Acta Biochim Biophys Sin 2006, 38: 385-392
Crystal Structure of the MAP3K TAO2 Kinase Domain Bound by an Inhibitor Staurosporine
Tian-Jun ZHOU1,2, Li-Guang SUN2, Yan GAO1, and Elizabeth J. GOLDSMITH1*
1 Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9038, USA;
2 Department of
Biochemistry and Molecular Biology,
Received: February 16, 2006
Accepted: March 25, 2006
This research was supported by National Institutes of Health (USA) grants DK46993 and GM53032 and grants I1128 and I1143 from the Welch Foundation
*Corresponding author: Tel, 1-214-6456376; Fax, 1-214-6456387; E-mail, Elizabeth.Goldsmith@UTSouthwestern.edu
Abstract Mitogen-activated protein kinase (MAPK) signal transduction pathways are ubiquitous in eukaryotic cells, which transfer signals from the cell surface to the nucleus, controlling multiple cellular programs. MAPKs are activated by MAPK kinases [MAP2Ks or MAP/extracellular signal-regulated kinase (ERK) kinases (MEK)], which in turn are activated by MAPK kinase kinases (MAP3Ks). TAO2 is a MAP3K level kinase that activates the MAP2Ks MEK3 and MEK6 to activate p38 MAPKs. Because p38 MAPKs are key regulators of expression of inflammatory cytokines, they appear to be involved in human diseases such as asthma and autoimmunity. As an upstream activator of p38s, TAO2 represents a potential drug target. Here we report the crystal structure of active TAO2 kinase domain in complex with staurosporine, a broad-range protein kinase inhibitor that inhibits TAO2 with an IC50 of 3 mM. The structure reveals that staurosporine occupies the position where the adenosine of ATP binds in TAO2, and the binding of the inhibitor mimics many features of ATP binding. Both polar and nonpolar interactions contribute to the enzyme-inhibitor recognition. Staurosporine induces conformational changes in TAO2 residues that surround the inhibitor molecule, but causes very limited global changes in the kinase. The structure provides atomic details for TAO2-staurosporine interactions, and explains the relatively low potency of staurosporine against TAO2. The structure presented here should aid in the design of inhibitors specific to TAO2 and related kinases.
Key words TAO2; MAP3K; inhibitor; staurosporine; crystal structure
Mitogen-activated protein kinase (MAPK) signaling modules are one of the most widespread signaling systems in eukaryotes that transfer signals from the cell surface to the nucleus, controlling multiple cellular programs such as embryogenesis, cell differentiation, cell proliferation and cell death. Each MAPK pathway consists of a central three-tiered protein kinase in which MAPKs are activated by dual phosphorylation on a motif of Thr-X-Tyr catalyzed by a family of dual specificity kinases known as MAPK kinases (MAP2Ks) or MAP/extracellular signal-regulated kinase (ERK) kinases (MEKs). MAP2Ks in turn are activated by a protein kinase superfamily referred to as MAPK kinase kinases (MAP3Ks) or MEK kinases (MEKKs). Finally, activated MAPKs phosphorylate various substrates in cytoplasm and nucleus to change the cellular program and regulate gene expression patterns [1-3]. Among the 12 or more homologous mammalian MAPKs that have been identified, three, ERKs, p38s and c-Jun N-terminal kinases (JNKs), have been well-studied. ERKs are activated by mitogenic stimuli, such as hormones, growth factors and phorbol esters, thus associated with proliferative processes. By contrast, p38s and JNKs are more potently activated in response to physical and chemical stresses such as osmotic shock, ultraviolet radiation, oxidative stress and inflammatory cytokines, and are linked both to reparative and apoptotic responses [1,3].
MAP3Ks represent the entry level of the MAPK signaling modules, and link MAP2K/MAPK components to a wide variety of upstream activators such as MAPK kinase kinase kinases (MAP4Ks), adaptor proteins and small guanosine triphosphate-binding proteins . TAO2 is a MAP3K level kinase, which was identified from rats by isolating mammalian cDNAs encoding proteins related to Ste20p, a yeast MAP4K that regulates the MAPK cascade in the pheromone-induced mating pathway of Saccharomyces cerevisiae [5,6]. The TAO2 human homolog, prostate-derived Ste20-like kinase, was identified in a screen for RNA overexpressed in human prostate carcinoma and shares over 90% sequence identity with rat TAO2 . TAO2 phosphorylates and thus activates the MAP2Ks MEK3 and MEK6 [6,8], which are direct activators of p38 MAPKs (p38a, p38b, p38g and p38d). Because p38 MAPKs are key regulators of inflammatory cytokines expression such as tumor necrosis factor-a and interleukin-1, their pathways are thought to be involved in human diseases such as asthma, arthritis, and other inflammatory or immunoresponsive diseases . As a result, p38 MAPKs are the target of most extensive activities in MAPK inhibitor development, and the testing of selective small-molecule inhibitors of p38a has progressed into animal and clinical trials . TAO2, as an upstream activator of p38, represents a potential drug target.
There are three members in the TAO family, TAO1 , TAO2 and TAO3 [11,12], and TAO2 is the best-studied one. We have recently determined the crystal structure of TAO2 kinase domain (1-320), solved in an active form . Here we report the crystal structure of TAO2 kinase domain in complex with an inhibitor staurosporine, which is the microbial alkaloid from Streptomyces sp. (Fig. 1) and a potent broad-range small-molecule inhibitor for a number of serine/threonine protein kinases. In an in vitro kinase assay, we determined that staurosporine inhibits the activity of TAO2 kinase domain towards myelin basic protein with an IC50 of 3 mM (data not shown). As observed in other protein kinases such as protein kinase A (PKA) , cyclin-dependent kinase 2 (CDK2)  and lymphocyte-specific kinase (Lck) , staurosporine is bound to the ATP binding site of TAO2. Both polar and nonpolar interactions contribute to the formation of the kinase-inhibitor complex. The TAO2-staurosporine structure provides the structural basis for the kinase-inhibitor interactions, which should help in the design of inhibitors specific to TAO family kinases.
Materials and Methods
Cloning, expression and purification
The details of the production and purification of TAO2 kinase domain
protein have been described elsewhere . Briefly, rat TAO2 kinase domain
(1-320) was cloned into Baculovirus and expressed in insect cells. The
TAO2 kinase domain protein was first purified with Ni2+-nitrilotriacetic
Crystallization, complex formation and data collection
The TAO2 kinase domain crystallized in a condition containing
Structure determination and refinement
The structure of the TAO2 (1-320)-staurosporine complex was determined with molecular replacement using active TAO2 (1-320) as the search model (Protein Data Bank code 1U5Q). The calculated difference Fourier map using this model reveals clear density for the bound staurosporine [Fig. 2(A)]. After several cycles of refinement of the TAO2 (1-320) model in crystallography and NMR system (CNS )  combined with manual rebuilding in o (a crystallographic model building program) , the staurosporine model was built into the density. This was followed by further refinement in CNS and o until convergence was reached. The crystal data and refinement statistics of TAO2 (1-320)-staurosporine are summarized in Table 1.
Results and Discussion
Overall structure of TAO2 (1-320)-staurosporine
The staurosporine-soaked TAO2 kinase domain crystals belong to P6522 space group with cell dimensions a=b=186.0 Å and c=94.6 Å. There are two TAO2(1-320)-staurosporine complexes in the asymmetric unit that are not related by non-crystallographic symmetry, but are almost identical in conformation. The electron density is clear for the bound staurosporine molecule [Fig. 2(A)]. Also, electron density is good throughout the TAO2 molecule, except for the first 11 amino acids and the His6-tag at the N-terminus, for which there is no density. Residues 63-65 (in the loop connecting strand 3 and helix C) and residues 302-312 (in the loop connecting helices J and K) are partially disordered. The final model is comprised of 618 of the total 640 residues in the two TAO2(1-320) monomers plus two molecules of staurosporine and 293 water molecules. The TAO2 (1-320)-staurosporine model has been refined with reasonable stereochemistry to free R factor and R factor of 27.0% and 21.0%, respectively (Table 1).
The inhibitor-bound TAO2 kinase domain was constitutively phosphorylated on serine181 at the activation loop, and adopts an active conformation as its apo form . TAO2(1-320) in the complex possesses the typical protein kinase two-domain architecture. The N-terminal domain is composed of an antiparallel five-stranded b-sheet and helix C plus two small helices at an N-terminal extension to the kinase core (labeled helices A an B) [Fig. 2(A)]. The C-terminal domain also possesses a standard structure, including six major helices, two b-ribbons, the catalytic loop, and the activation loop. In the truncated form (1-320) that was crystallized, TAO2 possesses two additional helices, J and K, which are in the C-terminal extension to the kinase core (277-320). As observed in other serine/threonine protein kinases such as PKA  and CDK2 , staurosporine binds in the ATP binding site of TAO2 between the domain interface, occupying a position where the adenosine moiety of ATP binds in the enzyme [Fig. 2(A)].
Interactions between TAO2 (1-320) and staurosporine
Staurosporine is a natural microbial alkaloid that was first
characterized in 1986 and shown to be a potent inhibitor of protein kinase C
. It was subsequently found that staurosporine is a potent and nonspecific
inhibitor of a number of protein kinases with IC50 values in the 1-100 nM range, such as PKA and CDK2 . The surface of the
staurosporine nucleus is highly hydrophobic (Fig. 1). This is
complemented by the large hydrophobic surface of the ATP-binding cleft of TAO2,
so that staurosporine bound to TAO2 makes extensive favorable van der Waal
contacts with residues surrounding staurosporine: Ile34 (Leu
The polar interactions between TAO2 and staurosporine involve three hydrogen bonds which are mimics of those observed in the TAO2 (1-320)-MgATP complex . These are the two bonds between N19, O30 of the lactam moiety of staurosporine and backbone carbonyl oxygen of Glu106 and amide nitrogen of Cys108, respectively, and one bond between N31 from the methylamino group of the glycosyl portion of staurosporine and the main-chain carbonyl oxygen of Gly155 [Fig. 2(B)].
Staurosporine-induced conformational changes
The TAO2 (1-320)-staurosporine complex was formed through diffusion
Conformational changes at the C-terminal extension
The full length TAO2 protein possesses 1235 residues in its polypeptide chain, and the kinase domain is located at its N-terminus . In the truncated form (1-320) that was crystallized, TAO2 possesses 44 residues as a C-terminal extension, which forms two helices, J and K. Helix K, which adopts a predominantly 3/10 conformation and spans the gap between the two domains of the kinase near the hinge region, is participating in ATP binding; Lys314 from helix K forms a hydrogen bond with the 2 hydroxyl of the ribose . In the complex of TAO2 (1-320)-staurosporine, a similar interaction is not possible due to lack of a homologous binding partner in the inhibitor molecule. So the charged tip of Lys314 turns away from the voluminous and hydrophobic indole carbazole ring I of staurosporine, whereas the aliphatic part of the residue makes van der Waals contacts with the inhibitor [Figs. 3(B) and 4]. In PKA, a structurally similar position to Lys314 of TAO2 is occupied by Phe327, which is from the C-terminal tail that crosses the two structural domains of the kinase . Phe327 was also displaced in the PKA-staurosporine-PKI (protain kinase inhibitor) complex to make room for the inhibitor, and makes direct contacts with the inhibitor through its hydrophobic side chain .
Comparison with TAO2 (1-320)-MgATP structure
The staurosporine molecule occupies a site in TAO2 that overlaps
with that of the adenosine moiety of ATP . The lactam ring of staurosporine
mimics the amino pyrimidine ring of adenine in its hydrogen bonding
interactions with the backbone carbonyl group of Glu106 and the amide NH group
Comparison with other protein kinases
In TAO2 (1-320), the position and conformation of
staurosporine in the ATP-binding cleft are highly similar to those observed in
PKA  or CDK2 . However, there are several differences among these
structures in inhibitor binding and the conformational changes induced. First,
a total of three hydrogen bonds were observed in the TAO2 (1-320)-staurosporine
structure compared to four in both the PKA and CDK2 complexes. The fourth bond
between the methylamine group of staurosporine and the side chain of Glu
Insights for the design of inhibitors specific to TAOs
Initial interest in protein kinases as drug targets was stimulated by the findings that many viral oncogenes encode structurally modified cellular protein kinases with constitutive enzyme activity . These findings are suggestive of the potential involvement of proto-oncogene-encoded protein kinases in human proliferative disorders. Thus, specific protein kinase inhibitors could block the disease pathologies resulting from aberrant protein kinase activity. In fact, after G protein-coupled receptors, protein kinases have become the second most important class of targets for drugs in the past 20 years. One of the successful cases is imatinib mesylate (Gleevec; Novartis Pharmaceuticals Inc., Cambridge, USA), which specifically inhibits the inactive form of Bcr-Abl tyrosine kinase, thereby exerting its treatment effects on chronic myeloid leukemia disease . Because p38a MAPK regulates the production of cytokines such as tumor necrosis factor-a and interleukin-1, its inhibitors might inhibit not only the production of these pro-inflammatory cytokines, but also their actions, interrupting the vicious cycle that often occurs in inflammatory and immunoresponsive diseases. The most extensive activity in MAPK inhibitor development is on p38a . It is believed that TAO2, an upstream regulator of p38, represents a potential drug target for the treatment of p38 MAPK-associated diseases such as arthritis, autoimmunity, and other diseases yet to be identified. Due to its broad spectrum of protein kinase inhibitory effects, staurosporine proved to be too toxic to use directly as a therapeutic agent, exhibiting a maximal tolerated dose in the order of 10 nM . Nevertheless, staurosporine could serve as a template for the design of inhibitors specific to TAO2. In fact, structure-based drug design has become an integral part of modern drug discovery. In particular, the efforts in designing specific TAO2 inhibitors should take advantage of the unique interaction in TAO2, observed between Lys314 of helix K and the 2 hydroxyl of the ribose of ATP, as a lysine at this position is present only in TAO family kinases .
In summary, the crystal structure of the active MAP3K TAO2 kinase domain in complex with staurosporine has been determined at 2.6 Å resolution. The inhibitor targets the ATP binding site of TAO2, and the binding mimics many features of MgATP recognition by the enzyme. Conformational changes occur locally to the inhibitor-binding pocket, whereas global changes are rather limited. The structure presented here thus provides a structural basis for the kinase杋nhibitor interactions, and should be helpful in the design of inhibitors specific to TAO2 and related kinases.
We thank Zhu CHEN and Melanie H. COBB (Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, USA) for providing TAO2 (1-320) expression vector.
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