Structural Analysis of Staphylococcus aureus Serine/Threonine Kinase PknB

Rakette, Sonja; Donat, Stefanie; Ohlsen, Knut; Stehle, Thilo

doi:10.1371/journal.pone.0039136

Cited by 30 publications

(46 citation statements)

References 52 publications

(99 reference statements)

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“…3A). The backbone of the model was almost identical to other published structures at similar resolution (1O6Y (2.2 Å) and 2FUM (2.9 Å)) and similar to the Stk1 structure (4EQM (3.0 Å)) from Staphylococcus aureus (Ortiz-Lombardia et al, 2003, Rakette, Donat et al, 2012, Wehenkel et al, 2006. The activation loop (residues 164-177) was not visible in the structure.…”

Section: Ipas Are Potent Biochemical Inhibitors Of Pknb and Require Asupporting

confidence: 76%

Repurposed kinase inhibitors and β-lactams as a novel therapy for antibiotic resistant bacteria

Wlodarchak

Teachout

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et al. 2017

Preprint

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Antibiotic resistant bacteria are an increasing global problem, and pathogenic actinomycetes and firmicutes are particularly challenging obstacles. These pathogens share several eukaryotic-like kinases that present antibiotic development opportunities. We used computational modelling to identify human kinase inhibitors that could be repurposed towards bacteria as part of a novel combination therapy. The computational model suggested a family of inhibitors, the imidazopyridine aminofurazans (IPAs), bind PknB with high affinity. We found that these inhibitors biochemically inhibit PknB, with potency roughly following the predicted models. A novel x-ray structure confirmed that the inhibitors bind as predicted and made favorable protein contacts with the target. These inhibitors also have antimicrobial activity towards Mycobacteria and Nocardia, and normally ineffective β-lactams can potentiate IPAs to more efficiently inhibit growth of these pathogens. Collectively, our data show that in silico modeling can be used as a tool to discover promising drug leads, and the inhibitors we discovered can synergize with clinically relevant antibiotics to restore their efficacy against bacteria with limited treatment options.

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Section: Ipas Are Potent Biochemical Inhibitors Of Pknb and Require Asupporting

confidence: 76%

Repurposed kinase inhibitors and β-lactams as a novel therapy for antibiotic resistant bacteria

Wlodarchak

Teachout

Procknow

et al. 2017

Preprint

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“…Genomic sequencing of this strain, which grew at a similar rate to wildtype (Fig. 6A), confirmed the successful deletion of prkP but also identified a trinucleotide deletion in the prkA gene (designated prkA *), effectively removing the complete codon of Gly18 that is part of a conserved glycine-rich loop important for ATP binding (48). Presumably, this mutation reduces the PrkA kinase activity to balance the absence of PrkP.…”

Section: Resultsmentioning

confidence: 66%

PrkA controls peptidoglycan biosynthesis through the essential phosphorylation of ReoM

Wamp

Rutter

Rismondo

et al. 2019

Preprint

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30Peptidoglycan (PG) is the main component of bacterial cell walls and the target for many 31 antibiotics. PG biosynthesis is tightly coordinated with cell wall growth and turnover and many 32 of these control activities depend upon PASTA-domain containing eukaryotic-like 33 serine/threonine protein kinases (PASTA-eSTK) that sense PG fragments. However, only a few 34 PG biosynthetic enzymes are actually direct kinase substrates. Here, we identify the conserved 35 ReoM protein as a novel PASTA-eSTK substrate in the Gram-positive pathogen Listeria 36 monocytogenes. Our data show that the phosphorylation of ReoM is essential as it controls 37 ClpCP-dependent proteolytic degradation of the essential enzyme MurA, which catalyses the first 38 committed step in PG biosynthesis. We also identify ReoY as a second novel factor required for 39 degradation of ClpCP substrates. Collectively, our data imply that the first committed step of PG 40 biosynthesis is activated through control of ClpCP protease activity in response to signals of PG 41 homeostasis imbalance.42 43 44 45The cell wall of Gram-positive bacteria is a complicated three-dimensional structure that engulfs 46 the cell as a closed sacculus. The main component of bacterial cell walls is peptidoglycan (PG), a 47 network of glycan strands crosslinked together by short peptides (1). PG biosynthesis starts with 48 the conversion of UDP-GlcNAc into lipid II, a disaccharide pentapeptide that is ligated to a 49 100 positive bacteria, which explains how PG biosynthesis is adjusted in these bacteria to meet PG 101 production and repair needs. 102 103 6 RESULTS 104 105Novel gpsB suppressor mutations in the lmo1503 (reoM) and lmo1921 (reoY) genes 106 A L. monocytogenes ΔgpsB mutant is unable to replicate at 42°C but forms suppressors 107 correcting this defect with high frequency (28). In a preliminary selection of gpsB suppressors, 108 the clpC and murZ genes, important for the stability of the UDP-N-acetylglucosamine 1-109 carboxyvinyltransferase MurA, were found to be mutated (28). We have characterized three more 110 shg (suppression of heat sensitive growth) suppressor mutants (shg8, shg10 and shg12) isolated 111 from a ΔgpsB mutant incubated on a BHI agar plate at 42°C. These three shg strains grew as fast 112 as the wild type when cultivated at 37°C or 42°C, whereas the parental ΔgpsB mutant grew at a 113 reduced rate at 37°C and did not grow at 42°C (Fig. 1A-B), as shown previously (32). 114Illumina sequencing of the shg8, shg10 and shg12 genomes identified one SNP in each strain that 115 was not found in the parental ΔgpsB strain. Strain shg8 carried a mutation in the uncharacterized 116 lmo1921 gene (herein named reoY, see below) that exchanged H87 into tyrosine; the same gene 117 was affected by the introduction of a premature stop codon after the 73 rd codon of reoY to yield 118 strain shg10. Strain shg12 carried a mutation in the ribosomal binding site (RBS) of the lmo1503 119 gene (renamed reoM), encoding another protein of unknown function and conta...

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“…However, the conformation of PknA corresponds to an inactive state, close to the autoinhibited structure of the kinase domain of Staphylococcus aureus PknB [Fig. (B)] (pdb entry 4eqm). Furthermore, the N‐terminus of PknA helix αC is rotated ∼17° outward of the active site pocket with respect to the same helix in PknB, hampering formation of the salt bridge Glu61–Lys42 that is a hallmark of active protein kinases [Fig.…”

Section: Resultsmentioning

confidence: 89%

“…See the text for details. ( B ) Lateral view of the superimposed catalytic domains of PknA (yellow, this study), M. tuberculosis PknB in complex with AMP‐PCP (pdb entry 1o6y, light brown), M. tuberculosis PknE (green; pdb entry 2h34), S. aureus PknB (gray; pdb entry 4eqm) and the human cAMP‐dependent kinase PKA in ternary complex with MgATP and an inhibitory peptide, shown as the paradigm of the active conformation of a Hanks‐type protein kinase (purple; pdb entry 1atp). The dashed lines indicate the different orientation of the αC helix axis when comparing PknA (yellow) and PknB (light brown).…”

Section: Resultsmentioning

confidence: 99%

The crystal structure of the catalytic domain of the ser/thr kinase PknA from M. tuberculosis shows an Src-like autoinhibited conformation

et al. 2015

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Signal transduction mediated by Ser/Thr phosphorylation in Mycobacterium tuberculosis has been intensively studied in the last years, as its genome harbors eleven genes coding for eukaryotic-like Ser/Thr kinases. Here we describe the crystal structure and the autophosphorylation sites of the catalytic domain of PknA, one of two protein kinases essential for pathogen's survival. The structure of the ligand-free kinase domain shows an auto-inhibited conformation similar to that observed in human Tyr kinases of the Src-family. These results reinforce the high conservation of structural hallmarks and regulation mechanisms between prokaryotic and eukaryotic protein kinases.

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Structural Analysis of Staphylococcus aureus Serine/Threonine Kinase PknB

Cited by 30 publications

References 52 publications

Repurposed kinase inhibitors and β-lactams as a novel therapy for antibiotic resistant bacteria

Repurposed kinase inhibitors and β-lactams as a novel therapy for antibiotic resistant bacteria

PrkA controls peptidoglycan biosynthesis through the essential phosphorylation of ReoM

The crystal structure of the catalytic domain of the ser/thr kinase PknA from M. tuberculosis shows an Src-like autoinhibited conformation

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