Mechanism of Substrate Specificity of Phosphatidylinositol Phosphate Kinases

Mechanism of substrate specificity of phosphatidylinositol phosphate kinases

Yagmur Muftuoglua, Yi Xuea, Xiang Gaoa,b, Dianqing Wua, and Ya Haa,1

aDepartment of Pharmacology, Yale School of Medicine, New Haven, CT 06520; and bDepartment of Neurosurgery and Institute of Neurosurgery, State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, People’s Republic of China

Edited by Jeremy W. Thorner, University of California, Berkeley, CA, and approved June 16, 2016 (received for review November 16, 2015) The phosphatidylinositol phosphate kinase (PIPK) family of enzymes kinase appeared later in evolution than the type I kinase, sug- is primarily responsible for converting singly phosphorylated phos- gesting that the type II kinase has a more specialized role in higher phatidylinositol derivatives to phosphatidylinositol bisphosphates. eukaryotes. The type III kinase (PIKfyve) is a much larger enzyme As such, these kinases are central to many signaling and mem- that has a complex domain structure, including an N-terminal brane trafficking processes in the eukaryotic cell. The three types FYVE zinc finger domain, which binds PI(3)P (14, 15). The type III of phosphatidylinositol phosphate kinases are homologous in se- kinase prefers PI(3)P as its substrate and is mainly involved with the quence but differ in catalytic activities and biological functions. Type I synthesis of PI(3,5)P2 in late endosomes (16). In vitro, the type III and type II kinases generate phosphatidylinositol 4,5-bisphosphate kinase demonstrates a weak activity toward PI, generating PI(5)P. from phosphatidylinositol 4-phosphate and phosphatidylinositol PIPKs do not share sequence homology with protein kinases. 5-phosphate, respectively, whereas the type III kinase produces Although crystal structures of the apo form of type I and type II phosphatidylinositol 3,5-bisphosphate from phosphatidylinositol kinases have been solved (17, 18), there are, as of yet, no structural 3-phosphate. Based on crystallographic analysis of the zebrafish type I data for the kinases’ complexes with substrate, hindering a deeper kinase PIP5Kα, we identified a structural motif unique to the kinase understanding of the catalytic mechanism. One particularly in- family that serves to recognize the monophosphate on the substrate. triguing question relates to how these kinases achieve signaling Our data indicate that the complex pattern of substrate recognition specificity; the kinases must distinguish highly similar PIP substrates and phosphorylation results from the interplay between the mono- and phosphorylate the substrates at distinct sites (19) (Fig. S1B). phosphate binding site and the specificity loop: the specificity loop Here, we report the X-ray structure of a type I kinase in complex functions to recognize different orientations of the inositol ring, with adenylyl-imidodiphosphate (AMP-PNP) and discuss the struc- whereas residues flanking the phosphate binding Arg244 determine ture’s implications for the kinase mechanism. We also describe whether phosphatidylinositol 3-phosphate is exclusively bound and the binding site for the singly phosphorylated PI, which coop- phosphorylated at the 5-position. This work provides a thorough erates with the specificity loop to influence substrate binding picture of how PIPKs achieve their exquisite substrate specificity. and phosphorylation.

lipid kinases | protein engineering | crystallography | substrate specificity Results and Discussion The Binding of ATP. Dimerization of PIP5Kα involves helix α4, hosphatidylinositol phosphate (PIP) kinases (PIPKs) are key whichisequivalenttothe“Chelix” of protein kinases that often Pplayers in the metabolism of phosphoinositides in eukaryotic cells. PIPKs are primarily responsible for the synthesis of doubly Significance phosphorylated phosphatidylinositol (PI) derivatives from singly – phosphorylated PIs (1 4). PIPK catalytic activities are important to Phosphatidylinositol phosphate kinases (PIPKs) generate two

the cell in part because they produce essential PI bisphosphates highly important phosphatidylinositol bisphosphates, PI(4,5)P2 BIOCHEMISTRY such as PI(4,5)P2, which is involved in a wide variety of signaling and PI(3,5)P2, which are central to many signaling and membrane pathways and membrane trafficking events, and PI(3,5)P2,which trafficking processes. The three types of PIPKs are homologous has a more specialized role in endosomes (5). In addition, these in sequence but demonstrate different substrate and catalytic kinases control the level of certain PI monophosphates such as specificities. In this study, we provide crystallographic and bio- PI(5)P, which appears to function as a stress signal (6). Mutations chemical evidence showing that the complex pattern of substrate in PIPKs have been linked to human diseases (7, 8), and, in can- recognition and phosphorylation results from interplay between cerous cells, the activities of PIPKs are often up-regulated, usually two structural elements: the specificity loop and the binding site as a consequence of overexpression (9, 10). for the monophosphate moiety of the substrate. This work pro- There are three subfamilies of PIPKs that share sequence vides the first complete understanding of how this family of lipid identity within the kinase domain (2). The type I kinase phospha- kinases achieves exquisite substrate specificity. The mechanistic tidylinositol 4-phosphate 5-kinase (PIP5K), localized mainly to the insights presented are timely because an increasing number of plasma membrane, is responsible for synthesizing the majority of studies implicate lipid kinases in major human diseases, including PI(4,5)P2 in the cell and accomplishes this process by phosphory- cancer and diabetes. lating the 5-hydroxyl group of PI(4)P (Fig. S1A). In vitro, the type I kinase also has a robust activity against PI(3)P, generating not only Author contributions: Y.M. and Y.H. designed research; D.W. helped conceptualize some of the foundational ideas; Y.M., Y.X., and X.G. performed research; Y.M., Y.X., and D.W. PI(3,4)P2 and PI(3,5)P2 but also the triply phosphorylated PI(3,4,5)P3 contributed new reagents/analytic tools; Y.M., Y.X., X.G., and Y.H. analyzed data; and (11, 12). Furthermore, the type I kinase can weakly phosphorylate Y.M. and Y.H. wrote the paper. PI to produce PI(5)P. In contrast, the type II kinase phosphati- The authors declare no conflict of interest. dylinositol 5-phosphate 4-kinase (PIP4K) is diffusively distributed This article is a PNAS Direct Submission. in the cytoplasm and nucleus. This enzyme prefers PI(5)P as sub- Data deposition: The atomic coordinates and structure factors have been deposited in the strate, phosphorylating it at the 4-position (13). In vitro, the type II Protein Data Bank, www.pdb.org (PDB ID codes 5E3S, 5E3T, and 5E3U). kinase also phosphorylates PI(3)P, generating PI(3,4)P2, albeit with 1To whom correspondence should be addressed. Email: [email protected]. much lower efficiency. Although both type I and type II kinases This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. produce PI(4,5)P2, their functions are nonredundant. The type II 1073/pnas.1522112113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1522112113 PNAS | August 2, 2016 | vol. 113 | no. 31 | 8711–8716 Downloaded by guest on September 30, 2021 plays a role in allosteric regulation (20, 21). We measured the identical conformation as that of protein kinase A (PKA) (22) initial rates of PI(4,5)P2 production from PI(4)P at different (Fig. 1 B and C), although the segment following the linker adopts ATP concentrations and found that the reaction follows simple a different fold in PIPKs to accommodate the unique substructure. Michaelis–Menten kinetics, with no cooperativity in ATP binding Lys171 (the “IIK” motif), Asp299 (“MDYSL”), and Asp378 (Fig. S2A). We solved the X-ray crystal structure of the zebrafish (“IID”) of PIP5Kα are functional equivalents of Lys72 (the PIP5Kα in complex with the ATP analog AMP-PNP by soaking “VAIK” motif of protein kinases), Asp166 (“HRD”), and Asp184 the analog into preformed crystals. Difference Fourier analysis (“DFG”) of PKA (underlined residues identify the amino acid of confirmed the presence of the analog bound at the predicted ATP interest). Lys171 is located within a β-strand (β5) that is structur- + binding site (Fig. S2B). Two manganese ions (Mn2 ), bound by ally equivalent to β3 of PKA, and its side chain points into the AMP-PNP, were clearly visible as 8σ peaks in the anomalous active site to bind the α-phosphate of ATP. Asp299, from a seg- Fourier map because of the anomalous diffraction of manganese ment equivalent to the catalytic loop, is positioned next to the at the wavelength of data collection (1.075 Å). The position of the γ-phosphate of ATP and could function as the general base to metal ions facilitated the building of AMP-PNP into the 3.3-Å receive the proton from the attacking 5-hydroxyl of PI(4)P. Asp378 + resolution electron density map. Improved crystallization condi- contributes to the coordination of the two Mn2 ions. tion also enabled us to extend the resolution of the apo structure The X-ray structure also reveals features within the ATP to 3.1 Å, which was used as the starting point for model building binding pocket that are unique to PIPKs. The side chain of Lys238 and refinement (Table S1 and Fig. S2 C and D). from the DLKGS motif points up toward the γ-phosphate and In PIPKs, the ATP binding site is flanked not only by the N- and interacts with it (Fig. 1B). The position of Lys238 superposes well C-lobes of the kinase but also by a substructure that is unique to with that of Lys168 in PKA. We propose that Lys238 provides a the family (blue in Fig. 1A). In the primary sequence, this unique positive charge to stabilize the reaction transition state, a role segment is inserted between the two lobes. The segment is com- traditionally assigned to Lys168 of PKA (23). Asp236 from the posed of two parallel β-strands (β8, β9) and two short helices DLKGS motif also plays a role in substrate binding by forming a (α4c, α5). Strands β8andβ9 form a continuous sheet with β10, hydrogen bond with the 2-hydroxyl of the ribose. Furthermore, the counterpart of the “catalytic loop” of protein kinases. The Asp236, together with Ser301 from the MDYSL motif, appears to “DLKGS” sequence motif, conserved in all PIPKs, is located at the contribute to metal coordination through a water molecule, thus tip of the substructure between β8andα4c. The substructure is one substituting the function of Asn171 in PKA. of the most conserved regions of the enzyme, because it harbors 5 In PIP5Kα, AMP-PNP binding causes the ends of β3andβ4to of the 22 invariant residues in the family: Asp236, Lys238, Gly239, move down slightly toward the ligand. The turn between the two Arg244, and Asp260. Gly239 and Asp260 mainly play a structural β-strands corresponds to the glycine-rich loop of PKA, which in- role: the flexible glycine is located at the turn between β8andα4c, teracts with the β-andγ-phosphate groups of ATP via its backbone whereas Asp260 maintains the shape of the turn by forming amides (20). In the structure of PIP5Kα in complex with AMP- hydrogen bonds with two backbone amide groups (Fig. S2C). PNP, however, the β3-β4 turn remains poorly defined in the Asp236, Lys238, and Arg244 contribute directly to substrate electron density map and may not contribute to ATP binding. binding and catalysis, described below. Compared with PKA, the two β-strands in PIP5Kα are also posi- The N1 and N6 atoms of the adenine ring of AMP-PNP are tioned further away from ATP, which generates a more open hydrogen-bonded to the linker (Asn222 and Leu224), which has an substrate binding site. This feature is partly attributable to Phe160

Fig. 1. Overall structure and ATP binding site. (A) Domain organization. The schematic compares PIPKs with protein kinases. The overall structure of the zebrafish PIP5Kα in complex with AMP-PNP is shown below the schematic with the N-lobe (gray), the C-lobe (light tan), and the PIPB domain (blue). Disordered 2− regions of the protein are indicated by dotted curves. Bound AMP-PNP and SeO4 are shown as stick models. (B) Structural details of the ATP-binding site of PIP5Kα. Hydrogen bonds are indicated by dotted lines. Red spheres represent water, and black spheres represent metal ions. (C) ATP-binding site of protein kinase A (PDB ID code 1ATP). Structural illustrations were all generated on PyMOL.

8712 | www.pnas.org/cgi/doi/10.1073/pnas.1522112113 Muftuoglu et al. Downloaded by guest on September 30, 2021 from β4, which is conserved in all PIPKs. The side chain of Phe160 bound selenate allows the 5-hydroxyl to move into an ideal position packs tightly against the adenine and ribose. In protein kinases, the to conduct nucleophilic attack on the γ-phosphate of ATP while corresponding residue often has a smaller side chain (e.g., Val57 in maintaining the lipid tails in the membrane bilayer. PKA), allowing the β-strand to be positioned closer to ATP. To experimentally test whether these residues are important for kinase function, we separately mutated them to alanine together The Phosphate-Binding Site. Singly phosphorylated PI derivatives are with seven other positively charged residues in the vicinity of the the preferred substrates for the PIPK family of enzymes. This fact active site. Of the four mutants that could be purified, K238A and suggests that PIPKs possess a structural feature to recognize the R244A completely abolished activity (Fig. 2B).Theeffectofthe monophosphate on the inositol ring and that the binding of the K238A mutation can be twofold, because the lysine also contributes phosphate group must be of sufficient affinity so that the enzyme can to ATP binding, in addition to the binding of lipid substrate. The distinguish PIP from PI, which is much more abundant in the cell (1). counterpart of Lys238 in protein kinases, Lys168 of PKA, also plays Crystals soaked with soluble forms of PI(4)P with shorter lipid tails a part in the binding of the non-ATP substrate (23). Arg244, on the did not reveal a convincing density in the active site. To identify the other hand, appears to play a dedicated role in binding the 2− phosphate binding pocket, we soaked selenate (SeO4 ), an isostere 4-phosphate of the lipid substrate. The R244A mutation does not 2− α of the phosphate ion (HPO4 ), into PIP5K crystals and collected hinder the intrinsic ATPase activity of the kinase (Fig. S3). We diffraction data at the selenium edge. The anomalous Fourier map named the unique substructure containing the “DLKGSxxxR” se- revealed four positive peaks. Two correspond to manganese, which quence as the PIP-binding motif (PIPBM) because of its conser- also has anomalous signal at the selenium wavelength. The other two vation within the PIPK family and its role in recognizing the correspond to selenate ions (Fig. 1A). One of the selenates (labeled monophosphate on PIP. as “Se1”) is bound to Lys238 and Arg244 at the active site, adjacent to the γ-phosphate of AMP-PNP (Fig. 2A). Mutation and Human Disease. A single mutation (D253N) within the Lys238 and Arg244, which coordinate Se1 at the active site, are kinase domain of human PIP5Kγ causes a severe form of absolutely conserved among PIPKs. They both come from the arthrogryposis called lethal congenital contracture syndrome substructure unique to the PIPK family: Lys238 is part of the type 3 (LCCS3) (8). LCCS3 is characterized by atrophy in the DLKGS sequence motif, whereas Arg244 is invariably three resi- patient’s spinal cord and multiple joint contractures. The γ isoform dues downstream. The possibility that Lys238 and Arg244 consti- of the type I kinase is highly expressed in the brain and is essential tute the binding site for the monophosphate on the inositol head for survival (24, 25). The disease phenotype appears to be related to group is supported by modeling PI(4)P into the active site of perturbed phosphatidylinositol 3-kinase (PI3K) signaling, which PIP5Kα (Fig. 2C): superposing the 4-phosphate group onto the could result from reduced PI(4,5)P2 production, because LCCS2, a BIOCHEMISTRY

2− Fig. 2. The monophosphate binding site. (A) Binding site for SeO4 . The difference Fourier map, shown in purple, is contoured at 5σ. Hydrogen bonds are indicated (dotted black lines). (B) Comparison of wild-type and mutant activities. R217A contains a mutation at the Se2 site, located in the N-lobe of the protein. In

the autoradiograph shown (B, Top), spots corresponding to the origin and the radioactive product, PI(4,5)P2, are indicated. (B, Middle)The amount of kinase used for the reaction. (B, Bottom) Quantification of the relative intensities of the PI(4,5)P2 spots, averaged over a triplicate of experiments. (C) Model of the dimeric PIP5Kα bound to PI(4)P embedded in membrane bilayer (gray box). Monomers are colored blue and green. The black oval depicts the twofold axis. (Right)The active site colored according to electrostatic potential. The active site appears open due to the disordered specificity loop. PI(4)P is depicted in stick model, and C1- C6 atoms of the inositol head group are labeled. PI(4)P is modeled into the active site by superimposing the 4-phosphate onto the Se1 bound at the active site. The 5-hydroxyl is near the γ-phosphate of ATP (green) for phosphorylation. The model projects the lipid tails into the membrane bilayer. (D) Autoradiograph comparing the activities of the D236N disease mutant and wild-type PIP5Kα. Approximately 10-fold more of the mutant was used than the wild type in this assay. (E) Model depicting the role of the specificity loop in recognizing different orientations (red arrows) of the lipid substrates.

Muftuoglu et al. PNAS | August 2, 2016 | vol. 113 | no. 31 | 8713 Downloaded by guest on September 30, 2021 condition similar to LCCS3, is caused by the mutation of a protein predicted that the 4- and 5-phosphates on the inositol head groups involved in PI3K activation (26). Asp253 of human PIP5Kγ corre- are recognized by residues on the activation loop (which corresponds to Asp236 in the zebrafish PIP5Kα, the first residue within sponds to the specificity loop of PIPKs) (29). (iii) The specificity the DLKGS sequence motif. We mutated Asp236 to asparagine loop: the ATP and phosphate binding sites dictate how the lipid and found that the mutant, although still capable of converting should be oriented for chemical reaction with ATP, whereas the PI(4)P to PI(4,5)P2 in vitro, has an activity three orders of magni- specificity loop functions to recognize the orientation of the lipid tude lower than that of the wild type (Fig. 2D). The X-ray structure instead of directly scrutinizing the lipid’s phosphorylation status. A suggests that the mutation disrupts a salt bridge between Asp236 summary of how various PIP substrates bind to the type I, type II, and Arg244, thus possibly affecting the conformation of the latter and type III kinases is provided in Fig. S5. (Fig. 2A). Arg244 is important for the recognition of PIP substrate. We tested this model of substrate specificity by trying to engineer The D236N mutation may also affect hydrogen bonding between the specificity of the type III kinase into the type I kinase PIP5Kα. Ser301 and the 2-hydroxyl of the ribose ring on ATP (Fig. 1B). Unlike type I or type II kinases, the type III kinase (PIKfyve) Because asparagine retains the hydrogen bonding potential of as- prefersPI(3)Pasitssubstrateandphosphorylates the 5-hydroxyl, partate, the loss of a negative charge, which should mostly impact skipping the 4-hydroxyl. Our proposed model predicts that residues the salt bridge with the positively charged arginine, is probably the responsible for a type III-like specificity must lie adjacent to the main reason for the 1,000-fold loss of catalytic activity we observed. phosphate binding site because the specificity loop of type III ki- Mutations in other PIPKs also play roles in human disease. For nase has the same “specificity switch” (Glu382 in PIP5Kα)asthe example, in the type II kinase PIP4Kα, there is a polymorphism type I kinase, suggesting that the orientation of PI(3)P bound to the (N251S) within the kinase domain that is associated with increased type I kinase is similar to that of PI(4)P (Fig. 3A). After studying risk for schizophrenia (27). The mechanism is unclear because the aligned PIPBM sequences, we identified two sequence motifs Asn251 is located near the amino terminus of α6, away from the that are conserved in type III kinases and adjacent to Arg244 in the ATP and lipid binding sites. In vitro, the N251S mutation does not 3D structure (Fig. 3A). In type III kinases, the “RxR” sequence affect kinase activity. The type III kinase PIKfyve harbors several motif introduces an arginine (which would be Arg242, according to mutations that cause fleck corneal dystrophy (7). Most of these PIP5Kα numbering) upstream of the universally conserved Arg244. induce frameshifts and truncations near the middle of this large This Arg242 replaces a mostly hydrophobic residue in type I and protein, completely eliminating the kinase domain that is located at type II kinases. The second sequence motif we identified in type III the carboxyl terminus. PIPKs, the “LD” motif, replaces Lys259 of PIP5Kα, a conserved residue in type I and type II kinases, with a leucine. The net con- Kinase Specificity. We propose that the phosphate binding site sequence of these substitutions is to shift the positively charged depicted in Fig. 2A is a general feature for the family. In order for binding pocket to the right, subtly altering the position of the the 4-phosphate of PI(4)P, the preferred substrate for the type I phosphate binding site relative to ATP. kinase, and the 5-phosphate of PI(5)P, preferred by the type II ki- We prepared a PIP5Kα mutant (RxR/LD) that incorporates nase, to interact with the same binding site while maintaining the these two type III-specific motifs and flanking residues: T241L/ reactive 5-hydroxyl of PI(4)P and 4-hydroxyl of PI(5)P close to ATP, Y242R/K243N/R245N/K259L/L261E. This mutant retains much of the inositol ring must flip 180° around a horizontal line passing the catalytic activity toward PI(3)P of the wild-type enzyme but through the center of the PIP (Fig. 2E). Type I and type II kinases becomes much less efficient in phosphorylating PI(4)P (Fig. 3B). In differ in amino sequence at a key position within the specificity loop agreement with previous studies of the human enzyme (11, 12, 30), (28). The function of residue appears to be to distinguish the two the zebrafish PIP5Kα generates PI(3,4)P2 and PI(3,5)P2 from lipid orientations by interacting with chemical groups on the sub- PI(3)P, which comigrate on thin-layer chromatography (TLC) plate strate that face the membrane, including the 1-phosphate, axial as an elongated spot just below PI(4,5)P2. PI(3,4)P2 is further 2-hydroxyl, and diglyceride (located to the right of the dotted lines phosphorylated by the type I kinase to triply phosphorylated E in Fig. 2 ). The chemical structure left of the dotted line for PI(4)P PI(3,4,5)P3, which migrates close to lyso-PI(4,5)P2. The phosphati- and for PI(5)P is completely identical and is recognized by the dylinositol bisphosphate (PIP2) product generated by the type III- structurally similar kinase core for the phosphoryl transfer reaction. like RxR/LD mutant does not have the elongated shape, and the The conformations of Lys238 and Arg244 in the type I kinase PI(3,4,5)P3 spot is also missing. Because the mutant is fully capable α β PIP5K differ from their counterparts in a type II kinase, PIP4K of phosphorylating PI(3,4)P2 (Fig. 3B), the missing PI(3,4,5)P3 in- A B [Protein Data Bank (PDB) ID code 1BO1; Fig. S4 and ]. This dicates that PI(3,4)P2 is not produced in the first phosphorylation difference appears to be attributable to crystallization instead of reaction. This finding is confirmed by running the TLC for a longer difference between subfamilies. In the reported structure of time, which shows that the PIP2 product generated by the mutant α C PIP4K (PDB ID code 2YBX; Fig. S4 ), the conformations of the migrates between PI(4,5)P2 and PI(3,4)P2. PI(3,4)2 is generated from lysineandargininearesimilartothoseinPIP5Kα. In this structure, PI(3)P by the type II kinase PIP4Kα (Fig. 3D). Deacylation of the there is also a phosphate ion modeled between Lys238 and Arg244. phosphorylated lipids and HPLC analysis of the soluble head groups 2− Compared with the bound SeO4 , the phosphate is shifted slightly confirmed the conclusion of the TLC experiment (Fig. 3E). Taken to the left and forms an additional interaction with the counterpart together, these results indicate that, after introducing the type of Lys259. Lys259, unlike Lys238 and Arg244, is conserved only in III-specific RxR and LD motifs, the kinase becomes type III-like, type I and type II kinases. Additional experiments, explained below, switching substrate specificity from PI(4)P to PI(3)P and phosphor- indicate that this residue contributes to the preference of type I and ylating the inositol ring exclusively at the 5-position. In cultured type II PIPKs to phosphorylate the hydroxyl adjacent to the HEK 293T cells expressing mutant zebrafish PIP5Kα,wealsoob- monophosphate already present on the substrate. served a consistent increase in the ratio of PI(3,5)P2 over PI(3,4)P2 The mechanism proposed above predicts three structural fea- compared with cells expressing the wild-type kinase (Fig. S6). tures that determine lipid substrate specificity: (i) The ATP binding The specificity of PIP5Kα toward PI(3)P can be modified also by site: although ATP is probably bound identically throughout the the E382A mutation within the specificity loop (Fig. 3A). In family, the lipid has to be arranged in such a way that the reactive agreement with the literature (28), PIP5Kα E382A prefers PI(5)P hydroxyl group is near the γ-phosphate of ATP. (ii) The PIPBM: over PI(4)P as its substrate (Fig. 3C). It should be noted that this motif underlies the preference of the family for singly phos- PIP5Kα E382A corresponds to the PIP5Kβ E362A mutant of the phorylated PIs. The inositol ring has to flip 180° to project the referenced study. The model proposed here predicts that the E382A phosphate, attached to hydroxyls at different positions on the ino- mutation causes the specificity loop to prefer a different lipid orien- sitol head group, into the common binding pocket. In PI3K, it was tation, which would render the kinase incapable of phosphorylating

8714 | www.pnas.org/cgi/doi/10.1073/pnas.1522112113 Muftuoglu et al. Downloaded by guest on September 30, 2021 model proposed here, however, it is tempting to speculate that the evolution took two paths where a promiscuous ancestral enzyme, which probably resembles the modern day type I kinase, developed more stringent and different catalytic activities by separately acquiring mutations within the specificity loop, giving way to the type II kinase and, within the PIPBM, giving way to the type III kinase. The activities gained were then selected for specialized signaling functions in the cell.

The Specificity Loop. The possibility that the specificity loop interacts with the inositol from the side opposite of the monophosphate is supported by a comparison of PIPKs with inositol polyphosphate kinases (IPKs) (32, 33) (Fig. S7 A and B). IPKs are the closest structural homologs to PIPKs. Both families have a hybrid structural arrangement, with an N-lobe resembling protein kinases and a C-lobe resembling ATP-grasp enzymes (34). Although this feature alone is not unique, in that α-kinase ChaK and SAICAR synthase are also hybrids (35, 36), PIPKs and IPKs share several unusual features that distinguish them from other members of the protein kinase and ATP-grasp superfamilies. Within the N-lobe, PIP5Kα lacks the equivalent of protein kinase’s “P-loop,” which binds ATP through backbone amide groups. IPKs lack not only the P-loop, but also the preceding β-strand, and sometimes the strand that follows the loop, as well (37). Within the C-lobe, the DLKGS and MDYSL sequence motifs of PIPKs are structurally similar to the “DxKxG” and “S(L/I)L” motifs found in IPKs and play identical roles in ATP binding and catalysis. PIPKs and IPKs more closely resemble protein kinases in the “crossing loops” than ATP-grasp enzymes. In PIPKs and IPKs, the linker between the N- and C-lobes is longer Fig. 3. Substrate specificity. (A) Sequence alignment of the monophosphate and forms a protruding loop. The loop has no clear function and binding sites and the specificity loops (boxed) of type I, II, and III PIPKs, with the maybeavestigialfeaturefromacommonancestor. RxR and LD motifs as well as the specificity-switch residues highlighted. The All IPKs have a helical segment called the “IP helices” that folds names of organisms are abbreviated as follows: Co, Capsaspora owczarzaki over the inositol substrate from the side of the kinase that corre- (XP_004364933.1 and KJE93967.1); Dr, Danio rerio (AAH95318.1); Hs, Homo sponds to the membrane binding surface of PIPKs (Fig. S7A). sapiens (AAH30587.1 and AAH18034.1 and Q9Y2I7.3); Mm, Mus musculus The helical segment is downstream of the β-strand that harbors (NP_032870.2 and AAH47282.1 and NP_001297553.1); Sm, Stegodyphus mim- the DxKxG sequence motif. The corresponding segment in PIPKs, osarum (KFM78861.1); and Sp, Schizosaccharomyces pombe (CAB10125.1). the β8-α4c loop, is too short to play a similar role. The specificity Residues are numbered according to the zebrafish PIP5Kα, and the locations of β α loop of PIPKs, disordered in the crystal structures, is near the 8- the RxR and LD motifs are shown in the context of the PIP5K structure. α B (B) Assay of wild-type PIP5K and the RxR/LD mutant. PI(3)P, PI(4)P, or PI(5)P was 4c loop (Fig. S7 ). Like the IP helices, the specificity loop harbors used as substrate (Left), and the lanes on the TLC plate are arranged in this multiple positively charged residues, including a pair of highly order. To obtain greater resolution, the TLC plates were run for 3 h. The locations conserved lysines. The N-terminal half of the loop is likely α-helical

of PI(4,5)P2,PI(3,5)P2, PI(3,4)P2, PI(3,4,5)P3,lyso-PI(4,5)P2, and lyso-PI(3,4)P2 or (19). To demonstrate that the specificity loop can fold back toward lyso-PI(3,5)P2 are indicated. The assay using PI(3)P or PI(3,4)P2 as substrate the β8-α4c loop to provide a similar side wall for the active site, we (Right) shows that the RxR/LD mutant is fully capable of phosphorylating introduced two cysteines into a cysteine-less PIP4Kα:onewithin BIOCHEMISTRY PI(3,4)P2, generating PI(3,4,5)P3.(C) Assay of wild-type PIP5K and the E382A mu- the β8-α4c loop and one within the specificity loop near the end of α tant with PI(3)P, PI(4)P, or PI(5)P as substrate. PIP5K E382A harbors the specificity the predicted α-helix (Fig. S7B). The cysteines are readily cross- switch mutation. (D) Detailed view of the migration of PI(3,4)P2,PI(3,5)P2,and α PI(4,5)P by TLC, as generated by wild-type PIP5Kα, the PIP5Kα RxR/LD mutant, or linkable by a disulfide bond, suggesting that their C atoms are less 2 than 7.5 Å apart. Importantly, cross-linking did not hinder the wild-type PIP4Kα. The yellow lines indicate the centroids of the spots for PI(3,4)P2 and PI(3,5)P2. The intensity of each spot as a function of the migration along the ability of the loop to recognize the correct lipid substrate (Fig. TLC plate (y axis) is shown on the right. The curve corresponding to the sample S7C): the cross-linked kinase prefers PI(5)P over PI(4)P as its of PI(3)P phosphorylated by wild-type PIP5Kα was deconvoluted by calculating substrate, whereas the cross-linked A371E mutant, the opposite of two Gaussian curves using the peak locations for PI(3,4)P2 (blue) and PI(3,5)P2 the E382A mutation for PIP5Kα, lost its ability to phosphorylate (red). (E) HPLC elution profiles of phosphorylation products generated by wild- PI(5)P but gained activity toward PI(4)P (28). The location of the type PIP5Kα, RxR/LD, E382A, and wild-type PIP4Kα. The measured radioactivity specificity loop relative to other elements within the active site of (gray dots) and the calculated Gaussian curves are shown. The internal standard the kinase makes it an ideal candidate to distinguish the orienta- of PI(3,4)P2 runs at 143.5 min (blue) and that of PI(3,5)P2 runs at 141.5 min (red). The run times of the peaks in each sample are indicated. tion of the PIP substrate. It is interesting that some members of the IPK family, with simpler structures flanking the IP helices, can also phosphorylate lipid substrates (38).

the 5-hydroxyl group because it no longer points toward ATP (Fig. Experimental Procedures S5). Therefore, in contrast with the RxR/LD mutation, E382A ex- Protein Purification. MutantsofPIP5Kα were generated from a construct – α clusively produces PI(3,4)P2 from PI(3)P (Fig. 3C). Furthermore, containing residues 49 431 of zebrafish PIP5K (17) using the QuikChange E382A is unable to generate the triply phosphorylated PI(3,4,5)P . site-directed mutagenesis kit (Agilent Technologies). For crystallization, 3 α These data illustrate again that the complex pattern of substrate wild-type PIP5K was purified as described (17), with 1mM DTT added to the chromatography running buffer during the gel-filtration step. Only selection and phosphorylation is the result of the interplay between freshly prepared protein (25 mg/mL) was used to set up crystallization the PIPBM and the specificity loop (Fig. S5). drops. For the radioactive kinase assay, protein preparations followed the The phylogenetic relationship among type I, type II, and type same protocol without the final step of gel filtration. All purified proteins III kinases is complex (31). Based on in vitro activities and the (from 0.25 mg/mL up to 2 mg/mL) were aliquoted, flash-frozen in liquid

Muftuoglu et al. PNAS | August 2, 2016 | vol. 113 | no. 31 | 8715 Downloaded by guest on September 30, 2021 nitrogen, stored at −80 °C, and thawed on ice before assay. Wild-type human Crystal Structure Determination. Crystallization sitting drops were set up at 4 °C PIP4Kα was generated from a construct containing residues 33–406 of human in 0.1 M Mes monohydrate (pH 6.5) and 12% wt/vol polyethylene glycol 20,000. PIP4Kα (pet28a). An His-tag and linker containing a thrombin cleavage site The crystallization trays were stored at 4 °C, and crystal growth occurred 3 d to were cloned into the N terminus (MGSSHHHHHHSSGLVPRGSHM). To express 3 wk later. Soaking was accomplished in a stepwise manner, adding 100 mM this protein, BL21 de3 Gold cells were grown to an optical density of 0.600, sodium selenate, 100 mM pH-adjusted sodium phosphate, or 1 mM AMP-PNP proteinexpressionwasinducedwith500μM isopropyl β-D-thiogalactoside with 2 mM MnCl2 to the crystallization solution. Cryoprotection was also ac- (IPTG), and the cultures were left shaking overnight at room temperature. complished stepwise, using 25% glycerol. The crystals were flash-frozen in Pellet was resuspended in 25 mL of buffer [10 mM Tris·HCl (pH 8)] per liter of liquid nitrogen following the soaking steps. Diffraction data were collected at culture and subjected to two freeze–thaw cycles. Lysozyme (25 mg per liter of Beamline X29A at the National Synchrotron Light Source. Data were indexed

culture), DNaseI (4 mg per liter of culture), and MgCl2 (200 μL of 1 M per liter and scaled using HKL2000 (39), refinement was conducted using CCP4i (40), of culture) were added to the resuspension, which was allowed to stir (30 and model building was performed using Coot (41). min at room temperature). After a third freeze–thaw cycle, keeping the resuspension on ice, NaCl (1.5 g per liter of culture) was added to stir the ACKNOWLEDGMENTS. We thank Howard Robinson for assistance during X-ray resuspension (30 min at 4 °C). After spinning down the resuspension diffraction data collection at Beamline X29A at the National Synchrotron Light [19,500 rpm using a J-20 rotor (Beckman Coulter) for 1 h at 4 °C], the su- Source. We thank Louise Lucast and Pietro De Camilli for help with HPLC, as well as Karen Anderson, David Calderwood, Anton Bennett, and Gary Rudnick for pernatant was incubated with cobalt resin (Talon Metal Affinity Resin; – · sharing their phosphorimager, fluorescent microscope, phase-contrast micro- Clontech) for 1 4hat4°C.Wash[20mMTrisHCl(pH8),0.5MNaCl,5mM scope, and imager. We also thank Benjamin Turk and Qianying Yuan for helpful · imidazole] and elution [20 mM Tris HCl (pH 8), 0.5 M NaCl, 200 mM discussions. This work was supported by Yale University (Y.H.), National Institutes imidazole] were performed at 4 °C, and aliquots were generated as of Health Grant GM112182 (to D.W. and Y.H.), and predoctoral fellowships from stated above. the Yale Gruber Foundation and the National Science Foundation (to Y.M.).

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8716 | www.pnas.org/cgi/doi/10.1073/pnas.1522112113 Muftuoglu et al. Downloaded by guest on September 30, 2021