1. Volume 25, Issue 8, Pages 1286-1294.e4 (August 2017)
Structural Insight into the Activation of PknI Kinase from M. tuberculosis via Dimerization of the Extracellular Sensor Domain 
Qiaoling Yan, Dunquan Jiang, Lanfang Qian, Qingqing Zhang, Wei Zhang, Weihong Zhou, Kaixia Mi, Luke Guddat, Haitao Yang, Zihe Rao 
Structure 
Volume 25, Issue 8, Pages e4 (August 2017)
DOI: /j.str
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2. Structure 2017 25, 1286-1294.e4DOI: (10.1016/j.str.2017.06.010)
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3. 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 β sheets and five large loops are labeled with β1–β10 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.
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4. 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 domain-swapped 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 D423-R538), 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 σ.
(F) Three hydrogen bonds stabilizing the DSB2 region at the dimer interface.
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5. 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 σ.
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6. Figure 4
Rapamycin-Induced Dimerization Activates PknI Autophosphorylation Activity
(A) A kinase assay using [γ-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).
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7. 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; ΔpknI, 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.
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