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The structure of Legionella pneumophila LegK4 type four secretion system (T4SS) effector reveals a novel dimeric eukaryotic-like kinase.

Flayhan A, Bergé C, Baïlo N, Doublet P, Bayliss R, Terradot L - Sci Rep (2015)

Bottom Line: The protein self-assembles through interactions mediated by helices αF and αG that generate a dimeric interface not previously observed in a protein kinase.The nucleotide-binding site of LegK4 contains an unusual set of residues that mediate non-canonical interactions with AMP-PNP.Nucleotide binding results in limited changes in the active site, suggesting that LegK4 constitutive kinase activity does not depend on phosphorylation of the activation loop but on the stabilizing effects of the dimer.

View Article: PubMed Central - PubMed

Affiliation: UMR 5086 BMSSI CNRS-Université de Lyon, Institut de Biologie et Chimie des Protéines, 7 Passage du Vercors, F-69367 Lyon Cedex 07, France.

ABSTRACT
Bacterial pathogens subvert signalling pathways to promote invasion and/or replication into the host. LegK1-4 proteins are eukaryotic-like serine/threonine kinases that are translocated by the Dot/Icm type IV secretion system (T4SS) of several Legionella pneumophila strains. We present the crystal structures of an active fragment of the LegK4 protein in apo and substrate-bound states. The structure of LegK4(1-445) reveals a eukaryotic-like kinase domain flanked by a novel cap domain and a four-helix bundle. The protein self-assembles through interactions mediated by helices αF and αG that generate a dimeric interface not previously observed in a protein kinase. The helix αG is displaced compared to previous kinase structures, and its role in stabilization of the activation loop is taken on by the dimerisation interface. The apo-form of the protein has an open conformation with a disordered P-loop but a structured activation segment in absence of targeted phosphorylation. The nucleotide-binding site of LegK4 contains an unusual set of residues that mediate non-canonical interactions with AMP-PNP. Nucleotide binding results in limited changes in the active site, suggesting that LegK4 constitutive kinase activity does not depend on phosphorylation of the activation loop but on the stabilizing effects of the dimer.

No MeSH data available.


Related in: MedlinePlus

LegK41–445 dimer organisation.(a) Side (upper panel) and top (lower panel) views of the dimer with chain A coloured in grey and shown as a surface and chain B coloured in blue and shown as ribbon. The cap and FHB domains of the two chains are coloured in pink and yellow, respectively. (b) Comparison of the experimental SAXS curve of apo-LegK41–445 (black) with theoretical SAXS curves of apo-LegK41–445 monomer (orange), dimer (magenta) and AMP-PNP•LegK41–445 dimer (blue). χ2 obtained with FOXS server51 are indicated. (c) Detailed view of the dimer interface coloured as in (a) with participating residues shown as ball and stick.
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f5: LegK41–445 dimer organisation.(a) Side (upper panel) and top (lower panel) views of the dimer with chain A coloured in grey and shown as a surface and chain B coloured in blue and shown as ribbon. The cap and FHB domains of the two chains are coloured in pink and yellow, respectively. (b) Comparison of the experimental SAXS curve of apo-LegK41–445 (black) with theoretical SAXS curves of apo-LegK41–445 monomer (orange), dimer (magenta) and AMP-PNP•LegK41–445 dimer (blue). χ2 obtained with FOXS server51 are indicated. (c) Detailed view of the dimer interface coloured as in (a) with participating residues shown as ball and stick.

Mentions: Both the apo and the substrate bound LegK41–445 crystal forms contain two molecules in the asymmetric unit forming a similar dimer. The interface is symmetric and buries around 900 Å2 as determined by the PDBePISA server39 (Fig. 5a). To determine if the dimer of LegK41–445 was relevant in solution we used inline size exclusion small angle X-ray scattering (SEC-SAXS)40. SAXS data indicates that the Rg of LegK41–445 is 36.1 Å with a Dmax of 111 Å (Fig. 5b, Supplementary Fig. S3). These values are in excellent agreement with the theoretical Rg and Dmax of respectively 34 Å and 111 Å, derived from the dimer observed in crystal structure. The small difference might be due to the disordered region of 45 residues present at the C-terminal portion of each monomer. Comparison of LegK41–445 experimental SAXS data with the theoretical curves obtained with the crystal structures demonstrate that the best fit was obtained with the dimer of apo-LegK41–445 (χ2 = 4.6) compared to the dimer of AMP-PNP•LegK41–445 (χ2 = 10.5) or to the apo-LegK41–445 monomer (χ2 = 41.1) (Fig. 5b). These data clearly show that in solution, LegK41–445 adopts the conformation observed in the crystal structure.


The structure of Legionella pneumophila LegK4 type four secretion system (T4SS) effector reveals a novel dimeric eukaryotic-like kinase.

Flayhan A, Bergé C, Baïlo N, Doublet P, Bayliss R, Terradot L - Sci Rep (2015)

LegK41–445 dimer organisation.(a) Side (upper panel) and top (lower panel) views of the dimer with chain A coloured in grey and shown as a surface and chain B coloured in blue and shown as ribbon. The cap and FHB domains of the two chains are coloured in pink and yellow, respectively. (b) Comparison of the experimental SAXS curve of apo-LegK41–445 (black) with theoretical SAXS curves of apo-LegK41–445 monomer (orange), dimer (magenta) and AMP-PNP•LegK41–445 dimer (blue). χ2 obtained with FOXS server51 are indicated. (c) Detailed view of the dimer interface coloured as in (a) with participating residues shown as ball and stick.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4588518&req=5

f5: LegK41–445 dimer organisation.(a) Side (upper panel) and top (lower panel) views of the dimer with chain A coloured in grey and shown as a surface and chain B coloured in blue and shown as ribbon. The cap and FHB domains of the two chains are coloured in pink and yellow, respectively. (b) Comparison of the experimental SAXS curve of apo-LegK41–445 (black) with theoretical SAXS curves of apo-LegK41–445 monomer (orange), dimer (magenta) and AMP-PNP•LegK41–445 dimer (blue). χ2 obtained with FOXS server51 are indicated. (c) Detailed view of the dimer interface coloured as in (a) with participating residues shown as ball and stick.
Mentions: Both the apo and the substrate bound LegK41–445 crystal forms contain two molecules in the asymmetric unit forming a similar dimer. The interface is symmetric and buries around 900 Å2 as determined by the PDBePISA server39 (Fig. 5a). To determine if the dimer of LegK41–445 was relevant in solution we used inline size exclusion small angle X-ray scattering (SEC-SAXS)40. SAXS data indicates that the Rg of LegK41–445 is 36.1 Å with a Dmax of 111 Å (Fig. 5b, Supplementary Fig. S3). These values are in excellent agreement with the theoretical Rg and Dmax of respectively 34 Å and 111 Å, derived from the dimer observed in crystal structure. The small difference might be due to the disordered region of 45 residues present at the C-terminal portion of each monomer. Comparison of LegK41–445 experimental SAXS data with the theoretical curves obtained with the crystal structures demonstrate that the best fit was obtained with the dimer of apo-LegK41–445 (χ2 = 4.6) compared to the dimer of AMP-PNP•LegK41–445 (χ2 = 10.5) or to the apo-LegK41–445 monomer (χ2 = 41.1) (Fig. 5b). These data clearly show that in solution, LegK41–445 adopts the conformation observed in the crystal structure.

Bottom Line: The protein self-assembles through interactions mediated by helices αF and αG that generate a dimeric interface not previously observed in a protein kinase.The nucleotide-binding site of LegK4 contains an unusual set of residues that mediate non-canonical interactions with AMP-PNP.Nucleotide binding results in limited changes in the active site, suggesting that LegK4 constitutive kinase activity does not depend on phosphorylation of the activation loop but on the stabilizing effects of the dimer.

View Article: PubMed Central - PubMed

Affiliation: UMR 5086 BMSSI CNRS-Université de Lyon, Institut de Biologie et Chimie des Protéines, 7 Passage du Vercors, F-69367 Lyon Cedex 07, France.

ABSTRACT
Bacterial pathogens subvert signalling pathways to promote invasion and/or replication into the host. LegK1-4 proteins are eukaryotic-like serine/threonine kinases that are translocated by the Dot/Icm type IV secretion system (T4SS) of several Legionella pneumophila strains. We present the crystal structures of an active fragment of the LegK4 protein in apo and substrate-bound states. The structure of LegK4(1-445) reveals a eukaryotic-like kinase domain flanked by a novel cap domain and a four-helix bundle. The protein self-assembles through interactions mediated by helices αF and αG that generate a dimeric interface not previously observed in a protein kinase. The helix αG is displaced compared to previous kinase structures, and its role in stabilization of the activation loop is taken on by the dimerisation interface. The apo-form of the protein has an open conformation with a disordered P-loop but a structured activation segment in absence of targeted phosphorylation. The nucleotide-binding site of LegK4 contains an unusual set of residues that mediate non-canonical interactions with AMP-PNP. Nucleotide binding results in limited changes in the active site, suggesting that LegK4 constitutive kinase activity does not depend on phosphorylation of the activation loop but on the stabilizing effects of the dimer.

No MeSH data available.


Related in: MedlinePlus