Data collection and refinement statistics.

α<p>Values in parentheses are for highest resolution shell.</p>β<p>r.m.s., root mean square.</p

Domain structure of <i>S. aureus</i> PknB.

<p>The kinase region (PknB_SA-KD) is shown in orange. TM: transmembrane domain, PASTA: penicillin-binding protein and serine/threonine kinase associated domains.</p

Analysis of conservation of PknB residues.

<p>Surface representation of PknB_SA-KD. The three views differ by rotations of 120° and 240°, respectively, around a vertical axis. The coloring is based on an alignment of 24 bacterial STKs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039136#pone.0039136.s006" target="_blank">Fig. S6</a>). Blue indicates highly conserved residues (100–91% conservation in dark blue, 87–70% conservation in blue, and 66–54% light blue). Residues that are highly conserved in most kinases but are different in PknB_SA-KD are colored in orange (83–70% conservation in orange, 66–54% conservation in light orange). In the right panel, the benzamidine bound to PknB_SA-KD in three of the six chains of the asymmetric unit is shown as a stick model. The benzamidine is not visible in the other two panels.</p

Stereo views of the activation site of PknB_SA-KD.

<p>(A). The PknB structure is shown in light orange. It was superimposed onto the kinase structures of active PKA (grey, PDB ID: 1ATP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039136#pone.0039136-Zheng1" target="_blank">[35]</a>) and inactive c-Src (purple, PDB ID: 2SRC <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039136#pone.0039136-Xu1" target="_blank">[36]</a>) using C-lobe residues 100–250. The highly conserved residues Lys39, Glu58, Asp151 and Phe152 of PknB_SA-KD are highlighted as red sticks. The latter two residues are part of the DFG-motif. Corresponding residues Lys72, Glu91, Asp184 and Phe185 of PKA, as well as the backbone of PKA and ATP, are colored in grey. Corresponding residues Lys295, Glu310, Asp404 and Phe405 of c-Src, as well as the backbone of c-Src and ATP, are colored in purple. Mn²⁺ ions in the PKA structure are shown as small gray spheres. (B). Close-up view of the β-sheet formed by β6 and β9 in active kinases such as PKA. The colors are the same as in A. The β-sheet in PKA is represented with triangles as the β-strands only consist of two residues each. The red part of PknB_SA-KD represents residues Ile129, Val130, Lys156 and Ala157, which are the residues that would form strands β6 and β9 in the active protein.</p

Analysis of PknB_SA-KD crystal contacts.

<p>(A). Footprint of contacts between a symmetry mate of molecule C (C′) and molecule A in the crystals. Molecule A is shown in surface representation. Areas within black lines indicate crystal contacts with molecule C′ (distance <4.5 Å). Areas within yellow lines indicate the residues involved in dimer formation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039136#pone.0039136-Lombana1" target="_blank">[22]</a>. The surface conservation is shown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039136#pone-0039136-g007" target="_blank">Fig. 7</a>, and the benzamidine is bound to chain A of PknB_SA-KD is shown as a red stick model. Molecule pairs B/B′ and C′/A form similar crystal contacts. (B). Detailed view into the dimer interface formed by the A/C′ dimer. Chains A and C′ of PknB_SA-KD are shown in orange and yellow, respectively. Chains A and B of dimeric <i>M. tuberculosis</i> PknB (PDB ID: 1MRU <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039136#pone.0039136-Young1" target="_blank">[24]</a>) are in grey. Residues Arg9, Tyr10, Leu32 and Asp75, which are involved in dimer formation, are represented with sticks. These residues were also used for superimposing the two dimers. (C). Overview of the orientation of the dimer of <i>M. tuberculosis</i> PknB and PknB_SA-KD. The colors are the same as in panel B. The N-lobes of PknB_SA-KD chain A and <i>M. tuberculosis</i> PknB chain A were superposed. (D). Crystal contact involving chains D and E in PknB_SA-KD. The orientation of chain D is the same as that of chain A in panel C.</p

The C- and R-spine regions of PknB_SA-KD.

<p>(A, B). Overview of the location of the two spines in PknB_SA-KD. The C-spine is colored in green, the R-spine in the background in blue. The two views differ by the indicated rotation to provide a better view of the orientation and location of the R-spine. (C). Detailed view of the residues belonging to the C-spine of PknB_SA-KD and the adenine of AMP-PNP as part of the spine are shown in green. The residues of the C-spine of PKA in a closed state (PDB ID: 1ATP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039136#pone.0039136-Zheng1" target="_blank">[35]</a>) are shown for comparison. (D). Detailed view of the R-spine residues of PknB_SA-KD in blue. Corresponding residues of PKA are shown in grey. While the spine is formed in PKA, it is interrupted by the Ser62 and placed away from ideal position in PknB_SA-KD. The structure of PKA in panels C and D was aligned with PknB_SA-KD C-lobe residues 100–250.</p

Activity test of PknB_SA-KD.

<p>PknB_SA-KD (25 ng) was incubated either with myelin basic protein (MBP; 1 µg) (A) or alone (B) together with γ³³-ATP, MnCl₂ and MgCl₂ for the time indicated. Position and size (kDa) of molecular weight markers are indicated on the left side. Phosphorylation of MBP (A) and autophosphorylation (B) are visualized by autoradiography using direct-exposure film. The phosphorylation rate is increasing as a function of time in both experiments, demonstrating that the purified PknB_SA-KD protein is active.</p

Overall structure of PknB_SA-KD in complex with AMP-PNP.

<p>The two views differ by a rotation of 45° around a vertical axis. The AMP-PNP ligand is located in the cleft between the N- and the C-lobe. Due to its high flexibility, the terminal phosphate group of AMP-PNP is not visible in the electron density and is therefore not shown here.</p

Structure-based sequence alignment.

<p>Structure-based sequence alignment of the <i>S. aureus</i> PknB kinase domain with the kinase domains of <i>B. subtilis</i> PrkC (no structure available), <i>M. tuberculosis</i> PknB (PDB ID: 1MRU <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039136#pone.0039136-Young1" target="_blank">[24]</a>), murine cAMP dependent Protein Kinase A (PDB ID: 1ATP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039136#pone.0039136-Zheng1" target="_blank">[35]</a>; PDB ID: 1CTP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039136#pone.0039136-Karlsson1" target="_blank">[34]</a>) and human tyrosine protein kinase c-Src (PDB ID: 2SRC <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039136#pone.0039136-Xu1" target="_blank">[36]</a>). The secondary structure of PknB_SA-KD is shown above the alignment and the numbering of the sequences corresponds to <i>S. aureus</i> as well. The HRD- and DFG-motifs and the glycine-rich loop are underlined in blue. The highly conserved residues Lys39 and Glu58 are marked in orange. Green triangles indicate the residues of the C-spine; magenta stars mark residues of the R-spine.</p