Advertisement

Identification and Structure Characterization of a Cdk Inhibitory Peptide Derived from Neuronal-specific Cdk5 Activator*

Open AccessPublished:March 12, 1999DOI:https://doi.org/10.1074/jbc.274.11.7120
      The activation of cyclin-dependent kinase 5 (Cdk5) depends on the binding of its neuronal specific activator Nck5a. The minimal activation domain of Nck5a is located in the region of amino acid residues 150 to 291 (Tang, D., Chun, A. C. S., Zhang, M., and Wang, J. H. (1997) J. Biol. Chem. 272, 12318–12327). In this work we show that a 29-residue peptide, denoted as the αN peptide, encompassing amino acid residues Gln145 to Asp173 of Nck5a is capable of binding Cdk5 to result in kinase inhibition. This peptide also inhibits an active phospho-Cdk2-cyclin A complex, with a similar potency. Direct competition experiments have shown that this inhibitory peptide does not compete with Nck5a or cyclin A for Cdk5 or Cdk2, respectively. Steady state kinetic analysis has indicated that the αN peptide acts as a non-competitive inhibitor of Cdk5·Nck5a complex with respect to the peptide substrate. To understand the molecular basis of kinase inhibition by the peptide, we determined the structure of the peptide in solution by circular dichroism and two-dimensional 1H NMR spectroscopy. The peptide adopts an amphipathic α-helical structure from residues Ser149 to Arg162 which can be further stabilized by the helix-stabilizing solvent trifluoroethanol. The hydrophobic face of the helix is likely to be the kinase binding surface.
      Cyclin-dependent kinases (Cdks)
      The abbreviations used are: Cdk, cyclin-dependent kinase; cyclin A-H6, C-terminal histidine-tagged cyclin A; GST, glutathione S-transferase; H6-Cdk5, N-terminal histidine-tagged Cdk5; MOPS, 3-(N-morpholino)propanesulfonic acid; Nck5a, neuronal Cdk5 activator; NOESY, nuclear Overhauser enhancement spectroscopy; TFE, 2,2,2-trifluoroethanol; TOCSY, total correlation spectroscopy; MLCK, myosin light chain kinase
      1The abbreviations used are: Cdk, cyclin-dependent kinase; cyclin A-H6, C-terminal histidine-tagged cyclin A; GST, glutathione S-transferase; H6-Cdk5, N-terminal histidine-tagged Cdk5; MOPS, 3-(N-morpholino)propanesulfonic acid; Nck5a, neuronal Cdk5 activator; NOESY, nuclear Overhauser enhancement spectroscopy; TFE, 2,2,2-trifluoroethanol; TOCSY, total correlation spectroscopy; MLCK, myosin light chain kinase
      are key regulatory enzymes in the eukaryotic cell cycle. The activation of a Cdk depends on its association with its specific cyclin partners. The activity of these enzymes is further regulated by an intricate system of protein-protein interactions and phosphorylation (
      • Morgan D.O.
      ). Members of the Cdk family are closely related by sharing a high level of amino acid sequence identity (40–70%). In contrast, cyclins are a family of molecules of diverse molecular mass and low sequence identity. Sequence alignments have shown that cyclins share a somewhat conserved region of approximately 100 amino acids in the center of the molecule, and this region is called the cyclin box (
      • Nugent J.A.
      • Alfa C.E.
      • Young T.
      • Hyams J.S.
      ). Recent crystal structures of cyclin A and cyclin H have shown that the cyclin box sequence forms a compact 5-helix domain called the cyclin fold (
      • Brown N.R.
      • Noble M.E.M.
      • Endicott J.A.
      • Garman E.F.
      • Wakatsuki S.
      • Mitchell E.
      • Rasmussen B.
      • Hunt T.
      • Johnson L.N.
      ,
      • Jeffrey P.D.
      • Russo A.A.
      • Polyak K.
      • Gibbs E.
      • Hurwitz J.
      • Massague J.
      • Pavletich N.P.
      ,
      • Kim K.K.
      • Chamberlin H.M.
      • Morgan D.O.
      • Kim S.-H.
      ), and that a region of cyclin A, C-terminal to the cyclin box also forms a cyclin fold. However, there is virtually no sequence similarity between the two cyclin fold domains. Theoretical predictions have suggested that other members of the cyclin family also contain two cyclin folds (
      • Brown N.R.
      • Noble M.E.M.
      • Endicott J.A.
      • Garman E.F.
      • Wakatsuki S.
      • Mitchell E.
      • Rasmussen B.
      • Hunt T.
      • Johnson L.N.
      ,
      • Bazan J.F.
      ).
      Unlike other Cdks, Cdk5 activity has been observed only in neuronal and developing muscle cells although the catalytic subunit of the enzyme is present in many mammalian tissues and cell extracts (
      • Hayes T.E.
      • Valtz N.L.M.
      • McKay R.D.G.
      ,
      • Ishiguro K.
      • Takamatsu M.
      • Tomizawa K.
      • Omori A.
      • Takahashi M.
      • Arioka M.
      • Uchida T.
      • Imahori K.
      ,
      • Lew J.
      • Winkfein R.J.
      • Paudel H.K.
      • Wang J.H.
      ,
      • Lew J.
      • Beaudette K.
      • Litwin C.M.E.
      • Wang J.H.
      ,
      • Philpott A.
      • Porro E.B.
      • Kirschner M.W.
      • Tsai L.-H.
      ). Recent experimental evidence has demonstrated that Cdk5 plays important roles in neurite outgrowth (
      • Nikolic M.
      • Dudek H.
      • Kwon Y.T.
      • Ramos Y.F.M.
      • Tsai L.-H.
      ), patterning of the cortex and cerebellum (
      • Oshima T.
      • Ward J.M.
      • Huh C.-G.
      • Longennecker G.
      • Veeranna
      • Pant H.C.
      • Brady R.O.
      • Martin L.J.
      • Kulkami A.B.
      ), and cytoskeletal dynamics (
      • Lew J.
      • Winkfein R.J.
      • Paudel H.K.
      • Wang J.H.
      ,
      • Paudel H.K.
      • Lew J.
      • Ali Z.
      • Wang J.H.
      ,
      • Nikolic M.
      • Chou M.M.
      • Lu W.
      • Mayer B.J.
      • Tsai L.-H.
      ). Loss of regulation of Cdk5 has been suggested to be involved in Alzheimer's disease (
      • Lew J.
      • Wang J.H.
      ). Active Cdk5 was first purified from brain extracts as a heterodimer with subunit molecular masses of 33- and 25-kDa, respectively (
      • Ishiguro K.
      • Takamatsu M.
      • Tomizawa K.
      • Omori A.
      • Takahashi M.
      • Arioka M.
      • Uchida T.
      • Imahori K.
      ,
      • Lew J.
      • Beaudette K.
      • Litwin C.M.E.
      • Wang J.H.
      ). The 33-kDa subunit was later identified as Cdk5, and the 25-kDa activator (neuronal Cdk5 activator, Nck5a) was a novel protein with no sequence similarity to any other known proteins. The 25-kDa subunit was later found to be a proteolytic product of a larger 35-kDa protein (
      • Lew J.
      • Huang Q.-Q.
      • Qi Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      ,
      • Tsai L.-H.
      • Delalle I.
      • Caviness Jr., V.S.
      • Chae T.
      • Harlow E.
      ). An isoform of Nck5a (Nck5ai) with 57% sequence identity to Nck5a has also been identified (
      • Tang D.
      • Yeung J.
      • Lee K.-Y.
      • Matsushita M.
      • Matsui H.
      • Tomizawa K.
      • Hatase O.
      • Wang J.H.
      ). Despite their functional similarity in terms of binding and activation of a Cdk, the Cdk5 activators share little sequence similarity to cyclins. Moreover, while the activation of the well characterized Cdks such as Cdk1 and Cdk2 by cyclins depends on the phosphorylation of the Cdk at a specific threonine residue, Cdk5 activation by its activator is phosphorylation-independent (
      • Qi Z.
      • Huang Q.-Q.
      • Lee K.-Y.
      • Lew J.
      • Wang J.H.
      ,
      • Poon R.Y.C.
      • Lew J.
      • Hunter T.
      ). Recently, the activation domain of Nck5a was precisely mapped to amino acid residues from Glu150 to Asn291 (
      • Poon R.Y.C.
      • Lew J.
      • Hunter T.
      ,
      • Tang D.
      • Chun A.C.S.
      • Zhang M.
      • Wang J.H.
      ). Extensive truncation and site-directed mutation studies of Nck5a, together with computer modeling, strongly suggested that the 142-residue activation domain of Nck5a adopts a cyclin fold structure (
      • Brown N.R.
      • Noble M.E.M.
      • Endicott J.A.
      • Garman E.F.
      • Wakatsuki S.
      • Mitchell E.
      • Rasmussen B.
      • Hunt T.
      • Johnson L.N.
      ,
      • Tang D.
      • Chun A.C.S.
      • Zhang M.
      • Wang J.H.
      ).
      In this work, we describe the discovery of a 29-residue Cdk inhibitory peptide which is derived from an internal fragment of Nck5a. This peptide is able to bind to and hence to inhibit the kinase activities of Cdk5·Nck5a and Cdk2·cyclin A complexes in a non-competitive manner. The solution structure of the peptide determined by two-dimensional NMR spectroscopy showed that a large part of the peptide adopts an amphipathic α-helical structure, and this helix is likely to be the main binding surface of the peptide to the enzyme complexes.

      DISCUSSION

      The minimal activation domain of Nck5a has previously been mapped to contain 142 amino acid residues spanning residues Asp150to Asn291 (
      • Poon R.Y.C.
      • Lew J.
      • Hunter T.
      ,
      • Tang D.
      • Chun A.C.S.
      • Zhang M.
      • Wang J.H.
      ). A number of theoretical and experimental studies have suggested that this minimal activation domain of Nck5a adopts a cyclin-fold (
      • Brown N.R.
      • Noble M.E.M.
      • Endicott J.A.
      • Garman E.F.
      • Wakatsuki S.
      • Mitchell E.
      • Rasmussen B.
      • Hunt T.
      • Johnson L.N.
      ,
      • Bazan J.F.
      ,
      • Tang D.
      • Chun A.C.S.
      • Zhang M.
      • Wang J.H.
      ). In this work, we have identified a 29-residue peptide, residues Gln145 to Asp173 of Nck5a, that can inhibit the kinase activities of the Cdk5·Nck5a and Cdk2·cyclin A complexes. Based on our earlier prediction, the sequence of this peptide encompasses the N-terminal α-helix of the cyclin fold (thus the peptide is termed the αN peptide) as well as some flanking amino acid residues at both ends of the helix (
      • Brown N.R.
      • Noble M.E.M.
      • Endicott J.A.
      • Garman E.F.
      • Wakatsuki S.
      • Mitchell E.
      • Rasmussen B.
      • Hunt T.
      • Johnson L.N.
      ,
      • Jeffrey P.D.
      • Russo A.A.
      • Polyak K.
      • Gibbs E.
      • Hurwitz J.
      • Massague J.
      • Pavletich N.P.
      ,
      • Tang D.
      • Chun A.C.S.
      • Zhang M.
      • Wang J.H.
      ). The inhibition of Cdk5 by the αN peptide supports an earlier study that a 50-amino acid fragment spanning residues 109 to 159 of Nck5a retains partial binding capability to Cdk5 (
      • Poon R.Y.C.
      • Lew J.
      • Hunter T.
      ). Knowing that Nck5a only weakly activates Cdk2 to the basal level, i.e. the activity observed for a Cdk2·cyclin A complex without Thr160 phosphorylation (
      • Qi Z.
      • Huang Q.-Q.
      • Lee K.-Y.
      • Lew J.
      • Wang J.H.
      ,
      • Poon R.Y.C.
      • Lew J.
      • Hunter T.
      ), it is surprising that the αN peptide inhibits Cdk2·cyclin A activity with an even higher potency than in the case with Cdk5·Nck5a inhibition (Fig. 3). In contrast, the corresponding peptide encompassing the N-terminal α-helix of cyclin A inhibits neither Cdk2 nor Cdk5 (Fig. 3). In this work, we have investigated the inhibition of Cdk5 and Cdk2 by the αN peptide, and it would be interesting to know whether the αN peptide can also inhibit other members of the Cdk family. Further work is in progress on this matter in our laboratories.
      Since the αN peptide was derived from an internal fragment of Nck5a, it is expected that it might act as a noncompetitive inhibitor with respect to the substrate of Cdk5 (Fig. 6). However, it is unusual that the αN peptide also functions as a noncompetitive inhibitor with respect to Nck5a (Figs. 4 and 5). Our results indicate that the inhibition of Cdk5 by the αNpeptide results from the formation of a ternary complex between the αN peptide and the Cdk5·Nck5a complex. Presumably, the αN peptide competes with the corresponding fragment in Nck5a for Cdk5 binding. This suggestion is in agreement with an earlier observation that the removal of 4 amino acid residues from the helical part of the peptide fragment from Nck5a completely abolished the ability of Nck5a to activate Cdk5 (
      • Tang D.
      • Chun A.C.S.
      • Zhang M.
      • Wang J.H.
      ). Comparison of the crystal structures of cyclin A in complex with Cdk2, and cyclin H, has indicated that the N-terminal helix of various cyclins may function as a relatively independent structural unit with respect to the tightly packed cyclin folds (
      • Jeffrey P.D.
      • Russo A.A.
      • Polyak K.
      • Gibbs E.
      • Hurwitz J.
      • Massague J.
      • Pavletich N.P.
      ,
      • Kim K.K.
      • Chamberlin H.M.
      • Morgan D.O.
      • Kim S.-H.
      ,
      • Fan J.-S.
      • Cheng H.-C.
      • Zhang M.
      ). However, this N-terminal helix is indispensable for the activity of cyclins (
      • Tang D.
      • Chun A.C.S.
      • Zhang M.
      • Wang J.H.
      ,
      • Dyson J.H.
      • Wright P.E.
      ,
      • Wishart D.S.
      • Sykes B.D.
      • Richards F.M.
      ,
      • Lee E.M.
      • Harlow E.
      ,
      • Andersen G.
      • Russo D.
      • Poterszman A.
      • Hwang J.R.
      • Wurtz J.M.
      • Ripp R.
      • Thierry J.C.
      • Egly J.M.
      • Morgan D.
      ), although the contacts between the helix and the kinase are not extensive (
      • Jeffrey P.D.
      • Russo A.A.
      • Polyak K.
      • Gibbs E.
      • Hurwitz J.
      • Massague J.
      • Pavletich N.P.
      ). Therefore, we hypothesize that the binding of the αNpeptide dislodges the corresponding N-terminal α–helix of Nck5a from Cdk5, thereby inhibiting the activity of the enzyme. The dislocation of the N-terminal α–helix does not lead to dissociation of the whole activator. Unlike the αN peptide, the control peptide derived from cyclin A inhibits neither Cdk2·cyclin A nor Cdk5·Nck5a (Fig. 3), suggesting a significant difference between the binding and activation of Cdk2 by cyclin A, on the one hand, and Cdk5 by Nck5a, on the other.
      The α-helical structure detected by CD spectroscopy for the αN peptide in aqueous solution (Fig. 6) qualitatively agrees with our earlier prediction that part of the αNpeptide could adopt an α-helical conformation (
      • Tang D.
      • Chun A.C.S.
      • Zhang M.
      • Wang J.H.
      ). The existence of multiconformational states of the peptide prevented us from a detailed structural determination of the peptide in aqueous solution. Hence, TFE and water were used as a co-solvent to study the structure of the αN peptide. The peptide segment from Ser149to Arg162 was found to adopt a stable α-helical conformation in aqueous TFE solution. Similar NOE patterns (especiallydNN NOE connectivities that were relatively well resolved) have also been observed for the αN peptide in pure water solution (data not shown), suggesting that the same α-helical conformation exists in this solution. It has been observed in numerous cases that TFE can either stabilize unordered α-helices in various peptide fragments in aqueous solution or promote the formation of α-helices in peptide fragments that have intrinsic propensities to form α-helix, but not induce new α-helical conformation (for example, see Refs.
      • Zhang M.
      • Yuan T.
      • Vogel H.J.
      ,
      • Dyson J.H.
      • Wright P.E.
      ,
      • Jasanoff A.
      • Fersht A.R.
      , and
      • Reymond M.T.
      • Huo S.
      • Duggan B.
      • Wright P.E.
      • Dyson H.J.
      ). Therefore, we suggest that the α-helical region observed in the αNpeptide would probably adopt a similar α-helical structure in Nck5a. The peptide region found to adopt an α-helical conformation has also been predicted to be an α-helix in the protein, and this α-helix aligns well with the N-terminal α-helix of the first cyclin-fold of cyclin A (Refs.
      • Brown N.R.
      • Noble M.E.M.
      • Endicott J.A.
      • Garman E.F.
      • Wakatsuki S.
      • Mitchell E.
      • Rasmussen B.
      • Hunt T.
      • Johnson L.N.
      ,
      • Jeffrey P.D.
      • Russo A.A.
      • Polyak K.
      • Gibbs E.
      • Hurwitz J.
      • Massague J.
      • Pavletich N.P.
      , and
      • Tang D.
      • Chun A.C.S.
      • Zhang M.
      • Wang J.H.
      , also see Fig. 1). The above notion is further underscored by the fact that the same α-helical structure was observed for the cyclin A peptide in solution as the corresponding N-terminal helix in the full-length cyclin A structures (
      • Fan J.-S.
      • Cheng H.-C.
      • Zhang M.
      ).
      A helical wheel presentation of the α-helix found in the αN peptide shows that the peptide is amphipathic with 4 Leu and 1 Phe on the hydrophobic face (Fig. 10). Indeed, deletion of part of the N-terminal end of the α-helix completely abolished the inhibitory effect of the peptide.
      K.-T. Chin, Y.-L. Kam, and M. Zhang, unpublished results.
      In an earlier study, we have also shown that mutations of the hydrophobic amino acid residues in the α-helix (Leu151, Leu152) to a polar amino acid residue (Asn) greatly reduced the Cdk5 activation ability of Nck5a (
      • Tang D.
      • Chun A.C.S.
      • Zhang M.
      • Wang J.H.
      ). In the crystal structure of the Cdk2·cyclin A complex, the corresponding N-terminal α-helix of cyclin A makes a significant amount of contacts with various regions (e.g.T-loop and α3 helix) of Cdk2 via hydrophobic interactions (
      • Jeffrey P.D.
      • Russo A.A.
      • Polyak K.
      • Gibbs E.
      • Hurwitz J.
      • Massague J.
      • Pavletich N.P.
      ). It is likely that the hydrophobic face of the peptide forms the major binding area between the αN peptide and Cdks. This hypothesis was supported by the result shown in Fig. 3 that an unrelated amphipathic MLCK peptide was able to inhibit both Cdk5 and Cdk2. Like the αN peptide, the α-helical structure of the MLCK peptide in solution can be promoted by TFE, and the MLCK peptide binds to calmodulin in an α-helical conformation with its hydrophobic face forming the main contact area with calmodulin (
      • Zhang M.
      • Yuan T.
      • Vogel H.J.
      ,
      • Ikura M.
      • Clore G.M.
      • Gronenborn A.M.
      • Zhu G.
      • Klee C.B.
      • Bax A.
      ,
      • Meador W.E.
      • Means A.R.
      • Quiocho F.A.
      ). The lower extent and potency of inhibitory activity observed with the MLCK peptide may originate from a large sequence difference in the α-helical region as well as the C-terminal random coil region between the MLCK peptide and the αN peptide.
      The structure of the αN peptide determined here and the interaction observed between the N-terminal α-helix of cyclin A and Cdk2 (
      • Jeffrey P.D.
      • Russo A.A.
      • Polyak K.
      • Gibbs E.
      • Hurwitz J.
      • Massague J.
      • Pavletich N.P.
      ) suggest that systematic alterations of the amino acid residues in the hydrophobic face of the α-helix and the C-terminal end of the αN peptide may enable us to find peptide inhibitors with higher specificity and/or potency toward various Cdks. We note that the present Cdk5 inhibitory peptide was discovered based on the unique regulatory property of the enzyme by its activator. It is, therefore, promising to develop the peptide into a Cdk5 specific inhibitor in contrast to the majority of ATP analog derived compounds, which acts as general kinase inhibitors. Also, the peptide in its present form can be used to screen for chemical compounds that can inhibit the activity of the Cdk5·Nck5a complex.

      Acknowledgments

      We thank Dr. Randy Poon for providing the expression construct of cyclin A and the cyclin A monoclonal antibody. We also thank Y. F. Leung for providing baculovirus-expressed histidine-tagged Cdk5, and Drs. James Hackett and David Smith for careful reading of the manuscript.

      REFERENCES

        • Morgan D.O.
        Nature. 1995; 374: 131-134
        • Nugent J.A.
        • Alfa C.E.
        • Young T.
        • Hyams J.S.
        J. Cell Sci. 1991; 99: 674-699
        • Brown N.R.
        • Noble M.E.M.
        • Endicott J.A.
        • Garman E.F.
        • Wakatsuki S.
        • Mitchell E.
        • Rasmussen B.
        • Hunt T.
        • Johnson L.N.
        Structure. 1995; 3: 1235-1247
        • Jeffrey P.D.
        • Russo A.A.
        • Polyak K.
        • Gibbs E.
        • Hurwitz J.
        • Massague J.
        • Pavletich N.P.
        Nature. 1996; 376: 313-320
        • Kim K.K.
        • Chamberlin H.M.
        • Morgan D.O.
        • Kim S.-H.
        Nat. Struct. Biol. 1996; 3: 849-855
        • Bazan J.F.
        Proteins Struct. Funct. Genet. 1996; 24: 1-17
        • Hayes T.E.
        • Valtz N.L.M.
        • McKay R.D.G.
        New Biol. 1991; 3: 259-269
        • Ishiguro K.
        • Takamatsu M.
        • Tomizawa K.
        • Omori A.
        • Takahashi M.
        • Arioka M.
        • Uchida T.
        • Imahori K.
        J. Biol. Chem. 1992; 267: 10897-10901
        • Lew J.
        • Winkfein R.J.
        • Paudel H.K.
        • Wang J.H.
        J. Biol. Chem. 1992; 267: 25922-25926
        • Lew J.
        • Beaudette K.
        • Litwin C.M.E.
        • Wang J.H.
        J. Biol. Chem. 1992; 267: 13383-13390
        • Philpott A.
        • Porro E.B.
        • Kirschner M.W.
        • Tsai L.-H.
        Gene Dev. 1997; 11: 1409-1421
        • Nikolic M.
        • Dudek H.
        • Kwon Y.T.
        • Ramos Y.F.M.
        • Tsai L.-H.
        Genes Dev. 1996; 10: 816-825
        • Oshima T.
        • Ward J.M.
        • Huh C.-G.
        • Longennecker G.
        • Veeranna
        • Pant H.C.
        • Brady R.O.
        • Martin L.J.
        • Kulkami A.B.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11173-11178
        • Paudel H.K.
        • Lew J.
        • Ali Z.
        • Wang J.H.
        J. Biol. Chem. 1993; 268: 23512-23518
        • Nikolic M.
        • Chou M.M.
        • Lu W.
        • Mayer B.J.
        • Tsai L.-H.
        Nature. 1998; 395: 194-198
        • Lew J.
        • Wang J.H.
        Trends Biochem. Sci. 1995; 20: 33-37
        • Lew J.
        • Huang Q.-Q.
        • Qi Z.
        • Winkfein R.J.
        • Aebersold R.
        • Hunt T.
        • Wang J.H.
        Nature. 1994; 371: 423-426
        • Tsai L.-H.
        • Delalle I.
        • Caviness Jr., V.S.
        • Chae T.
        • Harlow E.
        Nature. 1994; 371: 419-423
        • Tang D.
        • Yeung J.
        • Lee K.-Y.
        • Matsushita M.
        • Matsui H.
        • Tomizawa K.
        • Hatase O.
        • Wang J.H.
        J. Biol. Chem. 1995; 270: 26897-26903
        • Qi Z.
        • Huang Q.-Q.
        • Lee K.-Y.
        • Lew J.
        • Wang J.H.
        J. Biol. Chem. 1995; 270: 10847-10854
        • Poon R.Y.C.
        • Lew J.
        • Hunter T.
        J. Biol. Chem. 1997; 272: 5703-5708
        • Tang D.
        • Chun A.C.S.
        • Zhang M.
        • Wang J.H.
        J. Biol. Chem. 1997; 272: 12318-12327
        • Cheng H.-C.
        • Bjorge J.D.
        • Aebersold R.
        • Fujita D.J.
        • Wang J.H.
        Biochemistry. 1996; 35: 11874-11887
        • Wüthrich K.
        NMR of Proteins and Nucleic Acids. Wiley-Interscience, New York1986
        • Smallcombe S.H.
        • Patt S.L.
        • Keifer P.A.
        J. Magn. Reson. Sect. A. 1995; 117: 295-303
        • Bax A.
        • Davis D.G.
        J. Magn. Reson. 1985; 65: 355-360
        • Delaglio F.
        • Grzesiek S.
        • Vuister G.W.
        • Zhu G.
        • Preifer J.
        • Bax A.
        J. Biomol. NMR. 1996; 6: 277-293
        • Fan J.-S.
        • Cheng H.-C.
        • Zhang M.
        Biochem. Biophys. Res. Commun. 1999; (in press)
        • Blumenthal D.K.
        • Takio K.
        • Edelman A.M.
        • Charbonneau H.
        • Titani K.
        • Walsh K.A.
        • Krebs E.G.
        Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3187-3191
        • Zhang M.
        • Yuan T.
        • Vogel H.J.
        Protein Sci. 1993; 2: 1931-1937
        • Segel I.H.
        Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady State Enzyme Systems. Wiley-VCH Publishers, Inc., New York1975: 101-258
        • Dyson J.H.
        • Wright P.E.
        Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 519-538
        • Wishart D.S.
        • Sykes B.D.
        • Richards F.M.
        Biochemistry. 1992; 31: 1647-1651
        • Lee E.M.
        • Harlow E.
        Mol. Cell. Biol. 1993; 13: 1194-1201
        • Andersen G.
        • Russo D.
        • Poterszman A.
        • Hwang J.R.
        • Wurtz J.M.
        • Ripp R.
        • Thierry J.C.
        • Egly J.M.
        • Morgan D.
        EMBO J. 1997; 16: 958-967
        • Jasanoff A.
        • Fersht A.R.
        Biochemistry. 1994; 33: 2129-2135
        • Reymond M.T.
        • Huo S.
        • Duggan B.
        • Wright P.E.
        • Dyson H.J.
        Biochemistry. 1997; 36: 5234-5244
        • Ikura M.
        • Clore G.M.
        • Gronenborn A.M.
        • Zhu G.
        • Klee C.B.
        • Bax A.
        Science. 1992; 256: 632-638
        • Meador W.E.
        • Means A.R.
        • Quiocho F.A.
        Science. 1992; 257: 1251-1255