NleH Defines a New Family of Bacterial Effector Kinases

Structure

Article NleH Defines a New Family of Bacterial Effector Kinases Andrey M. Grishin,1,* Maia Cherney,1 Deborah H. Anderson,2 Sadhna Phanse,3 Mohan Babu,3 and Miroslaw Cygler1,4,* 1Department

of Biochemistry, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada Research, Saskatchewan Cancer Agency, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada 3Department of Biochemistry, Research and Innovation Centre, University of Regina, 3737 Wascana Parkway, Regina, SK S4S 0A2, Canada 4Department of Biochemistry, McGill University, 3655 Promenade Sir Willam Osler, Montreal, QC H3G 1Y6, Canada *Correspondence: [email protected] (A.M.G.), [email protected] (M.C.) http://dx.doi.org/10.1016/j.str.2013.11.006 2Cancer

SUMMARY

Upon host cell infection, pathogenic Escherichia coli hijacks host cellular processes with the help of 20–60 secreted effector proteins that subvert cellular processes to create an environment conducive to bacterial survival. The NleH effector kinases manipulate the NF-kB pathway and prevent apoptosis. They show low sequence similarity to human regulatory kinases and contain two domains, the N-terminal, likely intrinsically unfolded, and a C-terminal kinase-like domain. We show that these effectors autophosphorylate on sites located predominantly in the N-terminal segment. The kinase domain displays a minimal kinase fold, but lacks an activation loop and the GHI subdomain. Nevertheless, all catalytically important residues are conserved. ATP binding proceeds with minimal structural rearrangements. The NleH structure is the first for the bacterial effector kinases family. NleHs and their homologous effector kinases form a new kinase family within the cluster of eukaryotic-like kinases that includes also Rio, Bud32, and KdoK families.

INTRODUCTION Pathogenic Gram-negative bacteria evolved an arsenal of tools to allow them to invade and overtake their host. To this end, bacteria secrete a set of proteins, called effectors through a specialized secretion system (type III, type IV, type VI), a supramolecular syringe-like assembly, that spans two bacterial membranes and punctures the membrane of the eukaryotic host cell providing a passage for effectors (Gerlach and Hensel, 2007; Filloux et al., 2008). These diverse effectors are tailored precisely to the bacterial life cycle within the host. Nevertheless, there are common themes among effectors from different pathogens (Gala´n, 2009). Some of them are enzymes such as proteases, phosphatases, glycosylases, acetylases, and lipases, whereas others mimic host proteins and interact with regulatory host proteins to modify cell behavior (Dean, 2011). Phosphorylation plays a pivotal role in eukaryotic cell signaling, with kinases representing roughly 1.7% of all genes

in the human genome (Manning et al., 2002). Not surprisingly, the arsenal of bacterial effectors includes kinases and phosphatases that interfere with host signaling. Frequently, each genus of pathogenic bacteria contains at least one effector kinase: NleH1 and NleH2 (enteropathogenic and enterohaemorrhagic Escherichia coli; Gao et al., 2009; Hemrajani et al., 2010; Martinez et al., 2010), OspG (Shigella flexneri; Kim et al., 2005), SteC (Salmonella Typhimurium; Poh et al., 2008), LegK1–LegK4 (Legionella pneumophila; Ge et al., 2009; Hervet et al., 2011), YpkA (Yersinia pestis; Wiley et al., 2009), and YspK (Yersinia enterocolitica; Matsumoto and Young, 2006). No structure of a bacterial effector kinase has yet been reported. Some of these kinases (SteC, YpkA, LegK1, LegK3, and LegK4) show clear sequence similarity to the eukaryotic kinases and were likely acquired through lateral gene transfer (Lurie-Weinberger et al., 2010). However, other kinases such as NleH1, NleH2, OspG, YspK, LegK2 (Kim et al., 2005; Gao et al., 2009), and a putative kinase SboH (Salmonella bongori; Fookes et al., 2011) possess only basic kinase motifs and share some similarity to atypical Rio kinases (LaRonde-LeBlanc and Wlodawer, 2005; Figure 1). A large amount of sequencing data derived from an ocean metagenome, which among others identified 40,000 kinase genes, prompted a detailed bioinformatics analysis of the kinase superfamily (Kannan et al., 2007). Twenty distinct kinase families were identified, with eukaryotic protein kinases (ePKs) being just one of them. These families were further grouped into three main clusters: the first cluster contains ePK-like kinases, encompassing ePKs, Haspin, and PknB families; the second cluster includes metabolic kinases choline and aminoglycoside kinase, fructosamine kinase (FruK), and HSK2 families; and the third cluster contains KdoK, Rio, and Bud32 families. The small bacterial kinases NleH and OspG were not included in this analysis, partly due to their unknown functions at that time of this study and low abundance (present only as one to two genes in few pathogenic bacterial genomes and in some phages). NleH1 and NleH2 are very closely related bacterial effectors (84% sequence identity), which only recently have been shown to subvert the NF-kB pathway (Gao et al., 2009; Martinez et al., 2010; Wan et al., 2011; Pham et al., 2012) and prevent apoptosis (Hemrajani et al., 2010; Robinson et al., 2010). These effectors are comprised of a C-terminal kinase domain and an N-terminal segment of around 140 amino acids bearing signs of intrinsically disordered proteins.

250 Structure 22, 250–259, February 4, 2014 ª2014 Elsevier Ltd All rights reserved

Structure NleH Bacterial Effector Kinase

Figure 1. Comparison of Diverse Kinases Structure-based sequence alignment. Amino acid residues are colored using JalView according to ClustalX coloring scheme, with the color intensity indicating sequence conservation (Troshin et al., 2011). Hydrophobic residues are colored blue, His and Tyr are cyan, polar residues are green, Arg and Lys are red, Asp and Glu are magenta, Gly is orange, and Pro is olive. The segments (as defined by Hanks and Hunter, 1995), secondary structure elements, and kinase-specific insertions (SI) are shown. The Protein Data Bank (PDB) ID codes are indicated in brackets. HRK, haspin kinase.

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Structure NleH Bacterial Effector Kinase

The N-terminal segment was shown to bind the ribosomal protein 3 (RPS3), a known coactivator of NF-kB, which guides the latter to specific kB sites (Wan et al., 2007). RPS3 undergoes phosphorylation of Ser209 by IKK-b that induces nuclear translocation (Wan et al., 2007). NleH1 inhibits the IKK-b-dependent phosphorylation of Ser209 and translocation of RPS3 into nucleus, interfering with transcription activation (Gao et al., 2009; Wan et al., 2011; Pham et al., 2012). Intriguingly, NleH2, although very similar to NleH1, has a different effect on the NF-kB pathway: it stimulates phosphorylation of RPS3 and mildly activates the NF-kB pathway (Pham et al., 2012). However, when IKK-b is overexpressed, both NleH1 and NleH2 were shown to inhibit the NF-kB pathway (Royan et al., 2010; Pham et al., 2012). Although NIeH kinases do not appear to directly phosphorylate RPS3, their kinase activity is essential for their cellular function, as proven by the lack of the downstream effect of the kinase-dead mutants (Gao et al., 2009; Pham et al., 2012). The importance of the kinase activity was also shown for the homologous effector kinase OspG from Shigella (Kim et al., 2005); however, the phosphorylation targets of NleH and OspG remain to be identified. NleHs were also shown to bind to Na+-H+ exchanger regulatory factor 2 (NHERF2). This interaction involves the PSD-95/ Discs-Large/ZO-1 domain 2 (PDZ2 domain) of NHERF2 and the C terminus of the kinase domain (Martinez et al., 2010; Pham et al., 2012). In addition, the kinase domain of NleH interacted with the cytosolic N-terminal 40 residues of Bax inhibitor1, a host protein that protects cells from apoptosis induced by a number of proapoptotic compounds, such as universal kinase inhibitor staurosporin, inducers of endoplasmic reticulum stress brefeldin A and tunicamycin (Hemrajani et al., 2010), and Clostridium difficile toxin B (Robinson et al., 2010). Here, we describe the structure of the unusual kinase domains from NleH1 and NleH2 and show that they contain only the canonical kinase fold core but are missing the C-terminal segment of classic kinases. In addition, the canonical ‘‘activation loop’’ is significantly shortened and has the same conformation in ATP-free and ATP-bound states. The recombinant full-length NleH proteins, as well as their kinase domains, possess kinase activity. Furthermore, autophosphorylation activity was detected and several sites of phosphorylation were identified. Mutagenesis of putative catalytic residues confirmed their involvement in phosphotransfer. RESULTS AND DISCUSSION Kinase Activity and Autophosphorylation The C-terminal kinase domains NleH1KD(128–293) and NleH2KD(140–303) eluted from the size exclusion column as Figure 2. Protein Kinase Assay with [g-32P]ATP (A) Autophosphorylation of NleH2 full-length, NleH1 full-length. Phosphorylation of MBP by NleH1, NleH2, NleH1KD, and NleH2KD. Coomassie-stained SDS-PAGE gel. Lanes: 1, NleH2 (1 mg) autophosphorylation; 2, NleH2 (0.1 mg) + MBP (1 mg); 3, NleH1 (1 mg) autophosphorylation; 4, NleH1 (0.1 mg) + MBP (1 mg); 5, NleH1(40–293) (1 mg) autophosphorylation; 6, NleH2KD (1 mg) autophosphorylation; 7, NleH2KD (1 mg) + MBP (1 mg); 8, NleH2KD (0.1 mg) + MBP (1 mg); 9, NleH2KD (0.01 mg) + MBP (1 mg); 10, NleH1KD (1 mg) autophosphorylation; 11, NleH1KD (1 mg) + MBP (1 mg); 12, NleH1KD (0.1 mg) + MBP (1 mg). (B) Phosphorimage, corresponding to Figure 2A.

(C) Phosphorylation of the NleH2(1–147) N-terminal segment by NleH1KD and NleH2KD. Coomassie-stained SDS-PAGE gel (left) and phosphorimage (right). Lanes: 13, NleH2KD (1 mg) + NleH2(1–147) (1 mg); 14, NleH1KD (1 mg) + NleH2 (1–147) (1 mg). (D) Activity of NleH2 active-site mutants on MBP. Coomassie-stained SDSPAGE gel (left) and phosphorimage (right). Lanes: 15, NleH2KD(K169A) (1 mg) + MBP (1 mg); 16, NleH2KD(E183A) (1 mg) + MBP (1 mg); 17, NleH2KD(D249A) (1 mg) + MBP (1 mg), the arrow shows weakly phosphorylated MBP; 18, NleH2KD(D268A) (1 mg) + MBP (1 mg). See also Figure S2.

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Structure NleH Bacterial Effector Kinase

Figure 3. The Quadrupole TOF Spectra of Purified NleH1 and NleH2 and after Incubation with ATP and Mg2+ The NleH1 spectrum shows the presence of a low amount of non-phosphorylated species (MW 32532), and three peaks corresponding to mono-, di-, and triphosphorylated NleH1, differing in MW by 80 Da/phosphate group. Upon the incubation with ATP, the amount of native and monophosphorylated species decreased, while that of di- and triphosphorylated species increased. A similar trend was observed for NleH2. Upon treatment with ATP, the amount of mono-, di-, and triphosphorylated species increased. See also Tables S1 and S2.

sharp peaks with apparent molecular weights (MWs) of 19 kDa, corresponding to a monomer (Figure S1 available online). Dynamic light scattering results corroborated the monomeric state in solution. However, the full-length NleH1 and NleH2 and their N-terminal domains NleH1(1–137), NleH1(39–137), NleH2(1– 147), and NleH2(39–147), as well as NleH1(40–293), eluted from size exclusion column in the void volume, indicating protein aggregation (data not shown). This behavior is consistent with the prediction that the region 90–140 in NleH1/2 is disordered and lacks recognizable secondary structure, as estimated by online tools PONDR (Xue et al., 2010), FoldIndex (Prilusky et al., 2005), and Jpred (Cole et al., 2008). Using a radioactive in vitro kinase assay, we confirmed that the full-length NleH1 and NleH2 were active kinases through phosphorylation of myelin basic protein as an exogenous substrate (Hemrajani et al., 2010; Figures 2A and 2B). Likewise, the kinase domains of NleH1KD and NleH2KD were active in this assay (Figures 2A and 2B). Moreover, NleH1/2 were able to autophosphorylate (Figures 2A and 2B). The isolated kinase domains were also able to weakly autophosphorylate and to phosphorylate the N-terminal segments to some degree (Figure 2C). These results suggest that the main autophosphorylation sites on NleH1/2 are located within their N-terminal unstructured segments. The MWs of the recombinant NleH1 and NleH2 purified from E. coli corresponded to a mixture of non-, mono-, di-, and triphosphorylated species, as shown by electrospray ionization mass spectrometry (Figure 3). Incubation with ATP and Mg2+ led to a decrease of the nonphosphorylated species with a concomitant increase of the phosphorylated species (Figure 3). Subsequently, mass spectrometric analysis of trypsin-derived fragments of NleH2 identified multiple phosphorylation sites. Because the full-length NleH1 and NleH2 contained only up to three phosphate groups (see above), we conclude that many of these sites are of low abundance (Tables S1 and S2). We compared results from two sets of data obtained on two different instruments and with somewhat different protocols and methods of data analysis. We observed coverage of over 90% of the sequence and found that common phosphorylation sites be-

tween these two sets were Ser22, Ser30, Ser96, Thr101, Ser109, Tyr119, and Ser280. Except for Ser280, these sites are in the N-terminal segment preceding the kinase domain and are most likely the main autophosphorylation sites. Ser280 is located at the end of a short loop preceding helix F (segment IX). All of them (except Thr101) are conserved in NleH1 sequence. Structure of the NleH2KD and the Kinase Fold Attempts to solve the structure of NleH2KD by molecular replacement were unsuccessful, and the structure was solved by the single wavelength anomalous dispersion (SAD) method from SeMet-substituted protein crystal. The details of data collection and refinement are listed in Table 1. Twelve sequence regions (I–V, VIA, and VIB–XI) are present in all eukaryotic regulatory Ser/Thr kinases (Figure 1; Hanks and Hunter, 1995). The N-terminal lobe contains an antiparallel b sheet (segments I, II, and IV) and a-helix C (segment III), linked through b strand (segment V) to the C-terminal lobe that contains four b strands (segments VIB and VII) and several a helices (segments VIA, IX, X, and XI). The characteristic activation loop is within segment VIII. Finally, the C-terminal tail loops around the back of the protein and embraces the N-terminal domain. The NleH2KD (Figure 4A) lacks several segments of the classic kinase fold (Figure 4B) and represents a minimal kinase fold, encompassing only segments I to VII and IX of the classic fold (Figure 1). These segments contain the main secondary structural elements and the catalytic residues: a Gly-rich motif (also called the P loop), I149GKGGNAVV157, participating in ATP binding (segment I); invariant Lys169 correctly orienting the ATP b- and g-phosphates for catalysis (segment II); Glu183 that interacts with Lys169 (segment III); an intervening ‘‘catalytic loop’’ (segment VIB) with sequence FVD249TTETN254 replacing consensus H/YRDXKXXN, D268IS (DFGFA in protein kinase A [PKA]; segment VII); and a long a-helix (a-helix F, segment IX) at the C terminus, a putative PDZ-binding region of NleH2 (Martinez et al., 2010). An important difference of NleH2KD from eukaryotic kinases is the lack of an activation loop (segment VIII). The other a helices of the C-terminal lobe (segments X to

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Structure NleH Bacterial Effector Kinase

Table 1. Summary of Data Collection and Refinement Statistics KD

KD

NleH2 Apo NleH2KD (L-SeMet)

NleH1 , AMPPNP, Mg2+

Space group a,b,c (A˚)

P4212

P21

a,b,g ( )

90, 90, 90

90, 90, 90

Wavelength (A˚) Resolution (A˚)

0.97949

0.97871

0.97871

50.0–2.27

50.0–2.54

50.0–1.61

2.31–2.27

2.58–2.54

1.64–1.61

Data Set

Last resolution shell (A˚)

P4212

145.3, 145.3, 146.9, 146.9, 40.7, 98.1, 40.8 83.5 82.6 90.0, 90.3, 90.0

Observed hkl

864577

345540

232991

Unique hkl

41406

30085

41051

Completeness (%)a

99.7 (99.6)

100.0 (100.0) 99.7 (95.7)

Redundancya

20.9 (17.6)

11.5 (11.7)

5.7 (5.3)

Rsyma

0.145 (>1)

0.143 (>1)

0.052 (0.338)

I/(sI)a

28.4 (2.00)

23.36 (2.02)

CC1/2a,b

0.998 (0.429)

CC*a,b

1.000 (0.775)

1.000 (0.975)

Rwork

0.174

0.172

Rfree (%hkl)

0.220 (10)

33.5 Wilson B (A˚2) B-factor A˚2 (n atoms)

32.87 (5.4) 0.999 (0.905)

with its N6 amino group toward the protein floor made by Met201 and the aliphatic moieties of Lys203 and Ile204. The amino group is hydrogen bonded to the Asp202 carbonyl. The base is stacked on one side by Ile139, Val147, and Val157 and on the other side by Leu246 and Ile257. The N3 and N7 are hydrogen bonded to the protein via bridging water molecules. This environment is very similar to other kinases (Endicott et al., 2012). The a-phosphate forms hydrogen bonds with NHLys159 and the Asp258 side chain. The P loop Gly142 and Gly143 approach close to the b- and g-phosphates and NHGly143 forms a hydrogen bond with the g-phosphate. The b-phosphate also interacts with the OHThr243 and, through a water molecule, with the Lys141 carbonyl. The Mg2+ ion has tetragonal bipyramid coordination, liganded by the a- and b-phosphates, the carboxyl of Asp258 (DIS motif), and three water molecules, one of which forms the hydrogen bond with Asn244 and the other with the g-phosphate (Figure 5). This position of Mg2+ in NleH1 closely resembles the position of the primary Mg2+ in PKA (Zheng et al., 1993), although the conformation of b- and g-phosphates differs in these two enzymes (Figure 5).

0.206 (10) 58.6

23.4

Protein

38.3 (5,097)

15.28 (2,508)

Solvent

38.6 (299)

22.1 (283)

Ligand

NA

24.6 (62)

Allowed (%)

97.5

97.8

Generous (%)

2.3

1.6

Disallowed (%)

0.2

0.6

Bonds (A˚)

0.007

0.006

Angles ( )

1.017

0.997

PDB ID code

4LRK

4LRJ

Ramachandran plot

Rmsd

a

The information for the last shell of resolution is given in parentheses. b The correlation coefficients were calculated according to Karplus and Diederichs (2012).

XI) and the extended polypeptide that embraced the N-terminal lobe are absent in NleH2. Structure of the NleH1KD and the Binding of ATP NleH1 was crystallized with a noncleavable ATP analog AMPPNP and Mg2+. As expected, NleH1 and NleH2 are very similar, with a root-mean-square deviation (rmsd) of 0.9 A˚ for 98% of the Ca atoms, and they contain the same structural features (Figures 4A and 4C). A small difference in the orientation of N- and C-terminal lobes in the two proteins is likely due to the crystal environment. The pivot point for this rotation is around Asn215 in NleH2. ATP bound to NleH1 in a fashion similar to other kinases. The molecule sits in one end of a deep and narrow groove with the adenine at the end of the groove and the gamma phosphate near the middle (Figure 4C). The adenine base is oriented

Site-Directed Mutagenesis The roles of many residues in yeast PKA were tested by mutagenesis. The kcat of K116A (K72 in mouse PKA) decreased 850-fold, D210A (D166 in mouse PKA) 340-fold, E135A (E91 in mouse PKA) 20-fold, and the D228A (D184 in mouse PKA) was unstable (Gibbs and Zoller, 1991). We tested the homologous active-site residues in NleH2KD for their importance in catalysis. Four residues of NleH2KD, Lys169, Glu183, Asp249, and Asp268 (Lys116, Glu135, Asp210, and Asp228 in yeast PKA) were mutated to alanines (Figure S2) and the activity of the mutants was evaluated (Figure 2D). The K169A, D249A, and D268A mutants showed no activity detectable using our assay. The E183A mutant retained very low activity, paralleling the behavior of the corresponding mutation in PKA. Probable Substrate Binding Mode In the absence of a long activation loop, which is important for substrate binding, the question arises how do NleHs recognize their substrates? The crystal structure of NleH2KD with four independent molecules in the asymmetric unit provided some clues. The molecules form two pairs, with active sites facing each other in each pair. The substrate-binding site of molecule A is occupied by the planar b sheet of the N-terminal domain of molecule B in a fashion that could approximate the approach of a protein substrate (Figure 4D). The tip of the P loop (b1-b2 hairpin, 150GKGGNAV156) of molecule B, and in particular Gly153, is positioned near the g-phosphate of ATP in molecule A, as modeled based on the NleH1KD structure. In silico replacement of Gly153 with a serine brings its hydroxyl group within 4.3 A˚ of the g-phosphorus, and in a location very similar to that of the acceptor serine in a peptide substrate of the phosphorylase kinase (after superposition, 1.5 A˚ between the serine hydroxyls; Lowe et al., 1997). Two residue clusters on the surface of NleH1/2 near the active site are likely important for substrate recognition: a polar cluster comprised of (in NleH2) Asn154 (P loop), Gln175, and Glu179

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Structure NleH Bacterial Effector Kinase

Figure 4. The Minimal Kinase Fold (A) Superposition of NleH2KD (cartoon representation, wheat) and NleH1KD (ribbon, dark green). The regulatory and catalytic hydrophobic spines are shown as surfaces and colored blue and pink, correspondingly. The adenine moiety (shown as van der Waals spheres) complements the C-spine upon binding. (B) Superposition of NleH1KD (dark green) and PKA (light pink). The PKA activation loop is colored cyan, and the GHI domain is colored magenta. The AMPPNP in NleH1 is colored by atom type (cyan carbon), and the Mg2+ ion is colored blue. ATP bound to PKA is also colored by atom type (yellow carbon), while two Mn2+ ions are colored gray. (C) Comparison of active sites of NleH1KD (with AMPPNP and Mg2+; green cartoon and green carbon atoms) and apo-NleH2KD (wheat cartoon and yellow carbon atoms). (D) Possible NleH-substrate interaction. The P loop (sticks, colored by atom type with carbon atoms colored green) of one NleH2KD molecule is inserted into the active site of another NleH2KD molecule (wheat). The ATP molecule and Mg2+ ion are modeled based on NleH1KD. The P loop Gly153 was mutated in silico to Ser to represent the putative position of the Ser in the substrate. The putative substrate-binding residues are colored by atom type with carbon atoms colored cyan. See also Figure S1.

(segment III), and a hydrophobic cluster composed of Val248 (segment VIB), Tyr272, and Trp279 (segment VII; Figure 4D). Overall, the surfaces of NleH1 and NleH2 near their active sites are lined with identical residues. Therefore, these two kinases are expected to have very similar substrate specificity. The sequence identity between NleH1KD and NleH2KD is 88% and is the same for their N-terminal regions. Their most significant difference is an insertion of 10 residues in NleH2 at position 129, at the junction with the kinase domain. Based on these two observations, we conclude that the noted functional differences between them (Gao et al., 2009; Pham et al., 2012) result from the sequence differences in their N-terminal domains, corroborating the previous finding that mutations in residues 40–45 affect functional outcome (Gao et al., 2009).

Comparison with Eukaryotic Kinases Eukaryotic kinases are known to oscillate between an active and an inactive state. The active state is similar between kinases, while the inactive state is kinase-dependent (Huse and Kuriyan, 2002). Two structural features are characteristic of the kinase active state. The first is the characteristic conformation of the kinase domain, in which (1) the active site is free of autoinhibitory elements, (2) the N- and C-lobes of the kinase domain are poised at the right orientation relative to each other, (3) the active site residues are directed toward the active center, (4) the conserved Lys (strand b3) forms a salt bridge with Glu (helix aC), and (5) the DFG motif is in an ‘‘in’’-conformation (Huse and Kuriyan, 2002). The second is a cluster of four highly conserved hydrophobic residues assembled to interact with each other only in

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Structure NleH Bacterial Effector Kinase

family, we applied the MULTISEQ algorithm (Roberts et al., 2006) that uses sequence and structural information to build a phylogenetic tree (Figure 6). This algorithm was proven to be particularly useful for the analysis of remote homologs such as in aminoacyl-tRNA synthetases (Sethi et al., 2005). Accordingly, NleH belongs to a distinct kinase family that falls within the KdoK-Rio-Bud32 cluster (Figures 6 and S3). All these kinases lack the activation loop (segment VIII) and GHI-subdomain (segments X and XI) and show only small structural changes upon ATP binding. Interestingly, Rio was found to function as an adenosine triphosphatase rather than as a kinase, in which ATP hydrolysis is needed to release Rio from the maturing ribosome (Ferreira-Cerca et al., 2012). The divergence of NleH family occurred at a similar time as that of Rio and Bud32. Thus, NleH/OspG are ancient kinases that have acquired their current functions over the course of evolution. Figure 5. The Coordination of Mg2+ in NleH1 AMPPNP shown as thick sticks. Mg2+ is shown as a green sphere, waters are shown as red spheres, and dashed green lines show coordinating atoms. Side-chains of functionally important Lys159, Glu173, Asp239, Asn244, and Asp258, as well as P loop Gly142 and Gly143, are shown as sticks with cyan carbons. The corresponding residues from PKA kinase and the ATP molecule superimposed on NleH1 are shown as thin stick with gray carbons; the Mn2+ ions are colored gray and are shown as small spheres.

the active state, the so-called ‘‘regulatory R-spine’’ (Kornev et al., 2006, 2008). Comparison of NleH2KD with PKA showed that the conformation of NleH2 in its apo form was similar to the active conformation of PKA. Lys169 forms an ion pair with Glu183, and the R-spine, formed in NleH2KD by Phe187, Ala196, Phe247, and Ile269, is fully assembled (Figures 4A–4C). The NleH2KD structure and the active conformation of PKA can be superimposed with an rmsd of 1.7 A˚ for 96 Ca atoms (out of 160 in NleH2). Their catalytic machineries show even closer resemblance and superimpose with rmsd of 1.2 A˚. A second ‘‘catalytic C-spine’’ contains hydrophobic residues from the N-terminal and C-terminal lobes, connected together via the ATP adenine ring (Kornev et al., 2006, 2008). The C-spine is also conserved in NleH2 and is comprised of Val157, Val167 from the N-lobe, and Leu219, Val255, Leu256, Tyr257, Leu295, and Val299 from the C-terminal lobe (Figure 4A). Indeed, in NleH1KD the adenine moiety complements the C-spine. Comparison of apo-NleH2KD with ATP-bound NleH1KD showed that the binding of ATP and Mg2+ ion proceeds with minimal structural rearrangement. The rmsd (all atoms) for the catalytic machinery is 1.1 A˚ (Lys159, Glu173, Asp239, Asn244, and Asp258) and 1.0 A˚ for the P loop (Figure 4C). The absence of the long activation loop and confirmed activity of the kinase domains led to the conclusion that NleHs lacked the activation mechanism typical for eukaryotic protein kinases. Outside of eukaryotic-like kinase families, two other kinase clusters are prominent. The first contains predominantly kinases with metabolic functions—choline kinase (ChoK), aminoglycoside kinase (APH), and FruK. The second cluster contains Rio kinase (ribosome biogenesis), Bud32 (tRNA modification), and KdoK (lipopolysaccharide phosphorylation) (Kannan et al., 2007). To place NleH within the highly diverse kinase super-

EXPERIMENTAL PROCEDURES Cloning and Protein Expression Full-length genes and fragments of nleH1 and nleH2 were cloned from E. coli O157:H7 EDL933 genome into the pMCSG7 vector, a pET-21a derivative adapted for ligation-independent cloning (Eschenfeldt et al., 2009). Fragments of nleH genes corresponded to TEV-cleavable His6-tagged NleH1(1–137), NleH1(39–137), NleH1(40–293), NleH1KD(128–293), NleH2(1–147), NleH2 (39–147), and NleH2KD(140–303). The plasmids were transformed into BL21(DE3)Star (Invitrogen) for protein expression. The mutations NleH2KD(K169A), NleH2KD(E183A), NleH2KD(D249A), and NleH2KD(D268A)

Figure 6. Evolution Tree The evolution tree was derived by Multiseq (Roberts et al., 2006). The QH score served as a distance matrix for construction of the tree. See also Figure S3.

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Structure NleH Bacterial Effector Kinase

were generated using the Quik-Change site-directed mutagenesis method (Braman et al., 1996). An overnight culture of the expression strain was diluted 100 times with fresh terrific broth medium. Cells were grown at 37 C until the optical density 600 reached 0.6. Protein expression was induced by the addition of isopropyl b-D-1-thiogalactopyranoside to a final concentration of 1 mM. Cells were induced overnight at 20 C and harvested by centrifugation. Protein Purification Cells were resuspended in lysis buffer (50 mM HEPES pH 8.0, 400 mM NaCl, 10 mM imidazole, 0.5 mM Tris(2-carboxyethyl)phosphine [TCEP]) and disrupted by sonication. The cell lysate was centrifuged at 14,000 g at 4 C, and the supernatant was mixed with nickel-nitriloacetic acid (Ni-NTA) agarose beads (QIAGEN) and incubated at constant shaking for 2 hr at 4 C. The beads were washed with 10 column volumes (CV) of lysis buffer, 10 CV of lysis buffer containing 1 M NaCl, and 10 CV high lysis buffer containing 40 mM imidazole. The bound protein was eluted with lysis buffer containing 250 mM imidazole. The full-length NleH1 and NleH2, NleH1KD(128–293), NleH2(1–147), and NleH2(39–147) were loaded on Ni-NTA beads and washed as above. For on-column cleavage, TEV protease (in 50 mM HEPES pH 8.0, 50 mM NaCl) was added in a 1:100 (w/w) ratio. After overnight incubation at room temperature, the supernatant containing the target protein was collected and loaded on a Superdex 75 column (GE Healthcare) in 20 mM HEPES pH 8.0, 50 mM NaCl, 0.5 mM TCEP. After TEV cleavage, the resulting protein contained three additional N-terminal residues, Ser-Asn-Ala, instead of the N-terminal methionine, resulting in the MW shift of +141 Da. The L-selenomethionine derivative of NleH2KD(140–303) was expressed in the E. coli Met auxotroph strain DL41 (Doublie´, 1997). The overnight inoculum of transformed cells was grown in Luria-Bertani broth. The cells were collected by centrifugation, washed several times in sterile M9 minimal media, and resuspended in M9 minimal media supplemented with 5 g/L glucose, all amino acids except L-Met at 40 mg/L, L-SeMet (Sigma) at 50 mg/L, and vitamin solution (Hendrickson et al., 1990). Cells were grown and induced, and the protein was purified as described above. Protein Crystallization Purified NleH1KD(128–293) and NleH2KD(140–303) were concentrated to 10 mg/ml and mixed with AMPPNP and Mg2+ ions. The native and SeMet NleH2KD crystals were obtained by the hanging drop method by mixing 1 ml of protein solution with 1 ml of well solution containing 100 mM 2-(N-morpholino)ethanesulfonic acid (MES) 6.5, 200 mM (NH4)2SO4, 25%–35% polyethylene glycol (PEG) 5000 monomethyl ether (MME). The crystals were cryoprotected in 100 mM MES 6.5, 200 mM (NH4)2SO4, 25% PEG 5000 MME, 20% glycerol and flash frozen in liquid nitrogen. NleH1KD crystals were obtained with well solution containing 100 mM Tris 8.5, 20% PEG 3350, 100 mM NaCl. They were cryoprotected in 100 mM Tris 8.5, 20% PEG 3350, 100 mM NaCl, 20% glycerol. Structure Determination The diffraction data were collected at 100 K on a Mar300 charge-coupled device detector at the Canadian Macromolecular Crystallography Facility Insertion Device beamline at the Canadian Light Source (Table 1). Data were integrated and scaled with HKL3000 (Minor et al., 2006; Table 1). The NleH2KD structure was solved using the SAD method with Phenix_AutoSol (Adams et al., 2011). The initial model was refined with Phenix_Refine (Adams et al., 2011). The NleH1KD structure was solved by molecular replacement with Phaser (McCoy et al., 2007) using NleH2KD as a search model. Xtriage (Adams et al., 2011) identified twinning with the operator l,-k,h., and this information was included in the refinement with Phenix_Refine. Manual rebuilding was performed with COOT (Emsley et al., 2010). The final structures were validated with MolProbity (Chen et al., 2010). Kinase Assays The kinase activity was measured for NleH1, NleH2, NleH1(40–293), NleH1KD(128–293), and NleH2KD(140–303) as well as for mutants NleH2KD(K169A), NleH2KD(E183A), NleH2KD(D249A), and NleH2KD(D268A) with [g-32P]ATP. Proteins were kept in a buffer containing 50 mM Tris

pH 7.5, 50 mM NaCl, 5 mM MgCl2 and 1 mM dithiothreitol. Myelin basic protein (MBP) was used as an exogenous substrate. The 10 ml reaction mixture contained 0.01–1 mg of the kinase and 1 mg of MBP. The reaction was started by addition of a mixture of ATP with [g-32P]ATP to final concentrations of 25 mM ATP and 5 mCi/reaction of [g-32P]ATP (3000 Ci/mmol) and was carried out for 30 min at 37 C. Phosphorylation was monitored by separating the samples by SDS-PAGE and imaging the radiolabeled proteins using a phosphorimager screen, a Molecular Imager FX (Bio-Rad) using Quantity One software. Autophosphorylation was performed in the same manner with 1 mg of a kinase in 10 ml of the reaction mixture, in the absence of MBP. Mass-Spectrometry-Based Identification of Phosphorylation Sites MW determination of intact NleH1/2 was performed at the University of Alberta mass spectrometry service facility using reverse-phase high-performance liquid chromatography on a Poroshell C8 column followed by detection with UV absorption and mass spectrometry (RP-HPLC-UV-MS). Mass spectra were acquired in positive mode of ionization using an Agilent 6220 AccurateMass time-of-flight (TOF) HPLC/MS system. Data analysis was performed using the Agilent MassHunter Qualitative Analysis software package, version B.03.01 SP3. Identification of phosphorylation sites was performed at the UVic Proteomics Centre (University of Victoria) following their standard protocols (see Supplemental Experimental Procedures). As an independent assessment, we performed an analogous procedure at the University of Regina. The purified protein was resolved by SDS-PAGE. The band corresponding to the target protein was excised and the protein was digested with sequencing-grade trypsin (Promega). Samples of the peptide mixtures were analyzed using an LTQ-Orbitrap Velos mass spectrometer (ThermoFisher Scientific) with the higher-energy collision-induced dissociation fragmentation method using a nanospray ion source (Proxeon). A detailed protocol is provided in Supplemental Experimental Procedures. ACCESSION NUMBERS The RCSB Protein Data Bank accession numbers for the apo NleH2KD and NleH1KD with AMPPNP and Mg2+ reported in this paper are 4LRK and 4LRJ, correspondingly. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, three figures, and two tables and can be found with this article online at http://dx.doi.org/10.1016/j.str.2013.11.006. ACKNOWLEDGMENTS This work was supported by grant MOP-48370 from the Canadian Institutes of Health Research (CIHR) and Canada Foundation for Innovation to M.Cygler, the Natural Sciences and Engineering Research Council (NSERC; to M.B.), and CIHR training related to synchrotron techniques (CIHR-TRUST fellowship to A.M.G.). Diffraction data were collected at the beamline 08ID-1 at the Canadian Light Source, which is supported by NSERC, the National Research Council Canada (NRCC), CIHR, the Province of Saskatchewan, Western Economic Diversification Canada, and the University of Saskatchewan. The authors would like to thank Dr. R. Whittal and B. Reiz of the University of Alberta Mass Spectrometry Facility for their technical assistance with the HPLC-UVMS analysis. Received: September 14, 2013 Revised: November 4, 2013 Accepted: November 11, 2013 Published: December 26, 2013 REFERENCES Adams, P.D., Afonine, P.V., Bunko´czi, G., Chen, V.B., Echols, N., Headd, J.J., Hung, L.W., Jain, S., Kapral, G.J., Grosse Kunstleve, R.W., et al. (2011). The

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