPRK/ATP (QMZ) system
The 15-nsec-long MD simulation of the QMZ system reveals some new aspects not known from the previous simulations on the JST system. Particularly interesting is the G-loop movement that seems to be periodical. This motion is only observable at nanosecond or longer time scales. This is the reason why it was not detected in our previous JST simulation lasting only 3 nsec (Bártová et al. 2004). The G-loop flexibility is facilitated by its primary sequence, which includes three highly conserved glycine residues (GxGxxG) in protein kinases. The GxGxxG motif is the part of the protein kinase ATP PROSITE motif (PS00107) and majority of known protein kinases bear it (Hanks and Quinn 1991). The primary function of each G-loop motif residue can be deduced from comparison of presented results with the most extensively investigated member of the protein kinase family, the cAMP-dependent kinase (PKA) (Johnson et al. 2001). Similarly to S53-PKA (Aimes et al. 2000), the primary function of T14 is not anchoring the ATP {gamma}-phosphate transition state but predominantly regulation, i.e., T14 serves as an inhibitory site. The G11 and G13 are H-bonded with V18 and G16, respectively, making the secondary structure of the N-terminal {beta}-sheet ({beta}-strand-turn-{beta}-strand) structure. Any mutation of G11 and G13 must lead to improper orientation of ATP due to sterical hindrance between side chains and ATP ribose (G11) or ATP {beta}-phosphate (cf. Grant et al. 1996 and Hemmer et al. 1997). Mutational study results show a gradation of functional importance for glycines of the GxGxxG motif, the last G16 being the least important and the first G11 being the most important. Some amino acids introduced to G16 can be accepted due to indirect influence on ATP through interaction of their side chains with K33, but residues larger than S cannot be readily accommodated (Odawara et al. 1989). We conclude that the conserved motif GxGxxG is an evolutionarily optimized one because it guarantees G-loop flexibility, good accessibility of the active site, and order due to formation of the secondary structure.

The HHASPRK peptide is tightly bound to the substrate binding box during the entire simulation. While the HHA- residues are the most flexible ones, the K3 residue is very rigid, staying tightly bound to pT160 residue. The preference of CDK2 for (R/K)2 (either R or K residues at the P2 position) observed from kinetic experiments (Holmes and Solomon 1996) cannot be deduced from the 1QMZ crystal structure (Brown et al. 1999), because R2 makes no contact with the protein, having its side chain oriented to the bulk solvent. In contrast, the MD simulation offers a simple explanation for the above preference. It is based on the idea that R2 interacts with the ATP phosphate moiety and, consequently, it can also play a role in appropriate ATP alignment before the reaction.

The pT14-QMZ, pY15-QMZ, and pT14,pY15-QMZ systems
Our previous study on systems inhibited by phosphorylation (pT14-, pY15-, pT14,pY15-JST) suggested a mechanism for inhibitory phosphorylation. It was proposed that inhibitory phosphorylation of either T14 residue or both inhibitory sites T14 and Y15 together causes ATP misalignment and a G-loop shift resulting in the opening of the substrate binding box. On the other hand, Y15 inhibitory phosphorylation did not affect the G-loop position but it led to ATP phosphate moiety misalignment. It was speculated that the interaction of pY15 with (R/K)2 peptide residue may result in substrate misalignment (Bártová et al. 2004).

The simulations of QMZ systems inhibited by phosphorylation show that the phosphorylation in all cases causes ATP phosphate moiety misalignment and changes in the Mg2+ ion coordination sphere, namely, the loss of N132 (a residue conserved in all protein kinases) coordination and G-loop shift away from the ATP binding site. However, it is a positive fact that the previously predicted interaction (from simulations with JST) between the pY15 phospho-group and R2 residue is observed in the pY15-QMZ system. The ATP misalignment resulting in terminal phospho-group reconformation is demonstrated in Table 2Go by increasing the S0-O{gamma}...P{gamma}-ATP distance. All mentioned effects clearly explain the lost of kinase activity after inhibitory phosphorylation of the CDK2 G-loop, because correct coordination of the Mg2+ ion and appropriate orientation and conformation of the ATP phosphate moiety are crucial for the phospho-group transfer to the serine S0 hydroxyl from the peptide substrate. However, it is not possible to decide whether the ATP misalignment and changes in Mg2+ ion coordination sphere are caused by G-loop shift or by electrostatic repulsion between two negatively charged phosphate groups at pT14/pY15 and ATP. It seems that both effects are involved. The insertion of one negatively charged phosphate group at the T14 position causes the R2 positively charged side chain to interact with this group and the interaction with ATP phosphates is significantly weaker. The phosphorylation of the Y15 residue (or both residues altogether) causes the R2 positively charged side chain to interact preferably with this group and the interaction with ATP phosphates is lost. On the other hand, the inhibitory phosphorylations at T14 and/or Y15 do not affect interaction of K3 with the pT160 side chain.

The biological consequences and importance for CDK2 regulation of the GEGTYG G-loop motif, namely, the presence of TY inhibitory sites, have been sketched elsewhere (Bártová et al. 2004). The function of all GEGTYG motif residues in CDK2 can be summarized as follows: (1) G11, G13, and G16 form a structural motif ensuring the primary function, (2) T14 and Y15 serve as inhibitory sites, and (3) Y15 interacts with substrate backbone and preferred basic residue at the +2 substrate position. The G-loop functions, generalized for all protein kinases, may include (1) nucleotide alignment, (2) phosphorylation site with regulatory function, (3) formation of substrate binding box, and (4) conveying specificity for phosphorylation.


   Materials and methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
References
 
Molecular dynamic simulations of QMZ and QMZ phosphorylated at T14 and/or Y15 G-loop inhibitory sites pT14-QMZ, pY15-QMZ, and pT14,pY15-QMZ (cf. Table 3Go) were carried out using the SANDER module of the AMBER 6.0 software package (University of California, San Francisco) with the parm99 force field (Wang et al. 2000). The starting geometries for simulation were prepared from the X-ray structure (PDB ID code: 1QMZ [PDB] ) and the T14, Y15, T14/Y15 residues were phosphorylated in silico using InsightII. The MD simulation protocol was used as follows. At first, the protonation states of histidines were checked by WHATIF (EMBL, Heidelberg), H–3 was {varepsilon}-protonated and H–2 was double protonated to create an optimal H-bonds network. All hydrogens were added using the Xleap program from the AMBER 6.0 package. The structures were neutralized by adding 17, 15, 15, and 13 Cl– counterions for QMZ, pT14-QMZ, pY15-QMZ, and pT14, pY15-QMZ, respectively. Each system was inserted in a rectangular water box where the layer of the water molecules was equal to 10 Å. The optional closeness parameter, which is used to control how close the solvent atoms can come to the solute atoms, was reduced from the default value of 1.0–0.5 Å. This parameter helps to reduce "vacuum" shell between the solute and the water box and to increase the initial density (from ~0.86 to ~0.95 g•cm–3 in our cases). Then, each system was energy minimized prior to the production part of the molecular dynamics run in the following way. The protein was frozen and the solvent molecules with counterions were allowed to move during a 1000-step minimization and a 2-psec-long molecular dynamics run under NpT conditions. Then, the side chains were relaxed by several consequent minimizations with decreasing force constants applied to the backbone atoms. After the relaxation, the system was heated to 250 K during 10 psec and then to 298.15 K during 40 psec. The production parts were run for 15 nsec for QMZ and 10 nsec for all inhibited systems. The size of the studied systems was ~60,000 atoms. The simulation period was chosen as a compromise between the quality of configuration space sampling and the calculation length. The 2-fsec time integration step and particle-mesh Ewald (PME) methods for treating electrostatic interaction were used. All simulations were run under periodic boundary conditions in the NpT ensemble at 298.16 K and at a constant pressure of 1 atm. The SHAKE algorithm with a tolerance of 10–5 Å was applied to fix all bonds containing hydrogen atoms. The 8.0 Å cutoff was applied to treat non-bonding interactions. Coordinates were stored every 2 psec. All analyses of the MD simulations were carried out by the CARNAL and PTRAJ modules of AMBER 6.0 (University of California, San Francisco), by GROMACS (University of Groningen, The Netherlands), and by the program Retinal (Masaryk University, Czech Republic); for methodology see Kríz et al. (2004). Parametrization of the phosphorylated tyrosine residue was done according to the standard Cornell et al. (1995) scheme and is published elsewhere (Bártová et al. 2004).


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  		Table 3. Summary of trajectories’ characteristics.
 
All the discussed trajectories were stable during the whole production part of the molecular dynamics simulation. The trajectory stability was monitored and confirmed by the analysis of secondary structure elements (data not shown), radius of gyration, root-mean-squared deviation (for backbone atoms) from the crystal structure as a function of time, and total energy (Table 3Go).


   Acknowledgments
 
We thank MetaCenter (meta.cesnet.cz) for computer time. This work was supported by the Ministry of Education of the Czech Republic (Grant LN00A016). This financial support is gratefully acknowledged. Pavel Banás (Olomouc, CZ) is also gratefully acknowledged for phosphotyrosine parametrization. Our thanks are also addressed to R. Turland (UK) for language corrections.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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