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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 crystal structure.(a) Schematic representation of LegK41–445 protein sequence and the structurally assigned domains of LegK41–445. The “cap” domain (residues 1-65) is represented in pink, the kinase domain is separated in two lobes: the N-lobe (residues 66-148, green) and the C-lobe (residues 149-317, light blue) and the four-helix bundle (FHB) domain (residues 318-406) is coloured in yellow. (b) Purified LegK proteins were subjected to in vitro auto- and myelin basic protein (MBP) phosphorylation assays in the presence of [γ-32P]ATP. Phosphoproteins were separated by SDS-PAGE and visualised by autoradiography. (c) Ribbon representation of the crystal structure of LegK41–445 with domains coloured as in (a). (d) Topological diagram of LegK41–445, using the same colour code as in panels (a,c). The missing unstructured regions are represented as dashed lines.
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f1: LegK41–445 crystal structure.(a) Schematic representation of LegK41–445 protein sequence and the structurally assigned domains of LegK41–445. The “cap” domain (residues 1-65) is represented in pink, the kinase domain is separated in two lobes: the N-lobe (residues 66-148, green) and the C-lobe (residues 149-317, light blue) and the four-helix bundle (FHB) domain (residues 318-406) is coloured in yellow. (b) Purified LegK proteins were subjected to in vitro auto- and myelin basic protein (MBP) phosphorylation assays in the presence of [γ-32P]ATP. Phosphoproteins were separated by SDS-PAGE and visualised by autoradiography. (c) Ribbon representation of the crystal structure of LegK41–445 with domains coloured as in (a). (d) Topological diagram of LegK41–445, using the same colour code as in panels (a,c). The missing unstructured regions are represented as dashed lines.

Mentions: In the process of trying to crystallise LegK4 we observed partial degradation of the full-length protein during purification and identified a stable fragment encompassing residues 1 to 445 (LegK41–445). This fragment corresponds to the most conserved region in the LegK family15. We thus generated an expression vector corresponding to this fragment (Fig. 1a) and purified the protein. To determine if LegK41–445 was active, a phosphorylation assay was conducted with either the full length LegK4 or the LegK41–445 fragment. The results show that, as for the wild type, LegK41–445 was able to autophosphorylate using [γ-32P]ATP as substrate (Fig. 1b). Moreover, both wild type LegK4 and LegK41–445 were able to phosphorylate the myelin basic protein (MBP) in vitro in the presence of [γ-32P]ATP (Fig. 1b). Taken together, these experiments indicate that the C-terminal part (residues 446-961) of LegK4 is dispensable for kinase activity.


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 crystal structure.(a) Schematic representation of LegK41–445 protein sequence and the structurally assigned domains of LegK41–445. The “cap” domain (residues 1-65) is represented in pink, the kinase domain is separated in two lobes: the N-lobe (residues 66-148, green) and the C-lobe (residues 149-317, light blue) and the four-helix bundle (FHB) domain (residues 318-406) is coloured in yellow. (b) Purified LegK proteins were subjected to in vitro auto- and myelin basic protein (MBP) phosphorylation assays in the presence of [γ-32P]ATP. Phosphoproteins were separated by SDS-PAGE and visualised by autoradiography. (c) Ribbon representation of the crystal structure of LegK41–445 with domains coloured as in (a). (d) Topological diagram of LegK41–445, using the same colour code as in panels (a,c). The missing unstructured regions are represented as dashed lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: LegK41–445 crystal structure.(a) Schematic representation of LegK41–445 protein sequence and the structurally assigned domains of LegK41–445. The “cap” domain (residues 1-65) is represented in pink, the kinase domain is separated in two lobes: the N-lobe (residues 66-148, green) and the C-lobe (residues 149-317, light blue) and the four-helix bundle (FHB) domain (residues 318-406) is coloured in yellow. (b) Purified LegK proteins were subjected to in vitro auto- and myelin basic protein (MBP) phosphorylation assays in the presence of [γ-32P]ATP. Phosphoproteins were separated by SDS-PAGE and visualised by autoradiography. (c) Ribbon representation of the crystal structure of LegK41–445 with domains coloured as in (a). (d) Topological diagram of LegK41–445, using the same colour code as in panels (a,c). The missing unstructured regions are represented as dashed lines.
Mentions: In the process of trying to crystallise LegK4 we observed partial degradation of the full-length protein during purification and identified a stable fragment encompassing residues 1 to 445 (LegK41–445). This fragment corresponds to the most conserved region in the LegK family15. We thus generated an expression vector corresponding to this fragment (Fig. 1a) and purified the protein. To determine if LegK41–445 was active, a phosphorylation assay was conducted with either the full length LegK4 or the LegK41–445 fragment. The results show that, as for the wild type, LegK41–445 was able to autophosphorylate using [γ-32P]ATP as substrate (Fig. 1b). Moreover, both wild type LegK4 and LegK41–445 were able to phosphorylate the myelin basic protein (MBP) in vitro in the presence of [γ-32P]ATP (Fig. 1b). Taken together, these experiments indicate that the C-terminal part (residues 446-961) of LegK4 is dispensable for kinase activity.

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