Structural Insight into the Activation of PknI Kinase from M. tuberculosis via Dimerization of the Extracellular Sensor Domain

Article

Structural Insight into the Activation of PknI Kinase from M. tuberculosis via Dimerization of the Extracellular Sensor Domain Graphical Abstract

Authors Qiaoling Yan, Dunquan Jiang, Lanfang Qian, ..., Luke Guddat, Haitao Yang, Zihe Rao

Correspondence [email protected]

In Brief PknI is required for regulating M. tuberculosis growth within macrophage. Here, Yan et al. show that PknI_SD exists in a monomer-dimer equilibrium, with an arm region critical for dimer formation identified, and report rapamycin-induced dimerization of PknI_KJD fusion is able to activate kinase auto-phosphorylation activity, which, along with in vivo experiment results, suggest PknI probably functions as a dimer in regulating M. tuberculosis growth.

Highlights d

PknI_SD adopts two conformations in solution: monomer and dimer

d

Crystal structures of PknI_SD monomer and dimer, as well as PknI_KD, reported

d

Simulated PknI_KJD dimerization activates kinase autophosphorylation activity

d

PknI functions as a dimer in regulating M. tuberculosis growth

Yan et al., 2017, Structure 25, 1–9 August 1, 2017 ª 2017 Elsevier Ltd. http://dx.doi.org/10.1016/j.str.2017.06.010

Please cite this article in press as: Yan et al., Structural Insight into the Activation of PknI Kinase from M. tuberculosis via Dimerization of the Extracellular Sensor Domain, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.010

Structure

Article Structural Insight into the Activation of PknI Kinase from M. tuberculosis via Dimerization of the Extracellular Sensor Domain Qiaoling Yan,1 Dunquan Jiang,1 Lanfang Qian,1 Qingqing Zhang,1 Wei Zhang,1 Weihong Zhou,1 Kaixia Mi,4 Luke Guddat,5 Haitao Yang,6 and Zihe Rao1,2,3,7,* 1College

of Life Sciences, Nankai University, Tianjin 300071, China Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing 100101, China 3Laboratory of Structural Biology, School of Medicine, Tsinghua University, Beijing 100084, China 4CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Science, Beijing 100101, China 5School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia 6College of Life Sciences, Tianjin University, Tianjin 300072, China 7Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2017.06.010 2National

SUMMARY

Protein kinases play central roles in the survival of Mycobacterium tuberculosis within host. Here we report the individual high-resolution crystal structures of the sensor domain (in both monomer and dimer forms) and the kinase domain of PknI, a transmembrane protein member of the serine/threonine protein kinases (STPKs) family. PknI is the first STPK identified whose sensor domain exists in a monomer-dimer equilibrium. Inspection of the two structures of the sensor domain (PknI_SD) revealed conformational changes upon dimerization, with an arm region of critical importance for dimer formation identified. Rapamycin-induced dimerization of unphosphorylated fusions of PknI juxtamembrane and the kinase domain, intended to mimic the dimerization effect presumably imposed by PknI_SD, was observed to be able to activate auto-phosphorylation activity of the kinase domain. In vivo experiments using an M. bovis model suggested PknI functions as a dimer in the regulation of M. tuberculosis growth. INTRODUCTION Tuberculosis, caused by the infectious human pathogen, Mycobacterium tuberculosis, continues to be a major cause of morbidity and mortality worldwide. Survival of M. tuberculosis can be attributed to its ability to sense and adapt to the variable environments it encounters, especially in host cell niches. Signal transduction is one such response to varying environments that is exquisitely orchestrated by a suite of proteins to detect changes in external signals (e.g., hypoxia, pH change) (Magombedze et al., 2013; Elks et al., 2013; Sharma et al., 2004; Kreamer et al., 2015) and then trigger adaptive responses (Pereira et al., 2011). In prokaryotes, ‘‘two-component’’ systems are widely

known to play a predominant role in response to specific stimuli (Robinson et al., 2000; Haag and Bagnoli, 2016; Zhou et al., 2012; Bretl et al., 2011). Recent studies on eukaryote-like Ser/Thr protein kinases (STPKs) in bacteria have shown that they are critically involved in the regulation of stress responses and pathogenicity (Pereira et al., 2011; Liu et al., 2011; Hatzios et al., 2013). The genome of M. tuberculosis possesses 11 STPKs genes, nine of which are receptor-like transmembrane proteins that can be divided into three clades (PknA/B/L, PknD/E/H, and PknF/I/J) (Narayan et al., 2007). These STPKs are composed of an N-terminal kinase domain and a C-terminal extracellular sensor domain, plus a transmembrane helix in between. Comparative and phylogenetic analyses of pathogenic and nonpathogenic mycobacterial species reveals that pknA/B/L/G loci are inherited across the genomes, but pknI and pknJ are genes unique to pathogenic M. tuberculosis (Narayan et al., 2007), suggesting that pathogenic unique STPKs may be associated with bacterial virulence and pathogenicity. M. tuberculosis resides in a moderately acid environment (pH 6.2–4.5), such as phagosome or phagolysosome (MacMicking et al., 2003; Via et al., 1998; Mehta et al., 2016). Changes in pH act as an important host signal sensed by M. tuberculosis to regulate gene expression and establish chronic infection (Rohde et al., 2012). PknI is reported to be one of the proteins involved in sensing the host acidic environment and regulating the growth of M. tuberculosis therein (Gopalaswamy et al., 2009). Previous studies also showed that PknI has in vitro auto- and trans-phosphorylation activity at Ser/Thr residues (Gopalaswamy et al., 2004; Singh et al., 2006) and cooperates with DacB2 to maintain cell wall permeability and integrity (Kandasamy and Narayanan, 2015). However, the absence of structural information of M. tuberculosis PknI restrains our understanding of how the kinase is activated. Here we report the individual high-resolution crystal structures of PknI sensor domain (PknI_SD, in both monomer and dimer forms) and PknI kinase domain (PknI_KD). Inspection of the two structures of PknI_SD revealed conformational changes upon dimerization. Meanwhile, rapamycin-induced dimerization of unphosphorylated fusions of PknI juxtamembrane and kinase Structure 25, 1–9, August 1, 2017 ª 2017 Elsevier Ltd. 1

Please cite this article in press as: Yan et al., Structural Insight into the Activation of PknI Kinase from M. tuberculosis via Dimerization of the Extracellular Sensor Domain, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.010

Figure 1. Crystal Structures of PknI_SD (A) Schematic representation of M. tuberculosis PknI. JMD, juxtamembrane domain; TM, transmembrane helix. (B) Structure of the monomer. The ten b sheets and five large loops are labeled with b1–b10 and L1–L5, respectively. The disulfide bond between C443 and C448 is shown in stick representation and labeled DSB1. The arm region is colored magenta and the rest of the polypeptide colored green. The dark dashed lines represent the disordered region, i.e., residues 491–499 and 528–531. (C) A monomeric unit within the PknI_SD dimer. Here, the arm region is fully extended, allowing for interactions with the adjoining subunit see (D). The two disulfide bonds: C443-C448 and C491-C530 are shown in stick representation and labeled DSB1 and DSB2, respectively. (D) The dimer is formed by domain swapping of the arm region. The second subunit is colored green cyan and the arm region colored orange. The N and C termini of each protomer in structural representations (B–D) are labeled with red ‘‘N’’ and ‘‘C’’, respectively.

(Figure 1A). The trace on gel-filtration showed PknI_SD elutes as two peaks. Analytical ultra-centrifugation (AUC) analysis indicated that these two peaks represent the monomer (
22 kDa) and dimer (
40 kDa) forms of PknI_SD, respectively (Figures S1A and S1B). Crystals for the corresponding monomeric and dimeric fractions of PknI_SD from gel-filtration have also been obtained (Figures S1C and S1D), the structures of which were determined and confirmed the two different assemblies of PknI_SD (Table 1).

domain (PknI_KJD) to mimic the dimerization effect presumably imposed by PknI_SD was observed to be able to activate auto-phosphorylation activity of PknI_KD. Furthermore, in vivo experiments using an M. bovis BCG model suggested that PknI plays an essential role in slowing down the growth of M. tuberculosis within the host, especially under acidic conditions. Thus, this study, for the first time, offers new insight into the PknI signaling pathway by presenting the structural basis for PknI to activate its kinase activity. RESULTS The PknI Sensor Domain Adopts Two Conformations PknI is a transmembrane protein consisting of 585 amino acids subdivided into four subdomains: an intracellular Ser/Thr kinase domain (residues 1–260), the juxtamembrane domain (residues 260–345), a single transmembrane helix (residues 346–371), and an extracellular sensor domain (residues 372–585) 2 Structure 25, 1–9, August 1, 2017

Overall Structures of Monomeric and Dimeric PknI_SD The overall fold of monomeric PknI_SD consists of a twisted b barrel composed of ten b strands and five large loops (Figure 1B). Strand b1 is antiparallel with respect to b2 and b10, and these three strands together form part of the side of the barrel. The bottom of the barrel is sealed by loop 1 (L1) and the open side rim is composed of loops L2–L4, while L5 winds around the outside of the barrel, partially encircling three of the b strands (i.e., b1, b2, and b3) (Figure 1B). One intramolecular disulfide bond between C443 and C448 is observed. Residues 372–401 (at the N terminus of the domain), 491–500 (between b6 and b7), 528–531 (between b9 and b10), and 561–585 (at the C terminus of the domain) are not visible in the electron density map, and therefore were not included in the final model. Dimeric PknI_SD comprises two monomeric subunits stacked on top of each other. The overall fold of the monomeric subunits in the dimer resembles the monomer, with a root-mean-square deviation of 0.6 A˚ for all of the Ca atoms superimposed. There is, however, a striking difference at the N terminus involving loops L1, L2, and strand b1 (residues 402–431, designated as

Please cite this article in press as: Yan et al., Structural Insight into the Activation of PknI Kinase from M. tuberculosis via Dimerization of the Extracellular Sensor Domain, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.010

Table 1. Data Collection, Phasing, and Refinement Statistics PknI_SD (Dimer_SeMet)

PknI_SD (Monomer_Native)

PknI_KD (SeMet)

Space group

I23

P31

P212121

Wavelength (A˚) Resolution (A˚)

0.97916

0.97906

0.97854

2.20

2.20

1.6

a, b, c (A˚)

136.0, 136.0, 136.0

58.8, 58.8, 92.1

45.8, 113.5, 118.5

a, b, g ( )

90.0, 90.0, 90.0

90.0, 90.0, 120.0

90.0, 90.0, 90.0

Completeness (%)a

100 (100)

99.9 (100)

99.9 (100)

Redundancya

10.8 (11.2)

5.6 (5.5)

6.6 (6.8)

Rmergea,b

0.093 (0.824)

0.059 (0.958)

0.066 (0.621)

I/sIa

55.1 (4.2)

28.6 (1.8)

23.3 (2.0)

CC1/2

0.870

0.889

0.836

Data Processing Statistics

Phasing Selenium sites (a.u.)

6

–

12

Figure of meritc

0.42

–

0.36

Refinement Statistics Resolution (A˚)

48.10–2.20

44.56–2.20

37.29–1.60

No. of reflections

21,390

17,140

82,512

Rwork/freed(%)

22.35/25.66

22.07/26.54

16.54/20.00

Protein

2,408

2,229

3,885

Water

59

54

616

Average B factor (A˚2)

58.05

49.80

28.50

Bond lengths (A˚)

0.010

0.003

0.012

Bond angles ( )

1.253

0.627

1.119

No. of atoms

SD (RMSD)e

Ramachandran statistics (%)f Favored

98.07

96.03

97.59

Allowed

1.93

3.61

2.21

Outliers

0

0.36

0.20

a

Numbers in parentheses correspond to the highest-resolution shells. P P b Rmerge = jI  Ij = I ; I, intensity. P P c Figure of merit = hj PðaÞeia j= PðaÞi ; a, phase; P(a), phase probability distribution. a P P a d Rfactor = jjFo j  kjFc jj= jFo j : Fo, observed structure-factor amplitude; Fc, calculated structure-factor amplitude. hkl

hkl

e

Root-mean-square deviation from ideal values. f Validated by MolProbity.

the arm region hereafter). In the dimer structure, L1 and b1 adopt a fully extended conformation (Figure 1C), and simply exchange the interactions they make in the monomer for the same interactions in the dimer, constituting a symmetric domain swapping, a mechanism whereby b1 helps complete the b barrel in the opposing protomer (Figure 1D). Meanwhile, compared with the monomer, L2 in the dimer undergoes an allosteric change and is involved in different hydrogen bond interactions, which is henceforth designated as the hinge loop. Apart from the disulfide bond between C443 and C448, an additional one between C491 and C530 is also observed in the dimer structure (Figure 1C). A search for proteins sharing a similar 3D fold with monomeric PknI_SD was conducted using the Dali Server (Holm and Rosenstrom, 2010), leading to the finding that avidin-like proteins (tetrameric biotin-binding proteins) share the most resemblance

to PknI_SD with a Z score of 8, a value just into the significant range. Conformational Changes between the Two Structures of PknI_SD Superposing the monomer PknI_SD onto one protomer subunit of the dimer form, it can be seen that the shape of L2 changes from a sharp U to a rounded S (Figure 2A). The hinge loop, i.e., L2, in the dimer harbors three different hydrogen bonds, one within the protomer (R417-V492) and two between the opposing protomer subunits (R417-P431 and D423-R538) (Figure 2C), compared with the five hydrogen bonds (R417-Q429, Q420T427, Y422-V425, D423-V536, and Y424-R538) formed in the hinge region in the monomer (Figure 2B). Such an allosteric change stems from the presence of a proline-rich sequence Structure 25, 1–9, August 1, 2017 3

Please cite this article in press as: Yan et al., Structural Insight into the Activation of PknI Kinase from M. tuberculosis via Dimerization of the Extracellular Sensor Domain, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.010

Figure 2. Differences between the Monomeric and Dimeric Structures of PknI_SD (A) Superimposition of the two structures reveals a major difference in the conformation of the hinge loop (L2, residues 416–432). A close-up view of the superimposition of the hinge loops L2 from the monomer (cyan) structure and chain A of the dimer structure (magenta) is shown here, with L2 from chain B in the dimer colored green. (B) Stick representation of the hinge loop in the monomer. Five hydrogen bonds are formed in the hinge region: R417-Q429, Q420-T427, Y422-V425, D423-V536, and Y424-R538. (C) A close-up view of the occurrence of the double peptide bond flip within the hinge loop due to the presence of a proline-rich sequence, as well as two of the three hydrogen bonds formed by the hinge loop in the dimer. Residues are shown in stick representation and colored as in (A). Curved yellow arrows indicate the occurrence of the double peptide bond flip when two PknI_SD monomers (cyan) form a domainswapped dimer (green). In the dimer, the hinge loop harbors three different hydrogen bonds, one within the protomer (R417-V492) and two between the opposing protomer subunits (R417-P431 and D423R538), as opposed to the five hydrogen bonds shown in (B) for the monomer hinge loop. The hydrogen bond between D423 and R538 is not shown here due to the difficulty in properly presenting it. (D) Hydrophobic interactions at the domain-swapped interface in the dimer. The side chains of two residues (I413 and I415) in chain B (green) and nine residues in chain A (magenta) are represented as sticks. (E) 2Fo  Fc electron density map for residues 490– 501 in the dimer, contour level = 1.5 s. (F) Three hydrogen bonds stabilizing the DSB2 region at the dimer interface.

within L2, i.e., 426PTPQPP431, where two peptide bonds can flip from cis- to trans-conformation or vice versa. In the monomer structure, the Q429-P430 peptide bond is in the trans-conformation, as opposed to the cis-conformation in the dimer structure, while for the P430-P431 peptide bond the situation is reversed (Figure 2C). Thus, the clustering of proline residues in this region is an important feature that contributes to the ability of the polypeptide to adopt markedly different conformations. The dimer structure has a higher degree of order than that of the monomer. In the dimer structure, residues 491–500 and 528–531 can be built passably (Figure 2E), while these two regions are primarily disordered in the monomer (Figure 1B). As they are adjacent to the second disulfide bond, i.e., C491C530, we henceforth refer to this as the DSB2 region. This higher degree of order for the dimer could be attributed to the presence of the C491-C530 disulfide bond and three stabilizing hydrogen bonds (V492-R417, P494-N434, and N495-D457), while none of them exists in the monomer (Figure 2F). Overall Fold of PknI_KD Resembles That of Eukaryotic Ser/Thr Kinases The crystal structure of PknI_KD was determined to 1.6-A˚ resolution (Table 1), and structurally resembles eukaryotic Ser/Thr protein kinases that are featured with a bilobal arrangement consisting of an N lobe and a C lobe linked by a hinge region. 4 Structure 25, 1–9, August 1, 2017

In PknI_KD, the smaller N lobe contains six b strands, five of which constitute a curved b sheet. A kinked a helix is also observed between strands b4 and b5 (equivalent to the C helix in eukaryotic kinases). The major feature of the C lobe is a four-helix bundle (a3, a5, a7, and a9) flanked by four small a helices (Figure 3). The signature ATP binding, P-, catalytic and activation loops found in eukaryotic kinases are, in general, conserved in PknI_KD, too. The Arm Region Contributes to the Conformational Change of PknI_SD To specifically identify the critical residues of PknI_SD involved in its conformational switch, a series of truncation and site-directed mutagenesis experiments were performed. Four truncation constructs were made to investigate the role of the arm region: T1, T2, T3, and T4, with residues 372–402, 561–585, 372–402 plus 561–585, and with 372–421 removed, respectively (Figure S2F). The molecular masses of monomer and dimer, and the proportions they hold, were measured by multi-angle light scattering. Wild-type PknI_SD, T1, T2, and T3 samples showed monomer:dimer ratios of 53.4%:46.6%, 43.2%: 56.7%, 80%:20.0%, and 69.7%:30.3%, respectively (Figure S2E), indicating that residues 561–585 (C-terminal) are important for PknI_SD dimerization, while the 31-residue segment near membrane (N-terminal) appears to inhibit dimer

Please cite this article in press as: Yan et al., Structural Insight into the Activation of PknI Kinase from M. tuberculosis via Dimerization of the Extracellular Sensor Domain, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.010

Figure 3. Crystal Structure of PknI_KD (A) A cartoon representation of the overall fold of PknI_KD. The ATP binding P loop is colored green and catalytic loop colored magenta. The activation loop is shown in stick representation and colored by atom type with carbon in yellow. (B) 2Fo  Fc electron density map of the activation loop, contoured at 1.5 s.

formation. Only monomers were present in the T4 sample, with virtually no dimers found in solution, suggesting that residues 403–421 are of critical significance for the formation of the dimer. These experiments verified that the two quaternary conformations derived from the two respective crystal forms are indeed present in solution and the arm region is critically involved in stabilizing the dimer. To investigate the role of the proline-rich sequence, i.e., 426 PTPQPP431, both P430 and P431 were mutated to glycine. Mutants P430G and P431G favored the presence of monomers in solution in comparison with wild-type PknI_SD, showing an increase of
30% and
20% in monomer proportion, respectively. These data suggest that P430 and P431, the two residues forming a peptide bond flip, play important roles in defining the equilibrium between the monomer and dimer forms of PknI_SD (Figure S3A). Dimerization of PknI_KJD Activates its Kinase Activity Since only individual kinase and sensor domains were expressed and purified, it is not possible to directly test the effect of sensor-induced dimerization on the activity of the kinase domain. Nevertheless, a rapamycin system was reported to be utilized for the induction of kinase domain dimerization (Lombana et al., 2010; Banaszynski et al., 2005). Rapamycin can bind FKBP (FK506-binding protein, GEO: 30585002) with subnanomolar affinity, and FRAP (FK506-binding protein rapamycin associated protein, GEO: 19924298) subsequently binds to the FKBP-rapamycin complex, producing a heterodimer (Figure 4B). By fusing FRAP or FKBP to the C-terminal of PknI_KJD (i.e., PknI kinase domain and juxtamembrane domain), the dimerization effect imposed by the extracellular domain can be thus mimicked. The heterogeneously expressed PknI_KJD fusion proteins, KJD_A (FRAP fused to the C-terminal of PknI_KJD) and KJD_B (PknI_KJD fused with FKBP) were used to investigate the kinase activity of PknI (Figure 4A). As PknI_KJD was phosphorylated during expression in E. coli (data not shown), the available phosphorylation sites to be radiolabeled were reduced. To avoid phosphorylation interference derived from E. coli expression,

PknI_KJD fusion proteins were first subjected to dephosphorylation by PstP, a Ser/Thr phosphatase from M. tuberculosis. The dephosphorylated samples of KJD_A, KJD_B with or without rapamycin, as well as KJD_A and KJD_B mixture without rapamycin, exhibited no detectable auto-phosphorylation activity. While as expected, with the addition of rapamycin, the KJD_A and KJD_B mixture achieved a low but sufficient level of auto-phosphorylation activity (Figure 4A). Taken together, dimerization of PknI_KJD can activate the auto-phosphorylation activity of dephosphorylated PknI_KJD. Dimerization of PknI Is Critical for its Cell Growth Inhibition Function Structural analysis of the PknI_SD dimer showed that two residues, I413 and I415, both located within b1, form hydrophobic interactions with residues in the alternate subunit (Figure 2D). Mutagenesis experiments showed that mutating I413 to E, K, or G breaks these interactions, resulting in a conformational transition from dimer to monomer (Figure S3A). Therefore, mutant I413E was used to investigate the impact a monomeric PknI has on cell growth. Growth profiles of wild-type, pknI-knockout (DpknI), pknI-complemented (compknI), and monomeric pknIcomplemented (compknI_I413E) strains of M. bovis BCG were measured with 7H9 medium at both pH 5.6 and 7.0 (Figure 5). At pH 7.0, no obvious difference in growth kinetics has been seen for DpknI, compknI, or compknI_I413E strains, while wildtype strain demonstrated a slower growth rate (Figure 5B). However, at pH 5.6, there was a more profound divergence in growth between DpknI and wild-type strain, with the growth density of DpknI strain almost 2-fold higher than that of wild-type strain by day 9 (Figure 5A). Despite not fully restoring the inhibition effect of PknI on cell growth, compknI strain exhibited a greater level of mycobacterial growth slowdown at pH 5.6 than at pH 7.0. CompknI_I413E strain, on the other hand, appeared to be on par with DpknI strain in growth kinetics. These data combined suggest that PknI is involved in controlling the cell growth of M. tuberculosis by functioning as a dimer, which is more significant under acidic conditions, and the monomeric form of the extracellular sensor domain is unable to activate the intracellular Structure 25, 1–9, August 1, 2017 5

Please cite this article in press as: Yan et al., Structural Insight into the Activation of PknI Kinase from M. tuberculosis via Dimerization of the Extracellular Sensor Domain, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.010

Figure 4. Rapamycin-Induced Dimerization Activates PknI Autophosphorylation Activity (A) A kinase assay using [g-32P]-labeled ATP showed activated auto-phosphorylation activity for the PknI kinase domain. KJD_A is PknI_KJD fused with FRAP at the C terminus; KJD_B is PknI_KJD fused with FKBP; Rap refers to the dimer-inducer rapamycin. (B) Model for the ligand-induced heterodimer system. KJD_B is colored magenta and KJD_A colored green. RBD, rapamycin binding domain; JMD, juxtamembrane domain; KD, kinase domain. JMD is represented with dotted lines. (C) A model for PknI activation in host cells. The sensor and kinase domains of PknI whose structures have been determined in this study, as well as the transmembrane helix, are presented in cartoon representation. Regions where no structural data is presently available are signified by dotted lines. Yellow spheres labeled with ‘‘P’’ refer to phosphorylated Ser, Thr, or Tyr. Red stars refer to potential, yet unidentified, ligands. SD, sensor domain; JMD, juxtamembrane domain; KD, kinase domain. The two transmembrane helices in the dimeric PknI are positioned cross-wise in light of the compositional and functional similarities between STPKs and receptor tyrosine kinases (RTKs), which suggests that the transmembrane helix helices of STPKs possibly have similar traits and properties as those of RTKs that were shown by NMR and molecular dynamic simulation studies to form a crossed dimer within the membrane (Li and Hristova, 2010; Bocharov et al., 2017).

kinase domain, leading to a deficiency in PknI’s capability of slowing down mycobacterial growth. PknI Has a Broad Influence on the Phosphorylation State of the Whole Cell A whole-cell phosphoproteomic assay of both wild-type M. bovis BCG and DpknI strains was performed to measure the phosphorylation events of the whole cell that could possibly be attributable to PknI. The most notable difference observed between the wild-type and the DpknI strain was the breadth of phosphorylation that occurred. In the wild-type strain, 195 sites were phosphorylated, as opposed to 124 sites in the DpknI strain (Table S2), confirming that PknI is an active protein kinase that takes part in regulating a number of cellular processes, including 6 Structure 25, 1–9, August 1, 2017

cell wall synthesis, cell division, gene transcription, protein translation, transport, and protein secretion (Table S2B). The data also suggest that two-component systems, such as DevR-SigA (Gautam et al., 2014) (in response to hypoxia-stress) and oxyS (Domenech et al., 2001) (in response to oxidative-stress), as well as other STPKs, such as PknB and PknA, are influenced by or associated with the PknI pathway. The interplay between these different signaling pathways is a key factor in allowing M. tuberculosis to survive in the harsh environment of host cells. DISCUSSION Studies on eukaryotic receptor tyrosine kinases or receptorlike Ser/Thr kinases have revealed that the force causing

Please cite this article in press as: Yan et al., Structural Insight into the Activation of PknI Kinase from M. tuberculosis via Dimerization of the Extracellular Sensor Domain, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.010

Figure 5. Growth Kinetics for M. bovis BCG Strains Using Media at Two Different pH Values (A) Growth profile at pH 5.6. (B) Growth profile at pH 7.0. The four strains studied are: WT, wild-type M. bovis BCG strain; DpknI, pknI-knockout BCG stain; compknI, pknI-complemented pknI-knockout strain using the integrative plasmid pMV361; compknI_I413E, same as compknI except that I413 in the sensor domain was mutated to a glutamate to produce a monomeric mutant. Mean ± SD.

cytoplasmic domain dimerization and activation is derived from the dimerization of extracellular receptor (Lemmon and Schlessinger, 2010). However, to date, none of the sensor domains of prokaryotic STPKs has been reported to exist in a dimer form or in a monomer-dimer equilibrium, although intracellular kinase domains can form dimers by two mechanisms, either asymmetric dimerization (Mieczkowski et al., 2008; Zhang et al., 2006) or back-to-back dimerization via the N lobe region (Greenstein et al., 2007), as observed in the case of PknB, PknE, and PknD from M. tuberculosis (Lombana et al., 2010; Greenstein et al., 2007; Young et al., 2003; Gay et al., 2006). PknI is the first STPK identified so far whose sensor domain exists in a monomer-dimer equilibrium and can form a domain-swapped dimer. Interface analysis of the PknI_SD dimer by PDBePISA (Krissinel and Henrick, 2007) showed that the domain-swapping interface is the most stable and significant interface. In addition, site-directed mutagenesis of residues at the domain-swapped interface and in the hinge region (i.e., the proline-rich loop L2) resulted in a shift in the monomer-dimer equilibrium, suggesting that rather than being an artifact due to crystal packing, the domain-swapped dimer observed in the crystal structure does exist in solution. Moreover, in our study, in vivo experiments concerning the growth kinetics of monomeric mutant (I413E) and pknI-knockout strains of M. bovis BCG manifested the biological relevance of the domain-swapping interface in the PknI_SD dimer. Although the domain swapping causes the N termini of the two protomers in the PknI_SD dimer structure to be 75 A˚ apart, it is worth mentioning that there are still
30 amino acids unmodeled between the N-terminal of PknI_SD and the C-terminal of PknI transmembrane helix, implying a chance for the two N termini in a domain-swapped PknI_SD dimer to approach each other and connect to the C termini of the transmembrane helices. Therefore, despite the distance between the two N termini of the

dimer due to domain swapping, PknI_SD is still very likely to be able to mediate the dimerization of the kinase domains of PknI. The growth difference observed between M. bovis BCG wildtype and pknI-knockout DpknI in this study is more pronounced under acidic conditions than at neutral pH values, suggesting the importance of an acidic host environment in activating PknI (Figure 5). According to a proposed energetic model for the formation of 3D domain swapping, there is a high energy barrier between the closed monomers and a domain-swapped dimer or oligomer (Bennett et al., 1995), which can be reduced under certain circumstances, such as change of pH, mutation in the protein, and binding of a ligand (Liu and Eisenberg, 2002; Rousseau et al., 2001). We thus hypothesized that pH may be a factor that affects the PknI_SD monomer-dimer equilibrium. However, our experiment measuring the PknI_SD monomer:dimer ratios at different pH values (pH 4.0–8.0) by AUC showed only modest shift from monomeric PknI_SD toward the dimer form (Figure S4). It could possibly be that pH still needs the assistance of some other signals or a certain co-factor in vivo to mediate the switch between the monomer and dimer states of PknI to a greater extent. Eukaryotic receptor-like kinases, even when auto-inhibited, are thought to have a low, but sufficient level of kinase activity to trans-phosphorylate their partners (Lemmon and Schlessinger, 2010). This conjecture has been verified by a previous study on the PknD kinase showing that the dephosphorylated PknD_KJD monomer retains auto-kinase activity (Greenstein et al., 2007). However, in the case of PknI, there is arguably no detectable activity associated with dephosphorylated PknI_KJD monomer. Even after dimerization induced by rapamycin, the auto-phosphorylation activity of dephosphorylated PknI_KJD is still relatively low, just at about the same level as that of PknD_KJD monomer (Gopalaswamy et al., 2004), implying that Structure 25, 1–9, August 1, 2017 7

Please cite this article in press as: Yan et al., Structural Insight into the Activation of PknI Kinase from M. tuberculosis via Dimerization of the Extracellular Sensor Domain, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.010

PknI activates and maintains its kinase activity at a basic level via dimerization. Indeed, compknI_I413E M. bovis BCG that expressed only monomeric PknI_SD demonstrated growth kinetics quite similar to the pknI-knockout strain (DpknI), both of which grew faster than the wild-type, suggesting the necessity of dimeric PknI_SD for the proper function of PknI. Reports on receptor tyrosine kinases have revealed that a subset of them can form dimers or oligomers in the absence of ligand (Ward et al., 2007; Clayton et al., 2005). Nevertheless, the ligand is still required for full kinase activity (Lemmon and Schlessinger, 2010). Therefore, on top of dimerization, PknI presumably also undergoes ligand-induced conformational change to fully activate its kinase activity. Taken together, here we propose a mechanism by which PknI plays its part in assisting M. tuberculosis in invading macrophages. In the absence of stimuli, PknI exists in two states: a monomer form with no activity and a domain-swapped dimer form with a basic level of auto-phosphorylation activity. The monomer-dimer equilibrium of PknI is mediated by the double peptide bond flips within the hinge loop in the sensor domain. After M. tuberculosis has been engulfed by macrophages, internal signals, low pH for instance, could help overcome the energy barrier between the PknI_SD monomer and domain-swapped dimer, thus shifting the monomer-dimer equilibrium and leading to the formation of more dimeric PknI, which is intended to activate and maintain its kinase activity, albeit at a basic level. Once allosteric effects derived from extracellular ligand binding are transduced into the cytoplasm KJD through the transmembrane helix, PknI is fully activated, and in this moment well positioned to help M. tuberculosis escape host immune system attack and establish chronic infection by slowing down cell growth (Figure 4C). STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d d

d d

KEY RESOURCE TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS METHOD DETAILS B Genetic Manipulation B Protein Expression and Purification B Crystallization and Data Collection B Phasing, Model Building and Refinement B Multi-Angle Light Scattering B Analytical Ultracentrifugation B In Vitro Growth Kinetics B Kinase Assays B Phosphoproteomic Measurement of M. bovis BCG Strains QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND SOFTWARE AVAILABILITY

AUTHOR CONTRIBUTIONS Z.R. designed the research. Q.Y. and L.Q. performed the study. D.J., Q.Z., W.Z., W.Z., K.M., and H.Y. analyzed the data. Z.R., L.G., Q.Y., and D.J. wrote the paper. All authors read and approved the final manuscript. ACKNOWLEDGMENTS We are grateful to the scientific and technical staff at Beamline 17U and 19U of SSRF (China) and Beamline 5A of Photon Factory (Japan) for assistance with data collection. This work was supported by grants from the State Key Development Program for Basic Research of the Ministry of Science and Technology of China (973 Project grant numbers 2014CB542800, 2014CBA02003), and the National Natural Science Foundation of China (grant numbers 81330036, 81520108019). Received: March 6, 2017 Revised: April 27, 2017 Accepted: June 16, 2017 Published: July 13, 2017 REFERENCES Adams, P.D., Afonine, P.V., Bunkoczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W., et al. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221. Afonine, P.V., Grosse-Kunstleve, R.W., Echols, N., Headd, J.J., Moriarty, N.W., Mustyakimov, M., Terwilliger, T.C., Urzhumtsev, A., Zwart, P.H., and Adams, P.D. (2012). Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367. Banaszynski, L.A., Liu, C.W., and Wandless, T.J. (2005). Characterization of the FKBP.rapamycin.FRB ternary complex. J. Am. Chem. Soc. 127, 4715–4721. Bennett, M.J., Schlunegger, M.P., and Eisenberg, D. (1995). 3D domain swapping: a mechanism for oligomer assembly. Protein Sci. 4, 2455–2468. Bocharov, E.V., Bragin, P.E., Pavlov, K.V., Bocharova, O.V., Mineev, K.S., Polyansky, A.A., Volynsky, P.E., Efremov, R.G., and Arseniev, A.S. (2017). The conformation of the epidermal growth factor receptor transmembrane domain dimer dynamically adapts to the local membrane environment. Biochemistry 56, 1697–1705. Bretl, D.J., Demetriadou, C., and Zahrt, T.C. (2011). Adaptation to environmental stimuli within the host: two-component signal transduction systems of Mycobacterium tuberculosis. Microbiol. Mol. Biol. Rev. 75, 566–582. Chen, V.B., Arendall, W.B., 3rd, Headd, J.J., Keedy, D.A., Immormino, R.M., Kapral, G.J., Murray, L.W., Richardson, J.S., and Richardson, D.C. (2010). MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21. Clayton, A.H., Walker, F., Orchard, S.G., Henderson, C., Fuchs, D., Rothacker, J., Nice, E.C., and Burgess, A.W. (2005). Ligand-induced dimer-tetramer transition during the activation of the cell surface epidermal growth factor receptor-A multidimensional microscopy analysis. J. Biol. Chem. 280, 30392–30399. Domenech, P., Honore, N., Heym, B., and Cole, S.T. (2001). Role of OxyS of Mycobacterium tuberculosis in oxidative stress: overexpression confers increased sensitivity to organic hydroperoxides. Microbes Infect. 3, 713–721. Elks, P.M., Brizee, S., van der Vaart, M., Walmsley, S.R., van Eeden, F.J., Renshaw, S.A., and Meijer, A.H. (2013). Hypoxia inducible factor signaling modulates susceptibility to mycobacterial infection via a nitric oxide dependent mechanism. PLoS Pathog. 9, e1003789.

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Structure 25, 1–9, August 1, 2017 9

Please cite this article in press as: Yan et al., Structural Insight into the Activation of PknI Kinase from M. tuberculosis via Dimerization of the Extracellular Sensor Domain, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.010

STAR+METHODS KEY RESOURCE TABLE REAGENT or RESOURCE

SOURCE

IDENTIFIER

Bacterial and Virus Strains M. smegmatis mc2155

Gift from W.R. Jacobs

N/A

E.coli B834 (DE3)

Novagen

Cat#69041-3CN

E.coli HB101

Promega

Cat#L201B

Temperature-sensitive phage phAE159

Gift from W.R. Jacobs

N/A

Chemicals, Peptides, and Recombinant Proteins TiO2 Mag Sepharose magnetic beads

GE healthcare

Cat# 28-9440-10

EASY-Spray column

ThermoFisher

Cat#ES801

FRAP

Genewiz

N/A

FKBP

Gift from TJAB

N/A

pYU1471

Gift from W.R. Jacobs

N/A

Trypsin

Promega

Cat#V528A

L-(+)-Selenomethionine

Sangon Biotech

Cat# A601194

ATP Gamma 32P

PerkinElmer

Cat# NEG502A250UC

Critical Commercial Assays Fast Mutagenesis System

TransGen

Cat#FM111-01

MaxPlaxTM lambda packaging extract

Epicenter

Cat#MP5105

Crystallization Screens Index

Hampton Research

Cat#HR2-144

One Step Cloning Kit

Vazyme Biotech

Cat#C112-02

Crystal structure: PknI_KD

This paper

PDB ID:5XKA

Crystal structure: PknI_SD dimer form

This paper

PDB ID:5XLL

Crystal structure: PknI_SD monomer form

This paper

PDB ID:5XLM

Gift from W.R. Jacobs

N/A

pNSD: N-terminal 6His-tagged PknI SD

This paper

N/A

pGKD: N-terminal GST-tagged PknI KD

This paper

N/A

pGKJD: N-terminal GST-tagged PknI KJD

This paper

N/A

KJD_A: FRAP fused to C-terminal of pGKJD

This paper

N/A

KJD_B: FKBP fused to C-terminal of pGKJD

This paper

N/A

HKL 2000

(Otwinowski and Minor, 1997)

http://www.hkl-xray.com/hkl-2000

PHENIX

(Adams et al., 2010)

https://www.phenix-online.org/

Coot

(Emsley et al., 2010)

http://www2.mrc-lmb.cam.ac.uk/Personal/ pemsley/coot/

GraphPad Prism 5

GraphPad Software, Inc.

http://www.graphpad.com/ scientific-software/prism/

Deposited Data

Experimental Models: Organisms/Strains M. bovis BCG str. Pasteur 1173P2 Recombinant DNA

Softwares and Algorithms

CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for reagents may be directed to and will be fulfilled by the Lead Contact, Zihe Rao (raozh@nankai. edu.cn). EXPERIMENTAL MODEL AND SUBJECT DETAILS Strains used in Growth Curve Determination M. bovis BCG (str. Pasteur 1173P2) were cultured in Middlebrook 7H9 medium supplemented with albumin dextrose saline (ADS). e1 Structure 25, 1–9.e1–e4, August 1, 2017

Please cite this article in press as: Yan et al., Structural Insight into the Activation of PknI Kinase from M. tuberculosis via Dimerization of the Extracellular Sensor Domain, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.010

METHOD DETAILS Genetic Manipulation PknI Kinase and Sensor Domain Used for Structural Analysis The genes encoding the cytoplasmic kinase domain (residues 1-260, PknI_KD) and extracellular sensor domain (residues 372-585, PknI_SD) were amplified from the genome of M. tuberculosis H37Rv by polymerase chain reaction (PCR), respectively. Primers used for PCR are listed in the Table S1. DNA fragment of PknI_KD obtained from PCR was sub-cloned into pGEX6p-1 plasmid to produce an N terminal GST-tagged pGKD construct. In contrast, PknI_SD was constructed as an N-terminal 63His-tagged fusion protein (designated as pNSD) using the pET28a vector. Four truncation mutants of PknI_SD, T1, T2, T3, and T4 with residues 372-402, 561-585, 372-402 and 561-585, and 372-421 removed, respectively, were amplified using the full-length pNSD construct as a template. The four truncated constructs were also sub-cloned into the pET-28a vector. Single substitutions in PknI_SD were introduced with TransGen’s fast mutagenesis system following the manufacture’s protocol, resulting in PknI_SD mutants (I413G, I413E, I413K, I415G, I415E, I415K, P430G, P431G). Full-length pknI mutant I413E was also constructed by the use of TransGen’s fast mutagenesis system. PknI_KJD Fusions Used for Kinase Assay The coding sequence for the conjugate of PknI kinase domain and part of juxtamembrane domain (PknI_KJD), corresponding to the first 312 amino acids of PknI, was amplified by PCR from the genome of M. tuberculosis H37Rv. A pGEX6p vector linearized by PCR was fused with PknI_KJD via homogenous recombination with One Step Cloning Kit following the manufacture’s protocol to produce an N terminal GST-tagged pGKJD construct. The FKBP coding sequence was kindly provided by Tianjin International Joint Academy of Biotechnology & Medicine. The DNA sequence encoding the minimal functional domain of FRAP (FRB domain) was synthesized by Genewiz. The FKBP and FRAP coding sequences were separately conjugated to the C terminal of PknI_KJD domain in the pGKJD plasmid, resulting in PknI_KJD fusions (KJD_A and KJD_B). PstP Used for Kinase Assays The coding sequence for the only known phosphoserine/threonine phosphatase PstP from M. tuberculosis (corresponding to the first 268 amino acids of PstP), which was used to dephosphorylate the heterogeneously expressed PknI_KJD fusions, was amplified from the genome of M. tuberculosis H37Rv, and subcloned into pGEX6p-1. Construction of pknI-Knockout and Complemented M. Bovis BCG Strains A pknI-knockout strain was constructed using Mycobacteriophage-based specialized transducing phages (STPs) (Jain et al., 2014; Hu et al., 2015). Briefly, the upstream and downstream regions flanking the pknI gene were amplified from M. bovis BCG genomic DNA using upstream primer pair LL and LR, and downstream primer pair RL and RR, respectively (Figure S5 and Table S1). The corresponding PCR products were simultaneously inserted into the pYUB1471 vector using the PflMI restriction site to produce an allelic exchange substrate (AES), pYUB1471-DpknI, which confers hygromycin resistance. Temperature-sensitive phage phAE159 and pYUB1471-DpknI were both digested with PacI and then ligated. The ligated product was packaged in vitro via the lambda cos site present in the AES to produce a STP by using MaxPlax packaging extract (Epicenter Biotechnologies, USA). The resultant STP was then transformed into E. coli HB101 cells to screen for hygromycin-resistant clones which were intended for the isolation of successfully constructed STP plasmid that was subsequently transduced into M. smegmatis mc2155 at 30  C to obtain high titer STP phage stocks. Deletion-substitution mutation was performed by mixing high titer STP phage with M. bovis BCG at an MOI of 10:1 at 37  C. PknI gene deletion was confirmed by PCR using primers indicated in Figure S5 and Table S1. Complemented M. bovis BCG strain compknI was obtained by transforming the knockout strain with the integrative vector pMV361-pknI constructed by cloning full-length pknI into the integrative vector pMV361 using primer pair PknI_F and PknI_R. Complemented strain compknI_I413E was acquired in the same way, except that residue I413 in full-length pknI inserted into pMV361 was mutated to Glutamate. Protein Expression and Purification PknI Sensor Domain PknI_SD wild-type or mutants were all expressed in E. coli BL21 (DE3) with LB media supplemented with kanamycin (50 mg/ml) at 37  C until the OD600 reached
0.6. Protein expression was induced by the addition of 0.5 mM isopropyl b-D-1-thiogalactopyranoside at 16  C for
18 h. The selenomethionine-substituted PknI_SD protein was overexpressed in methionine auxotrophic E. coli strain B834 (DE3) with M9 minimal medium as previously described (Hendrickson et al., 1990) . All PknI_SD proteins were purified using the same protocol as follows: cells were harvested by centrifugation, and the obtained cell pellet was re-suspended in buffer R (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM b-mercaptoethanol, 0.5 mM EDTA and 10% glycerol), followed by lysis with sonication. Cell debris and unbroken cells were removed by centrifugation at 18,000 rpm for 40 min at 4  C. Supernatant containing soluble target protein was then loaded twice onto a 2 ml nickel-nitrilotriacetic acid affinity column (GE Healthcare) equilibrated with buffer R. The affinity column was subsequently washed in buffer W30 (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5% glycerol and 30 mM imidazole), followed by protein elution with buffer W300 (buffer W30 supplemented with 300 mM imidazole). PknI_SD proteins were further purified by anion exchange chromatography using a Resource S column (GE Healthcare) and gel-filtration chromatography using a Superdex200 column (GE Healthcare). All purified protein products were concentrated to 15 mg/mL in buffer C (30 mM HEPES, pH 7.0, 150 mM NaCl) prior to aliquoting and storage at -80  C until use.

Structure 25, 1–9.e1–e4, August 1, 2017 e2

Please cite this article in press as: Yan et al., Structural Insight into the Activation of PknI Kinase from M. tuberculosis via Dimerization of the Extracellular Sensor Domain, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.010

PknI Kinase Domain The expression of native or selenomethionine-substituted PknI_KD protein followed the same protocol used for wild-type or selenomethionine-substituted PknI_SD protein expression, respectively. GST affinity chromatography was chosen for the preliminary purification of PknI_KD protein (both native and selenomethioninesubstituted variant) which was constructed as a GST-fusion protein. Cells were harvested and re-suspended in buffer L (30 mM HEPES, pH 7.0, 150 mM NaCl, 10 mM b-mercaptoethanol, 0.5 mM EDTA and 10% glycerol), followed by lysis with sonication. Cell debris and unbroken cells were removed by centrifugation, and supernatant containing soluble target protein was then loaded onto a GST affinity column (GE Healthcare) and washed in 60 mL of phosphate buffer saline (PBS). The GST tag was removed with PreScission Protease (PPase), and PknI_KD was further purified by gel-filtration using a Superdex200 column equilibrated with buffer C. The protein was subsequently concentrated to 10 mg/mL prior to storage at -80  C until use. PknI_KJD Fusions and PstP PstP was expressed and purified using the same protocol as that for PknI_KD. Cell lysate containing the GST-tagged PstP protein was sequentially subjected to purification by GST affinity chromatography and gel-filtration chromatography. Expression and purification of KJD_A and KJD_B were carried out following the same protocols as those for PknI_KD. Briefly, target proteins were expressed in E. coli BL21 (DE3). Cells were harvested and lysed with sonication. Following centrifugation, supernatant containing soluble target protein was first purified on a GST affinity column. The GST tag was removed from the target protein prior to gel-filtration purification. Purified KJD_A and KJD_B products were dephosphorylated via incubation at 4  C for
7 hours with PstP at a ratio of 5:1 (w/w) with 5 mM MnCl2 present. PstP was removed by reloading dephosphorylated KJD_A and KJD_B samples onto a GST affinity column, and flow-through was collected for kinase assay. Crystallization and Data Collection Two PknI_SD crystal forms were found under two different conditions using the vapor diffusion method by mixing 1 ml of protein solution and 1 ml of reservoir solution. The first crystal form, crystallized from the elution peak corresponding to dimeric PknI_SD from gel-filtration (Figure S1C), was grown under a condition containing 1.1 M sodium malonate (pH7.0), 0.1 M HEPES (pH 7.0), 0.5% v/v Jeffamine ED-2001 (pH 7.0). Crystals of this form were obtained for both native and selenomethionine-substituted PknI_SD proteins. Crystals were cryoprotected before shipping for data collection. A SAD dataset was collected to 2.20 A˚ at the wavelength of 0.97916 A˚ at 100 K with a selenomethionyl derivative crystal of PknI_SD on Beamline 17U of Shanghai Synchrotron Radiation Facility (SSRF, China). The second crystal form of PknI_SD (Figure S1D), derived from the monomeric elution peak from gel-filtration, was acquired under a condition containing 1 M succinic acid (pH 7.0), 0.1 M HEPES (pH 7.0), 1% w/v polyethylene glycol monomethyl ether 2,000. A native dataset for this second crystal form was collected to 2.20 A˚ on Beamline 17U of SSRF (China). Crystallization of PknI_KD protein was also performed using the vapor diffusion method by mixing 1 ml of protein solution and 1 ml of reservoir solution. Crystals of selenomethionine-substituted PknI_KD were grown under a condition containing 0.1 M HEPES, pH 7.5, 3 M NaCl. A SAD dataset was collected to 1.60 A˚ on Beamline 19U of SSRF with a cryoprotected crystal. All datasets were processed with the HKL2000 suite of programs (Otwinowski and Minor, 1997) (Table 1). Phasing, Model Building and Refinement The SAD dataset collected from the first crystal form (dimer) of PknI_SD was used to calculate initial crystallographic phases with the single-wavelength anomalous dispersion (SAD) method using Phenix AutoSol Wizard (Adams et al., 2010; Terwilliger et al., 2009). Six selenium atoms were located, and refinement of heavy atom parameters and phase calculation resulted in a figure of merit of 0.42. Automated model building was then performed with Phenix.autobuild (Terwilliger et al., 2008). The atomic model obtained was subsequently subjected to iterative cycles of manual model adjustment with Coot (Emsley et al., 2010) and refinement with Phenix.refine (Afonine et al., 2012). The final structure model was refined to an Rfree value of 25.66%, with 317 residues and 59 water molecules located, while residues 372-401 and 561-585 were not included due to poor visibility in the electron density map. The structure of PknI_SD monomer was determined by molecular replacement with Phaser (McCoy et al., 2007) using a truncated monomeric model (the arm region removed) of PknI_SD dimer as a search template. The output model was then rebuilt by the use of Phenix.autobuild (Terwilliger et al., 2008). The final monomer structure model was refined to an Rfree value of 26.54%, with 288 residues and 54 water molecules located. Residues 372-401, 491-500, 528-531 and 561-585 were not visible in the electron density map, and thus were excluded from the model. The crystal structure of PknI_KD was also solved with the SAD method following the same procedure employed for the determination of PknI_SD dimer structure. The final structure model of PknI_KD was refined to an Rfree value of 20.00%, with 502 residues and 616 water molecules located, while residues 148-152 could not be traced in the electron density map and were excluded from the model. All atomic models were judged to have good stereochemistry according to the Ramachandran plot calculated by MolProbity (Chen et al., 2010). The phasing and refinement statistics are summarized in Table 1. Multi-Angle Light Scattering Standard multi-angle light scattering experiments were carried out at room temperature with a DAWN HELEOS II system which contains 18-angle static multi-angle light scattering detectors connected in-line to an Optilab rEX refractometer (Wyatt Technology e3 Structure 25, 1–9.e1–e4, August 1, 2017

Please cite this article in press as: Yan et al., Structural Insight into the Activation of PknI Kinase from M. tuberculosis via Dimerization of the Extracellular Sensor Domain, Structure (2017), http://dx.doi.org/10.1016/j.str.2017.06.010

Corporation) and an HPLC system (Agilent 1100) with a Superdex200 column attached. One hundred ml of samples at 5 mg/ml was injected at a flow rate of 0.5 ml/min onto the column equilibrated with 20 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM DTT. Sample molecular weights were determined using the Astra software package version 6 (Wyatt Technology Corporation). Analytical Ultracentrifugation Sedimentation Velocity analysis of PknI_SD was performed using a Beckman Coulter XL-I Optima analytical ultracentrifuge equipped with an An60Ti rotor. PknI_SD protein was first divided into five aliquots, which were subsequently incubated in five different solutions at pH 4.0, 5.0, 6.0, 7.0 and 8.0, respectively. The solutions were composed of 30 mM buffer (citrate acid pH 4.0, citrate acid pH 5.0, MES pH 6.0, HEPES pH 7.0 or HEPES pH 8.0), 150 mM NaCl. Each incubation with a starting concentration of 2 mg/ml were further diluted 100-fold in the five respective solutions and incubated for another 24 hours at room temperature. Afterwards, the samples were concentrated to 2 mg/ml, and sedimentation velocity data were collected every 6 min at 42,000 rpm using interference optics at 4  C. The data acquired were analyzed using Ultrascan v.7.2 software (Figure S4). The experiments were repeated three times. In Vitro Growth Kinetics The growth kinetics of M. bovis BCG strains at pH 5.6 and pH 7.0 were determined in 50 ml of Middlebrook 7H9 medium supplemented with ADS (albumin dextrose saline) in 100 ml flasks by monitoring the OD600 of cell suspension as described previously (Li et al., 2015). Briefly, logarithmic phase M. bovis BCG cultures (OD600 of
0.5) of all tested strains were diluted to an OD600 of 0.1 and then allowed to grow at 37  C with shaking at 110 opm. The OD600 of cell cultures for each BCG strain variant was monitored every 24 hours until it reached 2.0. Tests of growth kinetics were repeated three times. Kinase Assays Kinase assays were performed in buffer E (30 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM MnCl2, 0.5 mM DTT, and 5% glycerol). Each kinase sample was added to a final concentration of 4 mM. Excess rapamycin (at a final concentration of 10 mM) in DMSO was added to each reaction. The mixture was incubated for 30 min at 4  C, and the reaction was initiated by adding 1 ml of [g-32P] ATP (1mCi/ml) in 2 mM cold ATP. The final reaction volume was 30 ml. The reaction mix were incubated at 37  C for 60 min before being quenched with 53SDS-PAGE loading dye and separated by SDS-PAGE on 12% polyacrylamide gels. The gels were dried, and radio activity was quantified with a Typhoon FLA9500 Phosphor Imager (GE Healthcare). All assays were repeated three times. Phosphoproteomic Measurement of M. bovis BCG Strains Wild-type and pknI-knockout M. bovis BCG strains were grown in Middlebrook 7H9 medium supplemented with ADS, and culture samples were collected at the same growth stages (i.e. at the log phase with an OD600 of
0.6). A total of 2 mg of protein extract was dissolved in 50 mM ammonium bicarbonate, and subsequently reduced and alkylated by adding 25 mM DTT and 50 mM idoacetamide (final concentration). Proteins were further digested with sequencing grade-modified trypsin (Promega, Fitchburg, WI). The digested fragments were fractionated with 0.1% Trifluoroacetic acid (TFA) in different concentrations of acetonitrile (ACN) by HPLC. Phosphorylated peptides were enriched using TiO2 Mag Sepharose magnetic beads (GE healthcare) according to the manufacturer’s instructions. In LC-MS/MS analysis, digest products were separated by a 60 min gradient elution at a flow rate 0.300 ml/min with the EASY-nLC 1000 system which was directly interfaced with the Thermo orbitrap fusion mass spectrometer. The analytical column was pursed from Thermofisher (EASY-Spray column, 15cm350 mm ID, Thermofisher USA). Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 100% ACN and 0.1% formic acid. The orbitrap fusion mass spectrometer was operated with Xcalibur3.0 software in the data-dependent acquisition mode consisting of a single full mass spectrum scan in the Orbitrap (350-1550 m/z, 120,000 resolution) followed by top-speed MS/MS scans in the Ion-trap. The MS/MS spectra from each LC-MS/MS run were searched against the M. bovis BCG strain.fasta from UniProt database using Proteome Discovery searching algorithm (version 1.4). QUANTIFICATION AND STATISTICAL ANALYSIS Data of the monomer to dimer mass ratios (Figures S3 and S4) are represented as mean ± SD from three independent experiments. Statistical analysis was performed using Graphpad Prism (Version 5.01, Graphpad Software, Inc.) DATA AND SOFTWARE AVAILABILITY The atomic coordinates of PknI kinase domain and PknI sensor domain (both dimer and monomer forms) reported in this paper are deposited to the Protein Data Bank under the accession codes PDB 5XKA, 5XLL and 5XLM, respectively.

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