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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2006.00173.x |
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, [email protected]
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 [4]. 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 [7]. 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 [9]. 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 [10]. TAO2, as an upstream activator of p38, represents a
potential drug target.
There are three members in the TAO family, TAO1 [5], 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 [13].
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) [14],
cyclin-dependent kinase 2 (CDK2) [15] and lymphocyte-specific kinase (Lck)
[16], 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 [13]. 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 acid-agarose (
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 ) [17] combined with manual rebuilding in o (a
crystallographic model building program) [18], 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 [13]. 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 [14] and CDK2 [15], 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
[19]. 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 [20]. 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
hydrogen bonds which are mimics of those observed in the TAO2 (1-320)-MgATP
complex [13]. 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
of
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 [6]. 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 [13]. 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 [21]. Phe327 was also displaced in the PKA-staurosporine朠KI (protain kinase
inhibitor) complex to make room for the inhibitor, and makes direct contacts
with the inhibitor through its hydrophobic side chain [14].
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 [13]. 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
of Cys
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 [14] or CDK2 [15]. 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 [22]. 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 [24]. 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 [10]. 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 [25]. 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 [13].
In summary, the crystal
structure of the active MAP3K TAO2 kinase domain in complex with staurosporine
has been determined at 2.6 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.
Acknowledgments
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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