Crystal Structure of the Atypical Protein Kinase Domain of a TRP Channel with Phosphotransferase Activity

Molecular Cell, Vol. 7, 1047–1057, May, 2001, Copyright 2001 by Cell Press

Crystal Structure of the Atypical Protein Kinase Domain of a TRP Channel with Phosphotransferase Activity Hiroto Yamaguchi,1,2 Masayuki Matsushita,3 Angus C. Nairn,2 and John Kuriyan1,2,4 1 Howard Hughes Medical Institute 2 The Rockefeller University New York, New York 10021 3 Okayama University Medical School 2-5-1 Shikata Okayama 700-8558 Japan

Summary Transient receptor potential (TRP) channels modulate calcium levels in eukaryotic cells in response to external signals. A novel transient receptor potential channel has the ability to phosphorylate itself and other proteins on serine and threonine residues. The catalytic domain of this channel kinase has no detectable sequence similarity to classical eukaryotic protein kinases and is essential for channel function. The structure of the kinase domain, reported here, reveals unexpected similarity to eukaryotic protein kinases in the catalytic core as well as to metabolic enzymes with ATP-grasp domains. The inclusion of the channel kinase catalytic domain within the eukaryotic protein kinase superfamily indicates a significantly wider distribution for this group of signaling proteins than suggested previously by sequence comparisons alone. Introduction Transient receptor potential (TRP) channels are integral membrane proteins in which the ion-conducting pores are formed by six membrane-spanning helical segments that are similar to those of voltage-gated potassium channels and cyclic nucleotide-gated channels (reviewed by Harteneck et al., 2000). TRP channels take their name from the trp (transient receptor potential) mutation in Drosophila (Montell and Rubin, 1989), which results in the attenuated development of a depolarizing potential upon photoreceptor stimulation as a consequence of a defect in a calcium-entry mechanism (reviewed by Hardie and Minke, 1995; Ranganathan et al., 1995). A number of TRP channels are found in mammalian cells, and these are generally permeable to calcium ions (Harteneck et al., 2000). TRP channels are involved in the sustained entry of extracellular calcium into cells, following the activation of receptors that in turn activate protein kinases or heterotrimeric G proteins (reviewed by Barritt, 1999). TRP channels are divided into three families based on the nature of their cytoplasmic extensions and their mechanisms of activation (Harteneck et al., 2000). Short TRP channels (STRPC family) are typically activated by pathways involving various isoforms of phospholipase 4

Correspondence: [email protected]

C (PLC), although the details of this coupling remain unclear. OTRPCs (TRP channels related to osm-9) are activated by a variety of physical or chemical stimuli, such as heat or osmotic shock. Long TRP channels (LTRPCs) are distinguished by having particularly long extensions outside the channel segment. LTRPCs are implicated in important control mechanisms regulating growth, differentiation, and death, but their activation mechanisms are not well understood. The dearth of detailed information regarding how TRP channels are coupled to the activation of cell-surface receptors makes it particularly important to study the various mechanisms that modulate TRP channel activity. Recently, a novel TRP channel of the LTRPC subclass has been identified that differs from other members in having protein kinase activity. This novel channel was discovered by using a portion of PLC-␤1 as bait in a yeast two-hybrid screen of a rat brain library (Runnels et al., 2001) and also, independently, by analysis of EST and genome databases followed by RT–PCR (GenBank accession number 8131903; M. Matsushita et al., submitted). Named ChaK, for channel kinase, or TRP-PLIK, for phospholipase C-interacting kinase, this 1863-residue protein has a centrally located calcium channel domain that is highly similar to corresponding segments of members of the TRP family. The highest sequence similarity is to melastatin 1, an LTRPC channel that is expressed specifically in melanocytes but is suppressed in metastatic melanoma cells (Duncan et al., 1998). That this novel member of the TRP channel family might have protein kinase activity was suggested initially by the presence of strong sequence similarity between a ⵑ300 residue C-terminal region of ChaK and the catalytic domains of members of a family of atypical protein kinases known as ␣-kinases. The ␣-kinases were initially identified because of the experimentally observed ability of EF-2 kinase to phosphorylate elongation factor-2, and the subsequent discovery of sequence similarity to Dictyostelium myosin heavy chain kinase (Cote et al., 1997; Redpath et al., 1996; Ryazanov et al., 1997). The kinase domain of ChaK is 28% identical in sequence to that of mouse EF-2 kinase (Figure 1). Members of the ␣-kinase family share no detectable sequence similarity with the large family of “classical” eukaryotic protein kinases with specificity for serine, threonine, or tyrosine residues (Ryazanov et al., 1999). The combination of a Ca2⫹ channel and a protein kinase domain within a single polypeptide chain is unusual. Although no other known ion channel possesses a kinase domain within the same polypeptide chain, many channels are known to be regulated by phosphorylation (Levitan, 1999). The kinase domain of ChaK is active as a protein kinase when expressed by itself as a fusion protein with GST (Runnels et al., 2001). Expression of the kinase domain alone (residues 1580–1863) resulted in a low level of autophosphorylation on serine residues (M. Matsushita et al., submitted). Inclusion of a Ser/Pro-rich segment of ChaK that is immediately upstream of the kinase domain (residues 1385–1590) resulted in a much more vigorous level of autophosphory-

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Figure 1. Sequence Alignment of ␣-Kinase Domains A schematic diagram of the domain structure of ChaK (top), and a sequence alignment of the kinase domains of mouse ChaK, mouse and C. elegans elongation factor-2 kinases (EF2K) (GenBank accession numbers 3334172 and 3334171), and three subtypes of myosin heavy chain kinase (MHCK) of Dictyostelium (GenBank accession numbers 1170675, 3122317, and 585362). Shown above the alignment are the secondary structural elements found in the structure of the ChaK–AMP•PNP complex; ␣ helices (␣A–␣F) are shown as filled rectangular boxes, and ␤ sheets (␤1–␤14) are shown as arrows. The residue numbering of mouse ChaK is shown. The conserved sequence motif GxA(G)xxG (in a red box) and the phosphate binding P loop are indicated below the alignment. Conserved residues of functional importance are boxed in green for interaction with the adenosine moiety of nucleotide, in orange for interaction with the phosphate groups, and in blue for coordination of the zinc atom. The region shaded in gray between 1649 and 1709 cannot be aligned reliably.

lation on serine and threonine residues, most likely in the Ser/Pro-rich segment (M. Matsushita et al., submitted). GST-fusion proteins containing the ChaK kinase domain are also able to phosphorylate myelin basic protein on serine and threonine residues (M. Matsushita et al., submitted; Runnels et al., 2001). Mutation of residues in either of two segments within the kinase domain that are conserved among ␣-kinases (a glycine-rich segment and a zinc binding motif) significantly impaired the catalytic activity of ChaK (Runnels et al., 2001). When these mutations were introduced into the intact ChaK protein expressed in CHO-K1 cells, channel function was significantly reduced, indicating that the kinase activity is required for the proper function of ChaK (Runnels et al., 2001). Investigation of the mechanism of this novel kinase domain is clearly an important step toward working out how this channel functions. No structural information is available for any member of the ␣-kinase family, making such investigations difficult. We have therefore determined the crystal structure of the kinase domain of

ChaK. The central catalytic core of the kinase domain shows structural similarity to the classical protein kinases, which is unexpected because of the absence of significant sequence similarity between these two families of kinases. Analysis of the structures of nucleotide complexes of the ChaK kinase domain reveals a striking conservation of residues known to be important in the catalytic mechanism of classical protein kinases. Conserved residues that abrogate channel function and catalytic activity when mutated (Runnels et al., 2001) are seen to be important for peptide substrate recognition (the glycine-rich motif in the C-terminal region) and for structural stability (the zinc binding motif). Although the structural similarity between ChaK and classical protein kinases is particularly marked in the N-terminal lobe, the structure of the C-terminal lobe is closer in certain key features to the corresponding lobe of metabolic enzymes with ATP-grasp folds. This finding further strengthens the evolutionary link between protein kinases and metabolic enzymes that has been suggested previously (Grishin, 1999).

Structure of the Kinase Domain of a TRP Channel 1049

Figure 2. ChaK Kinase Domain Structure The structure of the ChaK kinase domain dimer is shown in stereo. Individual protomers are shown in red and blue. The zinc atoms are shown as yellow spheres. Both N and C termini are indicated as N and C.

Results and Discussion Overall Structure The structure of the kinase domain of ChaK (Table 1, Figure 2) bears a striking resemblance to that of classical protein kinases, as exemplified by cAMP-dependent protein kinase, PKA (Knighton et al., 1991a, 1991b) (Figure 3A). As in PKA, the domain consists of two lobes that bind nucleotide at the interface between them. The N-terminal lobe of ChaK is very similar in its topology to the corresponding lobe of PKA, but there are significant differences in the C-terminal lobe, which are discussed in a subsequent section. The kinase domain of ChaK forms a dimer in the crystal as a consequence of the exchange between monomers of an N-terminal 27-residue segment that is mainly helical (1551–1577; the dimerization segment) (Figure 2). The dimerization segment of one molecule interacts extensively with the kinase domain of the other molecule in the dimer, with a buried surface area of 1281 A˚2 out of a total surface area of 2945 A˚2 for this segment. Such “domain swapping” is not uncommon in multimeric proteins (Bennett et al., 1995) and is consistent with the observation that the kinase domain is a dimer in solution (data not shown). It has been suggested that TRP channels might form tetrameric assemblies, by analogy to voltage-dependent potassium channels (Harteneck et al., 2000), but further studies are required to establish whether the observed domain-swapped dimer is relevant for the biological function of ChaK. Each monomer is comprised of six ␣ helices (␣A–␣F) and fifteen ␤ strands (␤1-␤15) (Figure 3A, center). The N-terminal lobe consists mainly of a highly curved ␤ sheet (␤1-␤4/␤5-␤6-␤7-␤10-␤9). Significant sequence similarity in the ␣-kinase family starts from Trp 1600 in ChaK, in strand ␤3 (Figure 1). The loop between ␤5 and ␤6 is a phosphate binding P loop, which contains a semiconserved region, 1618GGGL. In the C-terminal lobe, the catalytic machinery of the kinase is constructed on a ␤ sheet platform (␤11-␤12/13-␤14/15-␤8) and presented to the interlobe cleft. A GxA(G)xxG motif that is highly conserved within the ␣-kinase family is located in the loop that connects this platform to the base of the C-terminal lobe (helices ␣E and ␣F). This region

has poor electron density in the structure of unliganded ChaK, suggesting that it is intrinsically flexible. In classical protein kinases, the so-called “activation loop” is in the equivalent position to this segment of ChaK (Figure 3A). Similarities between the Nucleotide Binding Sites of ChaK and PKA Suggest a Common Evolutionary Origin Classical protein kinases play central roles in eukaryotic signal transduction and have evolved into one of the largest protein families known to date. Starting with PKA (Knighton et al., 1991a, 1991b), the structures of many classical protein kinases have been determined, leading to considerable insights into the mechanism of nucleotide binding and catalysis (Johnson et al., 1998). Comparison of the structures of ChaK and PKA reveals that despite the lack of significant sequence similarity, key features of the active site are the same in the two proteins. This allows further studies on the ChaK kinase domain to be guided by the considerable body of knowledge regarding the mechanism of classical protein kinases. The structures of the ChaK kinase domain in complex with ADP and the ATP analog AMP•PNP show that nucleotides are bound at the interlobe cleft, as in classical protein kinases (Figures 3A and 4). Comparison of the polypeptide chain folds of ChaK and PKA in the region surrounding the nucleotide binding site reveals close correspondence in detailed topology (see Figure 5 for an “open book” representation of the nucleotide binding site that highlights these similarities in the chain fold). In addition, four sets of interactions between the nucleotide and the kinase domain involve residues that are similarly situated in ChaK and PKA in terms of their relative positions along the polypeptide chain (Figure 4). That ChaK and the classical protein kinases share a common evolutionary origin is indicated by the conservation of chain topology around the nucleotide binding site and the preservation of the order of the four interacting elements along the polypeptide chain (Figure 5). The first conserved interaction involves the formation of hydrogen bonds between nitrogen atoms of the adenine ring and main chain atoms in the loop connecting the two lobes of the kinase domain (Figure 4C). This

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Figure 3. Structural Comparison of the ChaK Kinase Domain with cAMP-Dependent Protein Kinase and Succinyl-CoA Synthetase (A) Segments of the ChaK kinase domain (center) are colored in a gradient from the N terminus (magenta) to the C terminus (blue). A zinc atom bound to the C-terminal lobe of the ChaK kinase domain is shown as a gray sphere. Secondary structural elements in cAMP-dependent protein kinase (PKA) (left, Protein Data Bank ID code 1cdk; Bossemeyer et al., 1993) and E. coli succinyl-CoA synthetase (right, Protein Data Bank ID code 1cqi; Joyce et al., 2000) are colored the same as in the ChaK kinase domain when they are in equivalent positions in the topology of the polypeptide chain fold. Functionally important segments are shown in distinct colors: the P loops (blue), the catalytic loop in PKA (magenta), the activation loop in PKA (orange), and the conserved glycine-rich segment in the ChaK kinase domain (red). (B) The zinc binding motif in the ChaK kinase domain. The polypeptide chain is shown as ribbons in the same color as in (A).

interaction in ChaK mimics a characteristic feature of classical protein kinases, in which the adenine group is similarly tethered. Small molecule inhibitors of classical protein kinases almost invariably exploit these hydrogen bonding interactions, further emphasizing their importance as major determinants of nucleotide orientation and specificity. The second feature that is conserved between ChaK and PKA involves another invariant feature of classical protein kinases. In the active conformation of these kinases, a strictly conserved lysine residue (Lys-72 in PKA) interacts with the ␣- and ␤-phosphates of ATP and is ion paired to a conserved glutamate side chain (Glu-91 in PKA) presented by helix ␣C. This lysine is absolutely required for catalytic activity; even the conservative substitution of this lysine by arginine reduces protein kinase activity very significantly (Robinson et al., 1996). In ChaK, Lys-1646 is located in precisely the same position along the polypeptide chain as Lys-72 in PKA (Figure 4B). Likewise, Glu-1672 of ChaK is located in the same

position on helix ␣C as is Glu-91 in PKA (data not shown). It should be noted, however, that instead of interacting with the ␣- and ␤-phosphates of AMP•PNP, Lys-1646 of ChaK forms hydrogen-bonding interactions with the ␣-phosphate group and the adenine ring and with Glu1718 (from strand ␤10) instead of Glu-1672 (presented by helix ␣C) (Figures 4B and 4C). The significance of this distinction from PKA is unclear because the presence of high phosphate concentrations in the crystallization condition appears to have displaced magnesium ions from the active site, perhaps leading to some alterations in active site geometry. The final two sets of interactions with the nucleotide that are similar between ChaK and PKA also involve interactions with the phosphates. The glycine-rich phosphate binding loop of ChaK, 1618GGGLR, presents backbone amide nitrogens toward the cleft so that they form hydrogen bonds with the ␤- and ␥-phosphate groups of AMP•PNP. This glycine-rich loop is located in the same position in the peptide chain as the corresponding

Structure of the Kinase Domain of a TRP Channel 1051

Figure 4. Nucleotide Binding in the ChaK Kinase Domain (A) The nucleotide binding site of ChaK (stereo view). Ball and stick models are shown for the conserved residues (box shaded in Figure 1) along with the bound nucleotide AMP•PNP (blue). Residues that interact with the adenine moiety are shown in green, and those that interact with the phosphate groups are in orange. (B) Superposition of PKA (orange) and the ChaK kinase domain (green) is shown in stereo. The side chains of catalytically important residues in the active site are shown along with the bound nucleotides. The bonds in PKA are drawn thinner than those in ChaK. (C) Comparison of hydrogen bonds and salt bridges to the bound nucleotide between PKA and the ChaK kinase domain.

element in PKA (Figures 3A and 5B). Three residues in ChaK that are absolutely conserved in ␣-kinases (Asp1765, Gln-1767, and Asp-1775) are located near the phosphate groups (Figures 1 and 4A). These are disposed very similarly to three essential residues of PKA (Asp-166, Asn-171, and Asp-184) and are located in the same positions along the chain in the two proteins (Figures 4B, 5, and 7). In PKA, Asn-171 and Asp-184 coordinate two magnesium ions that bridge the phosphate groups (Knighton et al., 1991a, 1991b), and two residues in ChaK, Gln-1767 and Asp-1775, most likely play equivalent roles. In one protomer of the ChaK–ADP complex, there is a magnesium ion between the ␣- and ␤-phos-

phates located 3.5 A˚ away from a carboxyl oxygen of Gln-1767. In the ChaK–AMP•PNP complex, a water molecule occupies virtually the same position as the magnesium ion. Although the general features of nucleotide binding are closely preserved between ChaK and PKA, there are also some significant differences between them that alter the detailed structures of the active sites. The residues that contact the base and the sugar of the nucleotide are generally different between the ChaK kinase domain and classical protein kinases (Figure 4C). In ChaK, a hydrophobic slot is created by two sets of residues in the interlobe cleft: Met-1721, Phe-1725, and Thr-

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Figure 5. Comparison of the ChaK Kinase Domain to Its Structural Neighbors (A) “Open book” ribbon diagrams in which the N- and C-terminal lobes of the kinase are opened up with respect to each other. Structural elements that are similar between cAMP-dependent protein kinase (PKA), ChaK, and the E. coli succinyl-CoA synthetase ␤ subunit are colored as in (B). Bound nucleotides are shown in orange. Three conserved residues that are important for catalysis are shown in blue. (B) Chain topology diagram showing alignments of the equivalently disposed structural elements in PKA, ChaK, and succinyl-CoA synthetase (SCS). The N- and C-terminal elements are shown in the upper and the lower panels, respectively. The nucleotide binding P loops are colored in orange and indicated. The bulged catalytic loop in PKA is also indicated.

Structure of the Kinase Domain of a TRP Channel 1053

Figure 6. The Conserved GxG(A)xxG Motif in ␣-Kinases Shown in red is the flexible loop (residues 1781–1799) that contains the conserved GxG(A)xxG motif in ␣-kinases. The C␣ atoms of the glycine and alanine residues are shown in cyan (the C-terminal glycine is at the bottom). The substrate-mimicking peptide from PKA shown in green (Ala-17 in a yellow sphere) is docked to the ChaK molecular surface by superposing the ternary complex structure of PKA to the ChaK kinase domain. The AMP•PNP in the ChaK complex structure is also shown in the cleft.

1774 (C␥ methyl group) on the lower, C-terminal side and Met-1617, Ala-1624, and Ile-1644 on the upper, N-terminal side (Figure 4A). None of these residues are pre-

served in PKA, and they are not strictly conserved amongst the ␣-kinases (Figure 1). These differences between ChaK and PKA are potentially of interest because the nucleotide binding sites of classical protein kinases are proving to be very important targets for drug development. Because of the importance of TRP channels in general and because of the essential role of the kinase domain of ChaK in its function, this domain is potentially an important target for the development of specific inhibitors. In the case of the classical protein kinases, the high level of sequence conservation in the vicinity of the nucleotide binding site has made it somewhat difficult to obtain highly specific inhibitors. The differences between the structure of ChaK and PKA suggest that it should be possible to obtain inhibitors that exploit the generally favorable binding properties of the ATP binding site but which selectively recognize ChaK (Cho et al., 2000). A Conserved C-Terminal Glycine-Rich Motif Is Likely to Be Involved in Peptide Substrate Recognition The C-terminal lobe of ChaK contains a GxA(G)xxG motif that is located between strand ␤15 and helix ␣E. This glycine-rich motif had been surmised to correspond to the glycine-rich phosphate binding loop of classical protein kinases. The structure of the ChaK kinase domain shows instead that the C-terminal GxA(G)xxG motif is located within the region that corresponds to the activation loop of classical kinases. In those kinases, the acti-

Figure 7. Comparison of the Structural Platforms for the Catalytic Residues between ChaK and Its Structural Neighbors Ribbon diagrams showing three ␤ strands and four conserved residues in cAMP-dependent protein kinase, P. polycephalum actin-fragmin kinase (Protein Data Bank ID code 1cja; Steinbacher et al., 1999), ChaK, and E. coli succinyl-CoA synthetase. Strands in the N- and the C-terminal lobes are shown as yellow and green arrows, respectively. The protruding catalytic loops in PKA and actin-fragmin kinase as well as the corresponding segment in ChaK are colored in red. Conserved residues and the bound nucleotides are labeled.

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Table 1. Crystallographic Data Diffraction Data and Phasing a X-ray Source

Wavelength (A˚)

Mean Redundancy

Completeness (%)

c

Rsym (%)

d

Riso (%)

e

dmin (A˚)

Unique Reflections

b

Crystals Native Yb(AcO)3-1 Yb(AcO)3-2 Yb(AcO)3-3 Yb(AcO)3-4 j EMP-1 k EMTS-1 k EMTS-2 Pb(AcO)2 AMP•PNP ADP

X25 X25 X4A X9B RU200 X9B X9B RU200 RU200 X4A X4A

1.1000 1.3838 1.2421 1.2421 1.5418 0.9795 0.9795 1.5418 1.5418 0.96487 0.96487

2.8 3.5 3.8 4.0 4.0 4.0 3.5 3.4 4.0 2.0 2.4

20775 10687 7865 7332 7448 7476 11065 12024 7153 53642 32145

3.4 3.6 3.2 4.0 4.1 4.1 4.0 3.9 3.0 4.9 4.0

97.4 98.7 91.0 98.7 99.0 100.0 98.5 99.8 95.8 94.1 97.0

4.9 7.7 7.3 9.5 7.2 5.7 5.1 9.5 7.9 6.8 5.1

— 24.9 31.6 33.3 22.7 14.5 17.4 20.1 14.6 — —

— 1.59/1.13 1.83/1.39 1.76/1.34 1.43/0.98 0.99/0.75 1.95/1.46 1.97/1.42 0.75/0.62 — —

(94.2) (98.0) (88.2) (98.3) (94.1) (100.0) (89.3) (99.2) (93.0) (92.1) (94.6)

(17.2) (22.8) (27.9) (41.8) (25.8) (12.8) (10.9) (22.4) (15.1) (18.5) (17.7)

Phasing Power

f

Rcullis

— 0.83 0.80 0.79 0.89 0.89 0.77 0.77 0.91 — —

Refinement Statistics Rms Deviations g

Reflections Used

h

Rwork (%)

i

Data Set

Nonhydrogen Atoms

Rfree (%)

Bonds (A˚)

Angles (⬚)

B Values (A˚2)

AMP•PNP ADP

4816 4808

51278 31871

24.0 21.5

29.4 24.9

0.007 0.008

1.4 1.4

3.02 2.50

Data for the outermost resolution shell are given in parentheses. a X-ray sources: RU200 is mirror-focused CuK␣ radiation from a Rigaku RU-200 rotating anode source; X4A, X9B, and X25 are beamlines at the National Synchrotron Light Source. b Values for redundancy represent ratios of the total number of measurements to the number of unique reflections. c Rsym ⫽ 100 • ⌺h⌺i|Iˆ(h) ⫺ I(h)i|/⌺h⌺i(h)i, where I(h)i is the ith measurement of reflection h, and Iˆ(h)i is the mean value of the N equivalent reflections. d Phasing power ⫽ rms (|FH|/E), where |FH| is the heavy-atom structure factor amplitude, and E is the residual lack of closure. e Riso ⫽ 100 • ⌺|FPH| ⫺ FP||/⌺FP, where |FPH| and |FP| are heavy-atom derivative and protein structure factor amplitudes, respectively. f Rcullis ⫽ ⌺||FPH ⫾ FP| ⫺ FH(calc)|/⌺|FPH ⫾ FP| for all centric reflections. g Number of reflections used in working set. h Rwork ⫽ 100 • ⌺||Fobs| ⫺ ||Fcalc||/⌺|Fobs|, where summation is over data used in the refinement. i Rfree is the same calculation including the 5% data excluded from all refinements. j ethylmercuric phosphate k ethylmercuricthiosalicylic acid

vation loop presents a ␤ strand for pairing with the peptide substrate, usually after the activation loop undergoes a conformational change that is induced by phosphorylation (Hubbard, 1997). While phosphorylation in this region is not known to be important for the ␣-kinase family, the location of this segment makes it likely that it too is involved in peptide substrate recognition (Figures 4A and 6). The structure of PKA has been determined in complex with an inhibitor that closely mimics a peptide substrate (Knighton et al., 1991a, 1991b). A structural alignment of the kinase domains of ChaK and PKA allows the substrate-mimicking inhibitor peptide to be docked onto the surface of ChaK (Figure 6). Although such a docking is only a rough guide to potential modes of substrate binding, it reveals that there is no obvious impediment to the docking of peptide substrates onto the ChaK surface in an extended conformation, as seen in classical protein kinases. In particular, there are no obvious features that would suggest that ChaK recognizes ␣-helical substrates, as had been surmised previously (Ryazanov et al., 1999). Given the model for the substrate, the ␥-phosphate of AMP•PNP in the ChaK structure is turned away from the site that would be occupied by the hydroxyl group of the substrate (Figure 6). This might be a consequence of distortions induced at the active site of ChaK by the crystallization conditions, as mentioned previously. The structures of classical protein kinases bound to AMP•PNP

have also revealed similar nonproductive conformations for the terminal phosphate (Hubbard, 1997; Sicheri et al., 1997). This model, based on PKA, suggests that a peptide substrate can be easily accommodated between the terminal phosphate group of the nucleotide in ChaK and the side chains of Asn-1731 and Asn-1795, replacing a string of water molecules observed in the ChaK crystal structure (Figure 4A). It is an intriguing possibility that the side chains of the asparagine residues might bind the peptide substrate via hydrogen bonds to the backbone amide and carbonyl groups, as seen in several peptide complexes of other proteins (Conti et al., 1998; reviewed by Madden, 1995). Replacement of the final glycine residue in the GxA(G)xxG motif by aspartate results in a severe reduction in kinase activity (Runnels et al., 2001). This mutation would introduce a negatively charged residue that would protrude directly between Asn-1731 and Asn-1795, colliding with the peptide substrate as modeled here (Figure 6). The aspartate mutation would also introduce a negative charge in the vicinity of the ␥-phosphate of ATP. Both of these factors are expected to impede catalysis. A Zinc Binding Module in the C-Terminal Lobe Is Important for Structural Integrity One distinction between ChaK and classical protein kinases is that the C-terminal lobe in ChaK contains a metal ion, most likely zinc. The metal ion is coordinated

Structure of the Kinase Domain of a TRP Channel 1055

by His-1751 (from ␣D), His-1808, Cys-1810 (in the linker between ␣E and ␣F), and Cys-1814 (from ␣F) (Figure 3B). The geometry of the coordination is tetrahedral, typical for Zn coordination in protein structures (Schwabe and Klug, 1994). The zinc atom is integrated into the hydrophobic core of the C-terminal lobe and is secluded from solvent. All four metal ligands are strictly conserved in ␣-kinases (Figure 1). The presence of a zinc binding module within the ChaK kinase domain was correctly predicted previously, but it had been wrongly surmised to be related to zinc binding FYVE domains (Runnels et al., 2001). In contrast to FYVE domains, which bind two zinc atoms (Mao et al., 2000; Misra and Hurley, 1999), only a single zinc atom is ligated to ChaK in a structural environment that is not obviously related to FYVE domains. The zinc binding module of ChaK is completely buried in the structure, and the coordination of zinc serves to pin down a “flap” that covers one portion of the hydrophobic core of the C-terminal lobe. The ability to properly coordinate zinc is expected to be crucial for the stability of the kinase domain. Replacement of the two cysteine residues in the zinc binding module by alanine, which would completely disrupt the zinc binding module, leads to complete loss of kinase activity as well as a reduction in currents generated by ChaK in wholecell recordings (Diggle et al., 1999; Runnels et al., 2001). Structural Neighbors and Evolutionary Relationships A search of the protein structure database using DALI (Holm and Sander, 1995) results in the identification of two distinct but structurally and functionally related sets of folds that are similar to the ChaK kinase domain. They are (1) members of the protein kinase structural superfamily and (2) ATP-grasp folds. The protein kinase superfamily is turning out to be much more extensive than had been expected based on sequence comparisons alone. There are several proteins that are similar in structure but not in sequence to classical protein kinases, but are also unrelated to ␣-kinases. One such atypical protein kinase is actinfragmin kinase from the slime mold Physarum polycephalum (Eichinger et al., 1996). The structure of the catalytic domain of P. polycephalum actin-fragmin kinase resembles closely that of classical protein kinases in its core region (Steinbacher et al., 1999). Actin-fragmin kinase is an isolated case, in that it does not have any known homologs in other organisms at present. Another interesting case but also an isolated one is that of aminoglycoside kinase, a bacterial enzyme that confers resistance to aminoglycoside antibiotics (Hon et al., 1997). Again, aminoglycoside kinase turns out to have a structure that is strikingly like that of classical kinases but without detectable sequence similarity. The N-terminal lobe of the ChaK kinase domain is very similar in structure to that of classical protein kinases, including actin-fragmin kinase and aminoglycoside kinase, whereas its C-terminal lobe resembles that of ATP-grasp proteins more closely. It appears as if the ChaK kinase domain were constructed by matching the N-terminal lobe of a classical protein kinase and the C-terminal lobe of an ATP-grasp protein. Figures 5A and 5B illustrate the “chimeric” nature of the ChaK kinase domain by comparing it to PKA and to E. coli succinyl-

CoA synthetase ␤ subunit (Joyce et al., 2000). In particular, the ChaK kinase domain does not have the so-called “catalytic loop”, one of the characteristic structural features of the protein kinase-like fold that distinguishes it from the ATP-grasp fold. Instead, residues that are important for catalysis, Asp-1765 and Gln-1767, are separated by only one residue on a continuous ␤ strand with a kink in the middle (Figure 7). The structural similarity between ATP-grasp fold proteins and classical protein kinases had been noted previously, but their resemblance was considered a consequence of evolutionary convergence from independent origins (Denessiouk et al., 1998). Recently, the structures of two other ATP-utilizing enzymes, phosphatidylinositol phosphate kinase II␤ and phosphoribosyl-aminoimidazole-succinocarboxamide synthase, have been solved (Levdikov et al., 1998; Rao et al., 1998). These structures also have a chimeric construction, as seen in the ChaK kinase domain. Phosphatidylinositol phosphate kinase catalyzes a phosphotransferase reaction that is chemically identical to the reaction catalyzed by protein kinases, and its structural and functional similarities to both ATP-grasp fold proteins and classical protein kinases suggests a possible evolutionary linkage among these proteins (Grishin, 1999). ChaK and other ␣-kinase family members are true protein kinases, and the structural similarities discussed here further suggest a direct evolutionary linkage between ATP-grasp fold proteins and classical protein kinases. Conclusion Given the paucity of information regarding the mechanisms that regulate various TRP channels, the determination of the three-dimensional structure of the functionally important kinase domain of the ChaK TRP channel is an important step forward. It now allows the structurebased design of new experiments aimed at answering mechanistic questions regarding substrate specificity and the regulation of kinase activity. At the same time, the structure provides a view of a member of the atypical ␣-kinase family. The structures of ChaK–nucleotide complexes demonstrate that catalytically important residues are well conserved between the ␣-kinases and classical protein kinases, allowing the considerable knowledge regarding the mechanisms of the latter class of kinases to be utilized in future investigations of ␣-kinases. Despite these similarities, aspects of the ChaK kinase domain structure are more similar to the architecture of metabolic enzymes with ATP-grasp folds. These and other differences between ChaK and classical protein kinases lead to the expectation that new inhibitors that are specific for the ␣-kinases can be developed. Experimental Procedures Protein Expression, Purification, and Crystallization A recombinant 343-residue ChaK kinase domain fragment (residues 1521–1863) was expressed in Sf9 insect cells with a hexa-histidine tag by using the Bac-to-Bac baculovirus expression system (Life Technologies). The expressed protein was partly purified from the lysate of infected cells on Q-Sepharose (Amersham Pharmacia Biotech) and Ni-NTA Superflow (Qiagen) chromatographic columns at 4⬚C. The eluate was concentrated and treated with TEV protease (Parks et al., 1994) at 4⬚C for 48 hr to remove the poly-histidine

Molecular Cell 1056

tag. The ChaK kinase domain fragment was further purified on a Superdex 200 column at 4⬚C. Thin needle-shaped crystals were obtained initially by vapor diffusion in 1–10 ␮l hanging drops that contained equal volumes of a 10 mg/ml protein solution and a solution of 100 mM HEPES•Na (pH 7.0), 5% (v/v) 2-propanol, and 5% (w/v) PEG4000 and were equilibrated at 20⬚C against a well solution containing 100 mM HEPES•Na (pH 7.0), 10% (v/v) 2-propanol, and 5% (w/v) PEG4000. Multiple rounds of macroseeding were performed to increase the width of the crystals from 20 ␮m to 120 ␮m. Single crystals harvested from the previous cycle were stored at 20⬚C in a solution containing 100 mM HEPES•Na (pH 7.0), 5% (v/v) 2-propanol, 2% (w/v) PEG4000, and 5 mM DTT. Typically, after 24 hr of storage, the crystals were further washed in the storage solution and seeded into a fresh cocktail solution of 0.5 mg/ml protein, 50 mM HEPES•Na (pH 7.0), 1.25% (v/v) 2-propanol, and 2% (w/v) PEG4000, and 2.5 mM DTT. The seeded protein solution was then equilibrated in a sitting drop against a well solution containing 100 mM HEPES•Na (pH 7.0), 10% (v/v) 2-propanol, 4% (w/v) PEG4000, and 5 mM DTT. A new construct (residues 1548–1863) was designed based on the structure of the unliganded form of the kinase domain so as to omit 28 disordered N-terminal residues. The truncated protein showed virtually the same biochemical characteristics as the original construct, and it was purified in the same manner. Crystals were obtained initially by vapor diffusion in 1–10 ␮l hanging drops containing 3:1 ratio mixture of a 10 mg/ml protein solution with 1 mM ADP•Mg or AMP•PNP•Mg2 and a solution of 100 mM HEPES•Na (pH 7.5) and 0.8 M ammonium dihydrogen phosphate that were equilibrated at 20⬚C against a well solution containing 100 mM HEPES•Na (pH 7.5) and 0.8 M ammonium dihydrogen phosphate. A few rounds of macroseeding with increased concentrations (2.5 mM–5 mM) of the nucleotides in protein cocktails enlarged the crystals to 300 ␮m in width. These crystals diffracted X-rays to significantly higher resolution than did the crystals obtained previously.

Structure Determination and Refinement All the data were measured from frozen crystals cryoprotected in 28% ethyleneglycol (the unliganded crystals) or in 30% ethyleneglycol (the nucleotide complex crystals). Both the unliganded and nucleotide complex crystals are in space group C2221, with one dimer per asymmetric unit, with unit-cell dimensions of a ⫽ 108.3 A˚, b ⫽ 138.2 A˚, c ⫽ 113.5 A˚ (the native unliganded crystal form); a ⫽ 109.0 A˚, b ⫽ 135.5 A˚, c ⫽ 113.2 A˚ (the ADP•Mg complex crystal form); or a ⫽ 109.3 A˚, b ⫽ 136.6 A˚, c ⫽ 113.3 A˚ (the AMP•PNP•Mg2 complex crystal form). Data sets were collected at home and at synchrotron beamlines (X4, X9, and X25) at the National Synchrotron Light Source, Brookhaven National Laboratory. Data processing, phase calculation, density modification, and model building were carried out in a standard manner (Bru¨nger, 1992; CCP4, 1994; Jones et al., 1991; La Fortelle and Bricogne, 1997; Otwinowski and Minor, 1997). The unliganded structure has been partially refined to 2.8 A˚ resolution. The current model contains 512 amino acid residues in total. Both termini (residues 1521–1550 and 1829–1863) as well as three internal loops (residues 1618–1623, 1657–1662, 1783–1797, and 1829–1863 in molecule A and residues 1619–1621, 1657–1662, and 1783–1794 in molecule B) are not included in the current model because of poorly defined electron density. This model was not analyzed further but was used to determine the structures of the nucleotide complexes by molecular replacement (Navaza, 1994). Subsequent refinement was performed against the 2.0 A˚ AMP•PNP data set and the 2.4 A˚ ADP data set using XPLOR (Bru¨nger, 1992) and REFMAC (Murshudov et al., 1999). The current models for the structures of the AMP•PNP and ADP complexes both contain 556 amino acid residues in total: residues 1551–1614 and 1617–1828 of molecule A and residues 1549–1828 of molecule B. Both models also contain 253 water molecules, two nucleotide molecules, and one DTT molecule. In addition, the ADP complex structure has one magnesium ion. The R values for both structures are somewhat higher than expected (Rfree ⫽ 29.4%, Rwork ⫽ 24.0% for the AMP•PNP complex; Rfree ⫽ 24.9%, Rwork ⫽ 21.5% for the ADP complex). The stereochemical parameters for the models are excellent, with no residues in the disallowed regions of the Ramachandran plots. Careful examination

of difference electron density maps showed that the modeling of discrete solvent features was essentially complete, but that the fit to electron density is poor in loop regions for which there was no interpretable density in the lower-resolution analysis of the apo structure. The model could not be improved in these regions, which are listed above and include 57 residues in loops and 38 residues at both termini. We attribute the high R values at least partially to this high level of disorder in the structure. Acknowledgments We thank M. Huse and S. Soisson for helpful discussions and X. Chen, M. Podobnik, S. Soisson, and staff at the beamlines X25 (H.A. Lewis), X4A (C. Ogata), and X9B (K. Rajashankar) for help with data collection at the National Synchrotron Light Source, a DOE facility. Assistance provided by H. Viguet for insect cell growth is gratefully acknowledged. Received December 21, 2000; revised February 26, 2001. References Barritt, G.J. (1999). Receptor-activated Ca2⫹ inflow in animal cells: a variety of pathways tailored to meet different intracellular Ca2⫹ signalling requirements. Biochem. J. 337, 153–169. Bennett, M.J., Schlunegger, M.P., and Eisenberg, D. (1995). 3D domain swapping: a mechanism for oligomer assembly. Protein Sci. 4, 2455–2468. Bossemeyer, D., Engh, R.A., Kinzel, V., Ponstingl, H., and Huber, R. (1993). Phosphotransferase and substrate binding mechanism of the cAMP-dependent protein kinase catalytic subunit from porcine heart as deduced from the 2.0 A structure of the complex with Mn2⫹ adenylyl imidodiphosphate and inhibitor peptide PKI(5–24). EMBO J. 12, 849–859. Bru¨nger, A.T. (1992). XPLOR v3.1, A System for X-Ray Crystallography and NMR (New Haven, CT: Yale University Press). CCP4 (Collaborative Computational Project 4) (1994). The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763. Cho, S.I., Koketsu, M., Ishihara, H., Matsushita, M., Nairn, A.C., Fukazawa, H., and Uehara, Y. (2000). Novel compounds, “1,3-selenazine derivatives” as specific inhibitors of eukaryotic elongation factor-2 kinase. Biochim. Biophys. Acta 1475, 207–215. Conti, E., Uy, M., Leighton, L., Blobel, G., and Kuriyan, J. (1998). Crystallographic analysis of the recognition of a nuclear localization signal by the nuclear import factor karyopherin alpha. Cell 94, 193–204. Cote, G.P., Luo, X., Murphy, M.B., and Egelhoff, T.T. (1997). Mapping of the novel protein kinase catalytic domain of Dictyostelium myosin II heavy chain kinase A. J. Biol. Chem. 272, 6846–6849. Denessiouk, K.A., Lehtonen, J.V., Korpela, T., and Johnson, M.S. (1998). Two “unrelated” families of ATP-dependent enzymes share extensive structural similarities about their cofactor binding sites. Protein Sci. 7, 1136–1146. Diggle, T.A., Seehra, C.K., Hase, S., and Redpath, N.T. (1999). Analysis of the domain structure of elongation factor-2 kinase by mutagenesis. FEBS Lett. 457, 189–192. Duncan, L.M., Deeds, J., Hunter, J., Shao, J., Holmgren, L.M., Woolf, E.A., Tepper, R.I., and Shyjan, A.W. (1998). Down-regulation of the novel gene melastatin correlates with potential for melanoma metastasis. Cancer Res. 58, 1515–1520. Eichinger, L., Bomblies, L., Vandekerckhove, J., Schleicher, M., and Gettemans, J. (1996). A novel type of protein kinase phosphorylates actin in the actin-fragmin complex. EMBO J. 15, 5547–5556. Grishin, N.V. (1999). Phosphatidylinositol phosphate kinase: a link between protein kinase and glutathione synthase folds. J. Mol. Biol. 291, 239–247. Hardie, R.C., and Minke, B. (1995). Phosphoinositide-mediated phototransduction in Drosophila photoreceptors: the role of Ca2⫹ and trp. Cell Calcium 18, 256–274.

Structure of the Kinase Domain of a TRP Channel 1057

Harteneck, C., Plant, T.D., and Schultz, G. (2000). From worm to man: three subfamilies of TRP channels. Trends Neurosci. 23, 159–166.

kinase: a protein kinase fold flattened for interfacial phosphorylation. Cell 94, 829–839.

Holm, L., and Sander, C. (1995). Dali: a network tool for protein structure comparison. Trends Biochem. Sci. 20, 478–480.

Redpath, N.T., Price, N.T., and Proud, C.G. (1996). Cloning and expression of cDNA encoding protein synthesis elongation factor2 kinase. J. Biol. Chem. 271, 17547–17554.

Hon, W.C., McKay, G.A., Thompson, P.R., Sweet, R.M., Yang, D.S., Wright, G.D., and Berghuis, A.M. (1997). Structure of an enzyme required for aminoglycoside antibiotic resistance reveals homology to eukaryotic protein kinases. Cell 89, 887–895. Hubbard, S.R. (1997). Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog. EMBO J. 16, 5572–5581. Johnson, L.N., Lowe, E.D., Noble, M.E., and Owen, D.J. (1998). The eleventh Datta lecture. The structural basis for substrate recognition and control by protein kinases. FEBS Lett. 430, 1–11. Jones, T.A., Zou, J.Y., Cowan, S.W., and Kjeldgaard (1991). Improved methods for binding protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119. Joyce, M.A., Fraser, M.E., James, M.N., Bridger, W.A., and Wolodko, W.T. (2000). ADP-binding site of Escherichia coli succinyl-CoA synthetase revealed by x-ray crystallography. Biochemistry 39, 17–25. Knighton, D.R., Zheng, J.H., Ten Eyck, L.F., Ashford, V.A., Xuong, N.H., Taylor, S.S., and Sowadski, J.M. (1991a). Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science 253, 407–414. Knighton, D.R., Zheng, J.H., Ten Eyck, L.F., Xuong, N.H., Taylor, S.S., and Sowadski, J.M. (1991b). Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphatedependent protein kinase. Science 253, 414–420. La Fortelle, E.D., and Bricogne, G. (1997). Maximum-likelihood heavy-atom parameter refinement in the MIR and MAD methods. Methods Enzymol. 276, 472–494. Levdikov, V.M., Barynin, V.V., Grebenko, A.I., Melik-Adamyan, W.R., Lamzin, V.S., and Wilson, K.S. (1998). The structure of SAICAR synthase: an enzyme in the de novo pathway of purine nucleotide biosynthesis. Structure 6, 363–376. Levitan, I.B. (1999). Modulation of ion channels by protein phosphorylation. How the brain works. Adv. Second Messenger Phosphoprotein Res. 33, 3–22. Madden, D.R. (1995). The three-dimensional structure of peptideMHC complexes. Annu. Rev. Immunol. 13, 587–622. Mao, Y., Nickitenko, A., Duan, X., Lloyd, T.E., Wu, M.N., Bellen, H., and Quiocho, F.A. (2000). Crystal structure of the VHS and FYVE tandem domains of Hrs, a protein involved in membrane trafficking and signal transduction. Cell 100, 447–456. Misra, S., and Hurley, J.H. (1999). Crystal structure of a phosphatidylinositol 3-phosphate-specific membrane-targeting motif, the FYVE domain of Vps27p. Cell 97, 657–666. Montell, C., and Rubin, G.M. (1989). Molecular characterization of the Drosophila trp locus: a putative integral membrane protein required for phototransduction. Neuron 2, 1313–1323. Murshudov, G.N., Lebedev, A., Vagin, A.A., Wilson, K.S., and Dodson, E.J. (1999). Efficient anisotropic refinement of macromolecular structures using FFT. Acta Crystallogr. D Biol Crystallogr. 55, 247–255. Navaza, J. (1994). AMoRe: an automated package for molecular replacement. Acta Crystallogr. A50, 157–163. Otwinowski, Z., and Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326. Parks, T.D., Leuther, K.K., Howard, E.D., Johnston, S.A., and Dougherty, W.G. (1994). Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase. Anal. Biochem. 216, 413–417. Ranganathan, R., Malicki, D.M., and Zuker, C.S. (1995). Signal transduction in Drosophila photoreceptors. Annu. Rev. Neurosci. 18, 283–317. Rao, V.D., Misra, S., Boronenkov, I.V., Anderson, R.A., and Hurley, J.H. (1998). Structure of type II␤ phosphatidylinositol phosphate

Robinson, M.J., Harkins, P.C., Zhang, J., Baer, R., Haycock, J.W., Cobb, M.H., and Goldsmith, E.J. (1996). Mutation of position 52 in ERK2 creates a nonproductive binding mode for adenosine 5⬘-triphosphate. Biochemistry 35, 5641–5646. Runnels, L.W., Yue, L., and Clapham, D.E. (2001). TRP-PLIK, a bifunctional protein with kinase and ion channel activities. Science 291, 1043–1047. Ryazanov, A.G., Ward, M.D., Mendola, C.E., Pavur, K.S., Dorovkov, M.V., Wiedmann, M., Erdjument-Bromage, H., Tempst, P., Parmer, T.G., Prostko, C.R., et al. (1997). Identification of a new class of protein kinases represented by eukaryotic elongation factor-2 kinase. Proc. Natl. Acad. Sci. USA 94, 4884–4889. Ryazanov, A.G., Pavur, K.S., and Dorovkov, M.V. (1999). Alphakinases: a new class of protein kinases with a novel catalytic domain. Curr. Biol. 9, R43–R45. Schwabe, J.W., and Klug, A. (1994). Zinc mining for protein domains. Nat. Struct. Biol. 1, 345–349. Sicheri, F., Moarefi, I., and Kuriyan, J. (1997). Crystal structure of the Src family tyrosine kinase Hck. Nature 385, 602–609. Steinbacher, S., Hof, P., Eichinger, L., Schleicher, M., Gettemans, J., Vandekerckhove, J., Huber, R., and Benz, J. (1999). The crystal structure of the Physarum polycephalum actin-fragmin kinase: an atypical protein kinase with a specialized substrate-binding domain. EMBO J. 18, 2923–2929. Accession Numbers Coordinates have been deposited in the Protein DataBank under ID codes 1IAJ (apo), 1IAH (ADP complex), and 1IA9 (AMP•PNP complex).