Structural Analysis of Staphylococcus aureus Serine/Threonine Kinase PknB

Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 Å resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation

Gestione energeticamente efficiente di bruciatori a gas metano

Il funzionamento efficiente dal punto di vista energetico di un impianto di processo termico riscaldato ametano dipende in larga misura dalla qualità e dalla costruzione dei bruciatori utilizzati. In base ad esempipratici viene dimostrato in che modo possa essere individuato un aumento inutile del consumo, verificatosi acausa di una errata regolazione del bruciatore o di pressioni oscillanti dei servo mezzi di alimentazione.Tali fenomeni possono essere evitati in maniera efficace e permanente, adottando delle contromisure, in parteanche semplici. Inoltre viene illustrata la diretta dipendenza dell’efficienza energetica dalla dimensione dellasuperficie del tubo radiante

Staphylococcal PknB as the First Prokaryotic Representative of the Proline-Directed Kinases

In eukaryotic cell types, virtually all cellular processes are under control of proline-directed kinases and especially MAP kinases. Serine/threonine kinases in general were originally considered as a eukaryote-specific enzyme family. However, recent studies have revealed that orthologues of eukaryotic serine/threonine kinases exist in bacteria. Moreover, various pathogenic species, such as Yersinia and Mycobacterium, require serine/threonine kinases for successful invasion of human host cells. The substrates targeted by bacterial serine/threonine kinases have remained largely unknown. Here we report that the serine/threonine kinase PknB from the important pathogen Staphylococcus aureus is released into the external milieu, which opens up the possibility that PknB does not only phosphorylate bacterial proteins but also proteins of the human host. To identify possible human targets of purified PknB, we studied in vitro phosphorylation of peptide microarrays and detected 68 possible human targets for phosphorylation. These results show that PknB is a proline-directed kinase with MAP kinase-like enzymatic activity. As the potential cellular targets for PknB are involved in apoptosis, immune responses, transport, and metabolism, PknB secretion may help the bacterium to evade intracellular killing and facilitate its growth. In apparent agreement with this notion, phosphorylation of the host-cell response coordinating transcription factor ATF-2 by PknB was confirmed by mass spectrometry. Taken together, our results identify PknB as the first prokaryotic representative of the proline-directed kinase/MAP kinase family of enzymes

Strukturanalyse der Serin/Threonin Proteinkinase B und des multiple peptide resistence factor von Staphylococcus aureus

Staphylococcus aureus (S. aureus) infections are becoming increasingly problematic. The bacteria gain resistance to new antibiotics in relatively short time periods. Besides the hospital associated methicillin-resistant S. aureus (HA-MRSA), infections with community-associated MRSA (CA-MRSA) are increasing. The only serine/threonine kinase PknB of S. aureus is composed of an intracellular kinase domain, a transmembrane helix and three extracellular penicillin-binding protein and serine/threonine kinase associated (PASTA) domains. PknB is able to perform phosphorylation and autophosphorylation in vitro. Transcriptome and functional analysis of S. aureus showed that PknB is involved in regulating purine/pyrimidine synthesis, cell wall metabolism and central metabolic functions. In peptide arrays using eukaryotic peptides and PknB, the kinase was identified to be a proline-directed kinase. The crystal structure of the S. aureus PknB kinase domain (PknBSAKD) was solved in complex with a non-hydrolyzable adenosine triphosphate (ATP) analog at 3.0 Å resolution. Different ATP analogs, divalent cations and crystallization techniques were tested to improve crystal quality. The PknBSAKD structure was compared to different eukaryotic and prokaryotic serine/threonine protein kinases. Similar to the full-length protein, PknBSAKD is able to perform phosphorylation and autophosphorylation in vitro. In contrast to the active state in solution, the kinase crystallized in an inactive conformation. The multiple peptide resistant factor (MprF) is a virulence factor of S. aureus. MprF is predicted to be composed of a large hydrophobic region, containing 14 transmembrane segments (TMS) and a hydrophilic C-terminal region. The hydrophilic portion and the six C-terminal TMSs form a synthase domain; the eight N-terminal TMSs harbor a flippase function. The synthase adds lysine to phosphatidylglycerol (PG); the flippase inserts this positively charged lysyl-phosphatidylglycerol (Lys-PG) into the outer membrane leaflet of S. aureus. This leads to a change in the membrane charge, and host defensins are thus no longer able to bind to the bacterial membrane. In this work, the initial constructs of the synthase of MprF contained all the C-terminal soluble part with varying N-terminal ends. In addition to the variations in the N-terminal end of the soluble part, the number of TMSs was varied. All these constructs were successfully cloned and expressed. Protein analysis indicated aggregation of the proteins. The linker region between the last TMS and the soluble part was in the main focus. Variations of only few amino acids influenced the behavior of the protein during purification. Inclusions of increasing numbers of N-terminal TMSs into the construct lead to a decrease in solubility.Infektionen mit Staphylococcus aureus (S. aureus) werden problematischer, da die Bakterien in immer kürzerer Zeit Resistenzen gegen neue Antibiotika entwickeln. Zusätzlich zur steigenden Anzahl von im Krankenhaus erworbenen Infektionen mit Methicillin resistenten S. aureus Stämmen (HA-MRSA) häuft sich die Anzahl der MRSA Fälle in der nicht hospitalisierten Bevölkerung (CA-MRSA). S. aureus besitzt eine einzige Serin/Threonin Proteinkinase. Diese besteht aus einer intrazellulären Kinasedomäne, einer Transmembranhelix und drei extrazellulären penicillin-binding protein and serine/threonine kinase associated (PASTA) Domänen. PknB ist dazu in der Lage sowohl andere Proteine als auch sich selbst in vitro zu phosphorylieren. Transkriptom- und Funktionalitätsstudien zeigten, dass S. aureus PknB an Regulation der Purin-/Pyrimidin-Synthese, dem Zellwand Metabolismus und zentralen metabolischen Funktionen beteiligt ist. Spezifitätsbestimmungen mittels Micorarrays, bei denen PknB und eukaryotische Peptide verwenden wurden, konnten zeigen, dass es sich bei PknB um eine prolingelenkte Kinase handelt. Die Kristallstruktur der Kinasedomäne von S. aureus PknB (PknBSAKD) wurde im Komplex mit einem nichthydrolysierbaren ATP (Adenosintriphosphat) Analogon bei einer Auflösung von 3.0 Å gelöst. Um die Kristallqualität zu verbessern, wurde PknBSAKD mit verschiedenen ATP-Analoga und unterschiedlichen Kristallisationstechniken kristallisiert. Die Struktur wurde mit anderen eukaryotischen und prokaryotischen Serin/Threonin Proteinkinasen verglichen. Ebenso wie das gesamte Protein ist PknBSAKD dazu in der Lage sowohl andere Proteine als auch sich selbst zu phosphorylieren. Im Gegensatz zu der aktiven Form der Kinase in Lösung ist die Kinasedomäne in einer inaktiven Konformation kristallisiert. Das Protein MprF (multiple peptide resistant factor) ist ein Virulenzfaktor von S. aureus. Voraussichtlich besteht es aus einem langen hydrophoben Teil, der 14 Transmembransegmente (TMS) besitzt, und einem hydrophilen C-terminalen Teil. Dieser hydrophile Teil bildet zusammen mit den sechs C-terminalen TMS die Synthase von MprF. Die acht N-terminalen TMS bilden die Flippase. Die Synthase verknüpft Phosphatidylglycerol (PG) mit Lysin zu Lysyl-Phosphatidylglycerol (Lys-PG), das dann von der Flippase in die äußere Schicht der Staphylococcenmembran integriert wird. Dies führt zu einer Änderung der Membranladung und verhindert so die Anlagerung von Defensinen. Die ersten MprF Konstrukte dieser Arbeit bestehen alle aus dem C-terminalen, löslichen Anteil von MprF, wobei das N-terminale Ende dieser Konstrukte variiert. Zusätzlich zu den verschiedenen N-terminalen Enden wurde auch die Anzahl der TMS variiert. Alle Konstrukte konnten kloniert und exprimiert werden. Die Analyse der Proteine ergab allerdings, dass sie aggregiert sind. Das Hauptaugenmerk lag auf dem Verbindungstück zwischen der letzten TMS und dem löslichen Anteil von MprF. Bereits kleine Veränderungen der Anzahl der Aminosäuren in dieser Region beeinflussten das Verhalten des Proteins merklich. Eine Verlängerung des Proteins durch zusätzliche TMS in N-terminale Richtung verringerte die Löslichkeit

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

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