Structure, Vol. 13, 541–550, April, 2005, ©2005 Elsevier Ltd All rights reserved. DOI 10.1016/j.str.2004.12.017

Structure and Inhibition of the Human Cell CycleCheckpoint Kinase, Wee1A Kinase: An AtypicalTyrosine Kinase with a Key Role in CDK1 Regulation

Christopher J. Squire, James M. Dickson,Ivan Ivanovic, and Edward N. Baker*School of Biological Sciences and Centre for

Molecular BiodiscoveryUniversity of AucklandAuckland 1001New Zealand

Summary

Phosphorylation is critical to regulation of the eukary-otic cell cycle. Entry to mitosis is triggered by thecyclin-dependent kinase CDK1 (Cdc2), which is inac-tivated during the preceding S and G2 phases byphosphorylation of T14 and Y15. Two homologous ki-nases, Wee1, which phosphorylates Y15, and Myt1,which phosphorylates both T14 and Y15, mediate thisinactivation. We have determined the crystal struc-ture of the catalytic domain of human somatic Wee1(Wee1A) complexed with an active-site inhibitor at1.8 Å resolution. Although Wee1A is functionally a ty-rosine kinase, in sequence and structure it mostclosely resembles serine/threonine kinases such asChk1 and cAMP kinases. The crystal structure showsthat although the catalytic site closely resembles thatof other protein kinases, the activation segment con-tains Wee1-specific features that maintain it in anactive conformation and, together with a key substitu-tion in its glycine-rich loop, help determine its sub-strate specificity.

Introduction

Typical eukaryotic cells cycle through four phases dur-ing cell division: G1, S (DNA synthesis), G2, and M (mi-tosis). This process must accurately replicate the par-ent cell’s genetic material in the S phase and distributeidentical chromosomal copies evenly between twodaughter cells at M phase. Progress through the cellcycle is coordinated by the actions of CDKs (cyclin-dependent kinases) that are activated by associationwith specific cyclin proteins (for reviews, see Pines,1993; Morgan, 1997). CDK activity is further regulatedthrough posttranslational modification of the CDKs,transcriptional control, the association of cofactors, in-tracellular localization, and the balance between syn-thesis and degradation of cyclins and other regulators(Morgan, 1995; Pavletich, 1999; Malumbres and Bar-bacid, 2001). Defects in this strict regulation of the cellcycle can result in the development of tumors and otherdiseases that involve diverse cellular processes includ-ing apoptosis, differentiation, angiogenesis, and inflam-mation.

In higher eukaryotes, entry to mitosis is triggered bythe key CDK known as Cdc2 (CDK1). The activity ofCDK1 is regulated both by association with B type

*Correspondence: ted.baker@auckland.ac.nz

cyclins and by activating and inhibitory phosphoryla-tion (Dunphy, 1994; Morgan, 1997). The association ofCDK1 with cyclin B during S phase facilitates the phos-phorylation of CDK1 at T14, Y15, and T161 (Atherton-Fessler et al., 1993; Draetta et al., 1988; Meijer et al.,1991; Parker et al., 1991; Solomon et al., 1992). T161phosphorylation allows stable association with cyclin Bthat is essential for CDK1 activation and the initiationof mitosis. On the other hand, phosphorylation of T14and Y15 is required to maintain CDK1 in an inactivestate during the preceding S and G2 phases.

The inhibitory inactivation of CDK1 is mediated byenzymes of the Wee1 kinase family, which thus play acritical role in the cell cycle by controlling the entryinto mitosis. In humans and other vertebrates, Y15 isphosphorylated by Wee1 kinase (Honda et al., 1992;McGowan and Russell, 1993; McGowan and Russell,1995; Parker and Piwnica-Worms, 1992; Watanabe etal., 1995), and another Wee1 family member, the mem-brane bound, dual-specificity Myt1 kinase, phosphory-lates both T14 and Y15 (Liu et al., 1997; Mueller et al.,1995). Late in G2, Cdc25 phosphatase dephosphory-lates T14 and Y15, activating CDK1 and thus promotingmitosis (Dunphy and Kumagai, 1991; Gautier et al.,1991; Sebastian et al., 1993).

Wee1 kinase is itself regulated by phosphorylationand degradation (Watanabe et al., 1995). Phosphoryla-tion of residues in its N-terminal domain, described be-low, generates a phospho-degron (signal for degrada-tion) that allows recognition by an F box protein andtargets Wee1A for ubiquitination and degradation (Wa-tanabe et al., 2004). Removal of Wee1 thus promotesentry into mitosis (Ayad et al., 2003; Watanabe et al.,2004). Phosphorylation of the C-terminal tail allows 14-3-3 peptides to bind to Wee1A, an event that influencesthe intracellular distribution of Wee1 and its level of ac-tivity (Wang et al., 2000; Lee et al., 2001). Recent evi-dence shows that Wee1 levels are also controlled bycomponents of the circadian clock, making it the keylink between the circadian clock and the cell cycle, andhence cellular proliferation (Matsuo et al., 2003).

The role of Wee1 in controlling entry to mitosis alsogives it considerable potential as a drug target. CellularDNA damage can initiate the G2 checkpoint, a pauseat G2 that prevents the cell from entering mitosis, pre-sumably giving time for other cellular mechanisms tomake repairs. Experiments in fission yeast indicate thatthe G2 checkpoint functions by maintaining Y15 phos-phorylation on CDK1 until DNA damage is repaired(O’Connell et al., 1997; Rhind et al., 1997). As a regula-tor of CDK1 Y15 phosphorylation, Wee1 is thus integralto this G2 checkpoint function, and Wee1 inhibitorshave potential use as G2-abrogating compounds. Asone example, studies using seven cancer cell lineslacking a functional p53 demonstrated that inhibition ofWee1 with a nanomolar inhibitor, PD0166285, effec-tively abrogated the G2 checkpoint (Wang et al., 2001).This allowed the p53−/− cells to proceed to prematuremitosis and cell death, suggesting that effective Wee1

Structure542

Tlinhibition might be used in this way to enhance conven-

tional cancer therapy. NpIn humans, two Wee1 kinases with different temporal

and spatial expression, Wee1A and Wee1B, have been wicharacterized (Nakanishi et al., 2000). The somatic

Wee1 kinase, Wee1A, is a 646 amino acid protein (Wa- αhtanabe et al., 1995) comprising three domains: a long

N-terminal regulatory domain, a central kinase domain, tNand a short C-terminal regulatory domain (Figure 1).

The N-terminal domain contains two phosphorylation tksites, at Ser53 and Ser 123, which provide the signal

that targets Wee1 for destruction. It also contains threeapotential PEST regions (Watanabe et al., 1995) and a

putative nuclear localization signal (Arg-Arg-Arg-Lys- PAArg). Phosphorylation of the penultimate residue in the

short C-terminal domain, Ser645, generates the 14- sm3-3 binding site. Although it has a strict specificity for

phosphorylation of Y15 in CDK1, the central kinase do- Asmain of Wee1A is not a typical tyrosine kinase. Se-

quence searches of the human genome do not place icWee1A in any of the identified tyrosine kinase subfami-

lies (Robinson et al., 2000), and comparisons with the abfull complement of protein kinases place Wee1A and

Myt1 in a separate family (Manning et al., 2002). vlHere we present the 1.8 Å crystal structure of the

kinase domain of Wee1A, complexed with a known in- i(hibitor of full-length Wee1A kinase, PD0407824 (Booth

et al., 2003). The structure, which is to our knowledge mtthe first for any Wee1 family member, reveals a close

structural relationship with prototypical serine/threo- dPnine kinases, coupled with Wee1-specific sequence

changes. These differences maintain Wee1A in an activewconformation and provide a basis for understanding the

substrate specificity of Wee1A. The structure also pro- mtvides a starting point for structure-based drug design

of Wee1 inhibitors for cancer therapy. f2SResults and Discussion(2Structure Determination

Sequence and structural alignments of other kinase do- cbmains led to the design of a construct comprising resi-

dues 291–575 of human Wee1A kinase, which was then ftexpressed in Sf9 insect cells. The crystal structure was

solved by single isomorphous replacement with anom- pWalous scattering (SIRAS) and refined at 1.8 Å resolution

to an R factor of 0.218 (Rfree = 0.237). Statistics for the Tsstructure determination and refinement are given in Ta-

bdpwadFigure 1. Domain Structure for Human Somatic Wee1 Kinase

(Wee1A) dThe N-terminal regulatory domain is shown as an open box with ethe known serine phosphorylation sites (S53 and S123) and nuclear wlocalization signal (NLS) indicated. The kinase domain is indicated rby a gray box, with the first and last residues of the construct ex-

Cpressed in this work indicated. The C-terminal serine residue impli-cated in 14-3-3β binding is also indicated (S642).

O

le 1. The final model comprises 258 amino acid resi-ues; the inhibitor PD0407824 (9-hydroxy-4-phenyl-6H-yrrolo[3,4-c]carbazole-1,3-dione); 2 Mg2+ ions, whichere identified as such by their coordination geometry;nd 144 water molecules. All 279 residues of the kinaseomain construct have been modeled except for resi-ues 436–455 and 570–575, which had no interpretablelectron density. The polypeptide has good geometry,ith 91.5% of nonglycine residues in the most favored

egions of the Ramachandran plot, as defined in PRO-HECK (Laskowski et al., 1993), and only one outlier.

verall Structurehe Wee1A catalytic domain displays a standard two-

obed kinase fold (Figure 2A). The mainly β sheet-terminal lobe is folded around a five-stranded anti-arallel β sheet and has the typical glycine-rich loop,ith consensus sequence GxGxxG (residues 306–311

n Wee1A), that is found in other kinases. The mainly-helical C-terminal lobe contains a characteristic four-elix bundle. The two lobes are joined by a linker pep-ide (residues 377–381) that connects strand β5 in the

lobe to helix αD in the C lobe; this has been showno allow relative movement of the two lobes in otherinases, for example cAMPK (Narayana et al., 1997).The active-site cleft is located between the two lobes

nd provides the binding site for the Wee1A inhibitorD0407824 (Figure 2B), an antagonist of the naturalTP substrate. The active-site region contains a shortection of extended polypeptide (the catalytic seg-ent) that includes the essential catalytic aspartatesp426, and a large loop, referred to as the activationegment, that provides a substrate binding platform

n protein kinases and can undergo conformationalhanges that control the activation state (Johnson etl., 1996). A sequence alignment of Wee1 family mem-ers from various species suggests structural conser-ation across the whole kinase domain (Figure 3). Theoop 436–455, not located in the structure, has beendentified from its sequence as a potential PEST regionWatanabe et al., 1995). PEST regions are found in

any short-lived eukaryotic proteins and play a role inheir degradation (Rogers et al., 1986). The solvated,isordered nature of this loop is consistent with itsEST annotation.A search of the current protein structure databaseith SSM (http://www.ebi.ac.uk/msd-srv/ssm/) revealsany protein kinases that can be superimposed onto

he Wee1A kinase domain with root-mean-square dif-erences in Cα positions of less than 2.0 Å over 220–40 residues. The closest structural matches are mostlyer/Thr kinases, including the active forms of CDK2

Jeffrey et al., 1995), Aurora kinase (Nowakowski et al.,002), and Chk1 kinase (Chen et al., 2000); these cellycle-associated kinases are all close metabolic neigh-ors to Wee1A. It is notable that the top hits are all

unctionally Ser/Thr kinases and that not until the four-eenth-highest hit is a Tyr kinase encountered. Althoughrotein kinases vary widely in amino acid sequence, theee1A sequence is also more closely related to Ser/

hr kinases than to Tyr kinases. This is illustrated by aimple BLAST comparison (Altschul et al., 1997) of the

Structure of Human Wee1 Kinase543

Table 1. Crystallographic Statistics

Crystal Native1 Native2 Hg

Data collection statisticsa

Resolution (Å) 1.81 (1.87–1.81) 2.25 (2.33–2.25) 2.60 (2.69–2.60)Reflections measured 734,175 189,188 750,884Unique reflections 34,834 19,169 12,493Completeness (%) 95 (92) 95 (77) 100 (99)Average I/σ 23 (2.6) 25 (4.9) 45 (13)Rmerge

b 0.082 (0.362) 0.079 (0.401) 0.081 (0.275)

SIRAS statistics (phasing to 3.0 Å resolution)

SOLVE FOM (3 × Hg) 0.32SOLVE z-score 12.07Resolve FOM 0.63

Refinement statistics

Resolution (Å) 47–1.81Reflections (F > 0.0) 34,800Total non-H atoms 2213Rcryst

c 0.218Rfree

d 0.237Residues in most favored regions of the Ramachandran 90.5

plot (%)Rmsd ideal bond length (Å) 0.022Rmsd ideal bond angles (o) 2.09

a Data for the highest resolution shell is given in parenthesesb Rmerge = S|Ii − <I>|/SIic Rcryst = S||Fobs|-|Fcalc||/S|Fobs|, calculated for the 95% data used in refinementd Rfree = same as for Rcryst but using only the 5% of the data excluded from refinement

Wee1A catalytic domain sequence with consensus se-quences for Ser/Thr kinases and Tyr kinases; expecta-tion values of 6e-40 and 3e-22, respectively, show thatWee1A shares features of both but is more Ser/Thr ki-nase-like. A dendrogram of 491 eukaryotic protein ki-nase domain sequences (Manning et al., 2002) likewiserecognizes Wee1 as a separate family, closest to sev-eral Ser/Thr kinase families. Taken together, the sequenceand structural comparisons, and the Myt1-Wee1 famil-ial relationship, suggest that the Wee1-family proteinsmay have evolved from a Ser/Thr kinase, a few key mu-tations converting Wee1 to a Tyr kinase that acts onCDK1.

Active SiteThe well-defined active-site cleft is bounded by thefive-stranded β sheet and glycine-rich loop from the Nlobe, which curve over it from above; the β5-αD con-necting loop (the hinge region), which defines the endof the cleft; and two parts of the C lobe, helix αD andthe catalytic segment, which make up the other wallof the cleft. The floor is formed by the N lobe helix αCand the region between β8 and β9 leading into the acti-vation segment. Many invariant or conservatively sub-stituted residues are found in the active site, particu-larly in the catalytic segment.

The catalytic segment, residues 422–433, extendsfrom strand β6 to the beginning of strand β7 and pres-ents the essential catalytic aspartate, Asp426, into theactive-site cleft. Its sequence, which is highly con-served (Figure 3), contains the active-site motif HxDand closely matches the protein kinase consensus se-quence IVHxDLKPxNIx. The residue preceding Asp426

is often an arginine in kinases that are activated byphosphorylation of residues in the activation segment,referred to as RD kinases (Johnson et al., 1996). InWee1A, however, the preceding residue is nonpolarMet425, which forms part of a hydrophobic cluster withVal487 and Leu495 that helps to anchor the activationsegment by interaction with Val475.

Superposition of the Wee1A structure onto the inhibi-tor or ATP bound structures of cAMPK and Chk1 ki-nases (Zheng et al., 1993; Chen et al., 2000) shows aclose overlay of the catalytically important residues(Figure 4A), including one of the two Mg2+ ions foundin the Wee1A structure. This Mg2+ is bound to Asn431from the catalytic segment and Asp463 from the startof the activation segment, together with three watermolecules, and has a coordination environment virtu-ally identical to that of the catalytically important Mg2+

ion in the cAMPK/ATP structure. The sixth coordinationsite is vacant in both structures.

One intriguing variation exists in the catalytic seg-ments. In cAMPK and Chk1, the carboxyl group of thecatalytic aspartate (Asp166 in cAMPK, Asp130 in Chk1)is hydrogen bonded to a threonine residue (Thr170 inChk1) that contributes to the specificity of Ser/Thr ki-nases versus Tyr kinases (Taylor et al., 1995). This helpsposition the aspartate side chain for its role in catalysis.In Wee1A, however, this threonine is substituted byAsp479 (a Wee1-specific substitution), precluding asimilar interaction with the catalytic Asp426. Instead,Asp426 is held in an appropriate geometry by hydrogenbonds with Asp479 NH and a network of water mole-cules, and by coordination to the Mg2+ ion bound toAsn431. A water molecule directly in front of Asp426

Structure544

Figure 2. X-Ray Structure of the Wee1A/PD0407824 Complex

(A) Ribbon diagram showing the canonical two-lobe protein kinase fold with inhibitor PD0407824 (ball-and-stick model) binding in the active-site cleft between the N and C lobes, hydrogen bonding to the hinge region. Two Mg2+ ions are shown as yellow spheres. β strands arecolored blue, α helices green, the catalytic (CS) and activation (AS) segments red. Figure prepared with Ribbons (Carson, 1997).(B) Electron density for the PD0407824 inhibitor bound to Wee1A, contoured at a level of 3 σ from an Fo − Fc omit map. Hydrogen bondslinking the inhibitor to the hinge region (residues 376–379) and to the two well-ordered water molecules, W614 and W724, are shown withbroken lines. Drawn with Ribbons (Carson, 1997).(C) Stereo Cα plot for the Wee1A catalytic domain structure. Every tenth residue is indicated with a small green sphere.

mimics a substrate tyrosine hydroxyl. A similarly placed Cwater molecule is found in cAMPK, where it is sug- 2gested to function as a nucleophile, initiating hydrolysis i(Madhusudan et al., 2002). r

dATP and Inhibitor BindingwCrystals of Wee1A were only obtained when the proteiniwas complexed with high-affinity inhibitors, suggestingNthat inhibitor binding may have induced some confor-

mational change and/or stabilization of the structure. a

onsistent with this, the inhibitor PD0407824 (FigureB) is bound deeply in the active-site cleft, largely bur-

ed within the protein, in a mostly hydrophobic envi-onment.

Inhibitor binding is marked by highly favorable hy-rogen bonding with surrounding protein groups, inhich the maleimido ring plays a key role. It is located

nternally, with its two oxygen atoms and interveningH hydrogen bonded to main chain and side chaintoms of residues 376–379 of the β5-αD loop, which

Structure of Human Wee1 Kinase545

Figure 3. Multiple Sequence Alignment of Wee1 Kinase Family Members

The sequence numbering is that of human Wee1A kinase, with the Wee1A secondary structure shown above the alignment. Invariant residues(white on red background) and conservatively substituted residues (red) in the Wee1 family are indicated. Unique Wee1 residues are markedwith an asterisk and are defined as those residues conserved in Wee1 but not found in protein kinase, serine/threonine kinase, or tyrosinekinase consensus sequences from the BLAST database (Altschul et al., 1997). The catalytic and activation segments are indicated with a redbar. Sequences shown are for Wee1A kinase of human (hu), mouse (mm), Carassius auratus (ca), Drosophila (dm), and Arabidopsis (at);Wee1B kinase of Xenopus (Weeb_xl); Mik1 kinase of S. pombe (Mik1_sp); and Myt1 kinase of human (Myt1_hu). Figure prepared using ESPript(Gouet et al., 1999).

forms the hinge region at the back of the cleft, and tothe first of two very well-defined water molecules,W614 and W724 (Figure 2B). Both waters are tetrahe-drally coordinated, completing a network of interac-tions between the inhibitor and the protein structure.W614 sits over the inhibitor phenyl ring, which occupiesa pocket bounded by hydrophobic side chain atomsof Val313, Lys328, and Ile374. Further stabilization ofinhibitor binding is provided by the packing of sidechains against the planar carbazole ring system: theconserved Wee1 residue Phe433 stacks against oneface of the ring system; Ile305 and Val313, which imme-diately precede and follow the glycine-rich loop, andAla326 from strand β3 together present a hydrophobicsurface to the other face. The glycine-rich loop itself,residues 306–311, makes no close contacts withPD0407824 and has higher B factors than the rest ofthe active-site region. The most exposed part of theinhibitor is the edge of the carbazole moiety, includingits pyrrole nitrogen.

Comparisons with other protein kinase inhibitor com-plexes (reviewed in Noble et al., 2004) show thatPD0407824 has particular features that enhance bind-ing in Wee1A. Thus, although the core structure of theprotein kinase inhibitor staurosporine is similar to thatof PD0407824, its IC50 for inhibition of Wee1A is morethan 100× higher (26 �M compared with 97 nM forPD0407824). By consideration of the mode of stauro-sporine binding found for Chk1 (Zhao et al., 2002), weattribute the stronger Wee1A inhibition by PD0407824to (1) the additional hydrogen bonds made by the sec-ond maleimido oxygen, and (2) improved hydrophobicinteractions and reduced steric repulsion resulting fromits rotatable phenyl substituent and lack of the tetrahy-dropyran ring of staurosporine (which would clashwith Phe433).

Attempts to cocrystallize Wee1A with staurosporine,or with ATP or ATP analogs, have been unsuccessful.This may be because of their lower affinity, but it mayalso reflect a change in the closure of the glycine-richloop over the PD0407824 inhibitor, which could affectcrystal packing. Differences of this type, accompanyingligand binding, have been seen for cAMPK (Narayanaet al., 1997).

Modeling ATP binding (Figure 4B) shows that the ad-enine moiety binds in the hydrophobic pocket formedby Ile305, Val313, Ala326, and Phe433, occupied by thecarbazole system in the inhibitor bound structure. Themodeling places the ATP adenine more shallowly in theactive site than PD0407824, however, with fewer con-tacts to the protein backbone, hydrogen bonding onlyto Glu377 O. This is entirely consistent with their rela-tive binding affinities, with ATP binding at least threeorders of magnitude more weakly than the PD0407824inhibitor (A.J. Kraker, personal communication). Two ly-sine residues, Lys328 and Lys428 (equivalent to Lys72and Lys168 in cAMPK), are positioned such that Lys328can hydrogen bond to the α- and β-phosphates, andLys428 to the γ-phosphate and the catalytic Asp426,like their analogs in cAMPK (Zheng et al., 1993). Lys428is not found in other Tyr kinases but is conservedamong Ser/Thr kinases.

Conformation of the Activation SegmentThe activation segment is a key functional region ofprotein kinases that extends from the back of the activesite, where a conserved Asp residue (Asp463 in Wee1A)binds the essential Mg2+ ion, and forms an irregular ex-tended loop that finishes with helix αEF in the C-ter-minal domain (Figure 2A). In many kinases this loop canundergo conformational changes, dependent on the

Structure546

Figure 4. Stereo Views of the Catalytic Sitesof Wee1A and cAMP Kinase

(A) Overlay of the Wee1A/PD0407824 andcAMP kinase/ATP structures. The Wee1A Cαribbon is shaded dark gray and the cAMPribbon is outlined in black (side chains areshaded similarly). Hydrogen bonds are indi-cated with broken lines. The experimentallylocated magnesium ions found in the twostructures are shown as larger spheres. Aftersuperposition, the catalytic sites have a root-mean-square difference in Cα atomic posi-tions of 0.76 Å for the residues shown.(B) Wee1A/ATP model. The ATP molecule,the tip of the glycine-rich loop, the side chainof Lys328, and a second magnesium (Mg2)were modeled on the basis of the cAMP ki-nase/ATP structure. The ATP base hydrogenbonds to Glu377 O at the back of the activesite while the phosphates coordinate the twomagnesium cations, Lys328, and Lys428.Both figures drawn with MolScript (Kraulis,1991).

phosphorylation state of certain residues (Johnson et gAal., 1996).

In Wee1A the activation segment is 25 residues in fHlength, residues 462–486. It is well ordered throughout

its whole length, with a conformation that corresponds eNvery closely to that of the activation segments in Chk1

(Chen et al., 2000), Aurora kinase (Nowakowski et al., ta2002), and phosphorylase kinase (Owen et al., 1995),

none of which appear to require phosphorylation for ac- bbtivation. Structural superpositions show that 21 Cα

atoms of the Wee1A activation loop can be superim- tposed on the corresponding atoms of Chk1, Aurora ki-nase, and phosphorylase kinase with rms differences s

aof 1.56, 1.55, and 1.62 Å, respectively. The activationloop does not occlude any part of the active site such m

ithat it could prevent either ATP or substrate peptidebinding. We conclude, therefore, that it is also in an 1

uactive conformation in our Wee1A structure.Like that in Chk1, the Wee1A activation segment is h

pstabilized in its active state by secondary structure andside chain interactions. In the N-terminal half of the ac- A

ftivation segment, a short β strand, β9 (468–470), pairswith β6 in the C-terminal domain, and the tip of the loop A

rhas consecutive β turns, 469–472 and 472–475, that arebuttressed against the C lobe by hydrophobic inter- B

actions through Ile470 and Val475. Side chain hydro-

en bonds between Thr468 and Ser472, and betweenrg469 and the C terminus of helix αC in the N lobe,

urther stabilize this portion of the activation segment.ydrophobic interactions involving side chains at eachnd of the activation segment (Leu464 and Val467 at its-terminal end and Leu483 at its C-terminal end) fur-

her anchor it to the C-terminal lobe, as does an invari-nt kinase ion pair between Glu486 and Arg557. A num-er of well-ordered water molecules form bridgesetween the activation segment and residues in both

he N-terminal and C-terminal lobes.Many of the side chains that stabilize the activation-

egment conformation are contributed by residues thatre invariant or highly conserved in all Wee1 familyembers and differ from the sequence patterns seen

n other protein kinases (Figure 3). Of the 25 residues,7 are strongly conserved in the Wee1 family and 7 arenique to it. This suggests that the conformation seenere is common to all Wee1 family members and hasarticular functional importance. Of particular note issp479, one of the Wee1-specific residues, which

orms a doubly hydrogen-bonded salt bridge withrg481, holding it in a position in which it can play a key

ole in substrate recognition and binding (see Substrateinding and Specificity).

Our structure suggests that regulation of Wee1A is

Structure of Human Wee1 Kinase547

achieved not by conformational changes in the activa-tion segment but through regions outside the catalyticdomain. This is consistent with previous experimentssuggesting that regulation of Wee1 kinases is achievedthrough its N- and C-terminal regulatory domains, bothby phosphorylation (Watanabe et al., 1995) and by 14-3-3 binding (Lee et al., 2001; Wang et al., 2000). Recentresults with both Xenopus and human Wee1 proteinsdemonstrate that phosphorylation of serine residues intheir N-terminal domains induces recognition by F box-containing proteins of the SCF E3 ubiquitin ligase com-plex and targets Wee1 for destruction (Ayad et al.,2003; Watanabe et al., 2004). This temporary removalof Wee1 leads to timely entry into mitosis.

Substrate Binding and SpecificityThe structure of the Wee1A kinase catalytic domainprovides a basis for understanding the specificity ofthis enzyme, which is so critical to the G2 checkpointand proper entry into mitosis in the cell cycle. The natu-ral substrate is CDK1, which is specifically phosphory-lated on Y15, located in its glycine-rich loop (sequenceGEGTYG, residues 11–16). Attempts to cocrystallizeWee1A or soak Wee1A crystals with 10- or 20-residuepeptides containing this target sequence were unsuc-cessful, presumably because binding depends also onparts of the CDK1 structure outside this region. Wetherefore docked an octapeptide with an appropriatesequence onto our Wee1A structure, as described inExperimental Procedures. In doing so, we assumedthat the substrate peptide has sufficient flexibility toconform to the contours of the Wee1A surface. Thepresence of three glycines and the known flexibility ofthis glycine-rich region in other protein kinases, includ-ing the very closely related CDK2, support this as-sumption.

The need to position Y15 for phosphorylation, withits hydroxyl replacing a water molecule from the Wee1Acrystal structure, imposes some constraints on wherethe peptide must bind. In this proposed binding mode(Figure 5A), the peptide occupies a shallow groove onthe Wee1A surface, passing between the side chainsof two arginine residues, Arg481 and Arg518, underGlu309 from the Wee1A glycine-rich loop, and docking

Figure 5. Model for Peptide Binding toWee1A

(A) The peptide Gly-Glu-Gly-Thr-Tyr-Gly-Val-Val (residues 11–18 of human CDK1) is shownas a stick model, color coded by atom type.Putative hydrogen bonds are shown as brokenlines. The peptide passes between Arg518and Arg481, partly hidden below it, and a hy-drogen bond to T14 helps position Y15 forphosphorylation by Wee1A. Glu309 wouldlimit the approach of substrate for phos-phorylation at T14. Figure drawn with SYBYL(Blanco, 1991), with solvent-accessible sur-faces calculated with a probe radius of 1.4 Åand colored by electrostatic potential (nega-

tive potential blue, positive potential red, and neutral regions green, as indicated by the color-bar legend).(B) Specific difference between Wee1 kinases and other tyrosine kinases in the peptide binding region, shown by comparison of Wee1A(green) with the insulin receptor tyrosine kinase IRK3 (mauve). In typical tyrosine kinases, the arginine residue equivalent to Arg481 hydrogenbonds to a nearby loop, causing a surface bulge. In Wee1A, however, Arg481 is sequestered by a Wee1-specific salt bridge with Asp479 thatinstead creates a shallow groove, assisted by an invariant leucine. Drawn with Ribbons (Carson, 1997).

V17 of the peptide into a hydrophobic pocket. The Y15

side chain is positioned in the active site for phosphoryltransfer, whereas the preceding T14 side chain is ori-ented outwards and can hydrogen bond to Arg518.From the Wee1A structure, two Wee1-specific featuresseem likely to play important roles in substrate specific-ity. First, Arg481 in the activation loop is the site of akey structural deviation from other tyrosine kinases, inwhich an equivalent arginine forms a surface bulge as-sociated with a conserved PxR (or PxK) sequence motifin the activation segment (Figure 5B). In Wee1A, in con-trast, Arg481 is sequestered by a salt-bridge interactionwith the Wee1-specific Asp479. This removes the bulgeand results in the surface groove in which the substratepeptide is docked in Figure 5A. Second, for phosphory-lation of T14 the peptide would have to approach moreclosely the glycine-rich loop of Wee1A. The presenceof the Glu309, another Wee1-specific feature, at the tipof this loop may inhibit this closer approach in the sameway that phosphorylation of Thr14 or Tyr15 in CDK1is believed to inhibit substrate binding through sterichindrance (Endicott et al., 1999).

ConclusionWee1 kinase plays a key role in cell cycle regulation byinactivating the cell cycle kinase Cdc2 (CDK1) throughphosphorylation of Y15 on CDK1. This inactivation isimportant in preventing entry into mitosis until DNAdamage is repaired, giving Wee1 an important functionin the G2 checkpoint. Our crystal structure of the hu-man Wee1A catalytic domain shows that Wee1 is anatypical tyrosine kinase in that it groups most closelywith Ser/Thr rather than Tyr kinases. Its specificityfor CDK1 Y15 phosphorylation appears to arise fromWee1-specific features in its activation loop, coupledwith the presence of Glu309 in the glycine-rich loop ofWee1A, which may disfavor phosphorylation of the pre-ceding CDK1 T14 residue by steric hindrance.

The importance of substitutions in the glycine-richloop in controlling activity is a recurring theme in pro-tein kinases and arises because of the need for thisflexible loop to fold over ATP for stable binding. InCDK1 and CDK2, phosphorylation of the glycine-richloop residues T14 and Y15 (equivalent to Glu309 andPhe310 in Wee1A) causes inactivation by disfavoring

protein-substrate binding and/or productive ATP bind-

Structure548

aing (Endicott et al., 1999; Bartova et al., 2004). By anal-cogy, we propose that the Wee1-specific presence of

Glu309 in the glycine-rich loop influences substratespecificity in a similar way. S

The Wee1A crystal structure, in complex with a nano- Tmolar inhibitor, provides a starting point for structure- p

dbased drug design of inhibitors that might be targetedlat Wee1A itself or at other Wee1 family members. Inhi-ebition of human Wee1A has been shown to induce pre-1mature entry into mitosis, before DNA damage hasT

been repaired at the G2 checkpoint (Wang et al., 2001), idemonstrating the potential use of such inhibitors as uenhancers of conventional cancer therapy. Our struc- a

cture defines features of Wee1A sequence and surfaceutopology that could be targeted by inhibitor moleculescin this and other Wee1 family members, and the bindingcmode of the PD0407824 molecule provides a guide toe

positions that might be appropriate for medicinal pchemistry or combinatorial chemistry substitutions i(provisional US patent application filed May 29, 2003, o

sUS Serial No. 60/473887).p(

Experimental Procedures FT

Construct Design, Expression, and Purificationa

The construct used for the structure analysis, comprising residues291–575 of human somatic Wee1A kinase (Wee1A291–575), was cho-sen on the basis of molecular-modeling experiments. Superpo-

Msition of seven tyrosine kinase structures downloaded from the

AHOMSTRAD website (Mizuguchi et al., 1998) led us to choose ap-Spropriate start and end points that would not compromise the in-(tegrity of the catalytic domain.sHuman U937 cells (T cell lymphoma) were used as a source oftmRNA for human Wee1A291–575 cDNA production. Amplicons usingMthe above primers were generated from U937 cDNA via the poly-imerase chain reaction using Platinum Pfx DNA Polymerase (Invitro-Pgen). The amplicons were purified using a High Pure PCR kitw(Roche), restricted with NcoI and EcoRI, and ligated into the NcoI/mEcoRI sites of the pFastBacHTb vector. The resultant recombinanttplasmids produced an in-frame fusion of the human Wee1A se-dquence with the vector His-tag and rTEV cleavage sequences3downstream of the polyhedrin promoter. Human Wee1A291–575 wascpurified from Sf9 insect cells by 6×His affinity chromatographyWusing Talon resin (CLONTECH), rTEV cleavage, dialysis, affinityschromatography, and, finally, size-exclusion chromatography on apSuperdex 200 HR 10/30 column (Pharmacia).bt

Crystallization and Data Collection (A 50 µl aliquot of Wee1A291–575 protein (concentration w7 mg/ml)was mixed with 2.5 µl of PD0407824 (9-hydroxy-4-phenyl-6H-pyr-

Crolo[3,4-c]carbazole-1,3-dione) at a 4 mM concentration in dimethyl

csulfoxide. Crystals were grown in hanging drops by mixing equal

tvolumes of the protein complex and the reservoir solution, com-mprising 100 mM HEPES (pH 7.5), 4.3 M NaCl. Crystals formed inc2–3 days, growing to approximately 0.5 mm in their longest dimen-Tsion within 3 weeks. The crystals were dipped briefly in a solutionWcontaining 85% (v/v) reservoir and 15% (v/v) glycerol, and flash-bfrozen in liquid nitrogen for data collection. Data to 1.8 Å resolutionswere collected at −170°C at the undulator beam line 17-ID of APSCusing a 165 mm MarResearch CCD detector with Pt/Pd-coated

ULE mirrors. The crystals belong to space group P41212 with unitcell dimensions a = b = 69.78, c = 157.02 Å. An additional 2.25 Åresolution native data set was collected at −160°C on a Rigaku

Arotating anode generator equipped with Supper-like total reflectionmirrors and a Mar345 image-plate detector. A mercury derivative

Wcrystal was produced by supplementing a crystallization drop withA1 mM HgCl2 and soaking a crystal for 10 min. The derivative crystalfwas soaked in cryoprotectant without the addition of HgCl2 and

flash-frozen for data collection on the rotating anode instrument b

bove. Data were collected to 2.6 Å resolution. All data were pro-essed with the Denzo/HKL package (Otwinowski and Minor, 1997).

tructure Determination and Refinementhe structure was determined by SIRAS (single isomorphous re-lacement with anomalous scattering) using the HgCl2 derivativeata and the rotating anode native data. Three mercury atoms were

ocated and refined with respect to their positions, thermal param-ters, and occupancy using SOLVE (Terwilliger and Berendzen,999). The SOLVE phases were input to RESOLVE (Terwilliger, 1999;erwilliger, 2000) for density modification of the resulting map. Annitial Wee1 model was built automatically into the RESOLVE mapsing MAID (Levitt, 2001). This resulted in a backbone trace forpproximately 90% of the secondary structure elements. Sidehains were manually built into the experimental electron densitysing O (Jones et al., 1991). Further refinement used the 1.8 Å syn-hrotron data and iterative cycles of CNS simulated annealing,onjugate gradient minimization, and B factor refinement (Brüngert al., 1998). Throughout refinement, 2Fo − Fc maps (including ex-erimental phases) and the SIRAS map were used for model build-

ng. CNS density-modified maps (solvent flipping) and compositemit maps further aided the manual building process. All mapshowed clear density for the inhibitor, allowing manual docking andositional refinement. Finally, 144 water molecules and 2 Mg2+ ions

identified by octahedral coordination geometry) were located ino − Fc maps and were refined. The final refinement cycles usedLS and CGMAT refinement in REFMAC (Murshudov et al., 1997)nd eased restraints.

olecular ModelingTP was modeled into the Wee1A active site using the programsYBYL (Blanco, 1991), GOLD (Jones et al., 1995), and MODELLER

Marti-Renom et al., 2000). The glycine-rich loop of the Wee1Atructure was modified to conform to the more closed conforma-ion of the cAMPK/ATP/peptide structure (Zheng et al., 1993) usingODELLER. Side chains in the active site and an additional Mg2+

on were also positioned as in the cAMPK structure. TheD0407824 inhibitor and water molecules were removed, chargesere assigned to the remaining atoms using SYBYL (Gasteigerethod), and an ATP molecule was then docked into the apo struc-

ure using GOLD. GOLD assigned atom types by default, and aistance restraint between Glu377 O and ATP N6 (min 2.4 Å, max.2 Å, spring constant 5.0) was included in the GOLD docking pro-ess to position the adenine moiety of ATP in the back of theee1A active-site cleft. GOLD defined the active site by a cavity

earch within 10 Å of Glu377. Multiple GOLD solutions consistentlylaced the ATP molecule in the same site, with the ATP phosphatesound to the active-site magnesium ions. The top solution was

hen subjected to minimization in SYBYL using the Powell methoddefault parameters).

An octapeptide GEGTYGVV, comprising the target sequence ofDK1, was manually docked into the Wee1A/ATP model. Theentral tyrosine, corresponding to CDK1 Y15, was placed besidehe catalytic Asp426 with its hydroxyl replacing a bound waterolecule and its location also guided by that observed in a peptide

omplex of the insulin receptor tyrosine kinase (Hubbard, 1997).he remainder of the peptide was placed to complement theee1A surface in a conformation similar to that found for peptide

inding to the closely related cAMPK (Zheng et al., 1993). The re-ulting Wee1A-peptide model was then energy minimized usingNS conjugate gradient minimization with the X-ray term excluded.

cknowledgments

e thank Erli Zhang for collecting data at the IMCA-CAT station atPS; John Booth, Al Kraker, Dan Ortwine, and Charles Hasemann

or many useful discussions; and Pfizer Global Research, Ann Ar-or, Michigan, for support of the research.

Structure of Human Wee1 Kinase549

Received: July 14, 2004Revised: December 21, 2004Accepted: December 22, 2004Published: April 12, 2005

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ccession Numbers

he coordinates and structure factor amplitudes for the Wee1A ki-ase domain structure have been deposited in the Protein Dataank with the entry code 1X8B.