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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

Nucleotide binding to LegK41–445.(a) Cartoon representation of superimposed active sites of apo-LegK41–445 (gold) and AMP-PNP•LegK41–445 (blue). Side chains of important residues are shown as ball and stick and coloured as the cartoon (carbon), red (oxygen) and blue (nitrogen). (b) Structural superimposition of apo-LegK41–445.(gold) and AMP-PNP•LegK41–445 illustrating the movement of the cap domain and the N-lobe. (c) Comparison of the architectures of the C- (yellow surface) and R-spines (red surface) in the apo (upper panels) and nucleotide bound states (lower panels) of LegK41–445 and mouse PKA mutant R194A (pdb code 4DFY).
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f4: Nucleotide binding to LegK41–445.(a) Cartoon representation of superimposed active sites of apo-LegK41–445 (gold) and AMP-PNP•LegK41–445 (blue). Side chains of important residues are shown as ball and stick and coloured as the cartoon (carbon), red (oxygen) and blue (nitrogen). (b) Structural superimposition of apo-LegK41–445.(gold) and AMP-PNP•LegK41–445 illustrating the movement of the cap domain and the N-lobe. (c) Comparison of the architectures of the C- (yellow surface) and R-spines (red surface) in the apo (upper panels) and nucleotide bound states (lower panels) of LegK41–445 and mouse PKA mutant R194A (pdb code 4DFY).

Mentions: In the AMP-PNP•LegK41–445 structure, one AMP-PNP molecule and two Mg2+ ions were present per LegK41–445 monomer (Fig. 4a). The activation segment, including the catalytic loop, the magnesium binding loop, the activation loop and the P+1 loop, adopts almost the same conformation as in the apo form (overall rmsd = 0.4 Å) (Fig. 4a). The nucleotide binds to LegK41–445 in a canonical manner, between the N- and C-lobe, with the adenine moiety buried in a hydrophobic pocket formed by Phe102, Phe147, Ile148 and Val212. The N1 and N6 amino groups of the adenine are hydrogen bonded to the Ile148 amide and to the Phe147 carbonyl, respectively while the N3 and N7 amino groups interact with surrounding bridging water molecules. Interestingly, the adenine is stacked by the Phe102 side chain. In the vast majority of kinases, this position is occupied by an alanine as part of the consensus “VAIK” sequence. In the case of kinase suppressor of Ras (KSR) 1, mutation of this residue to phenylalanine ablates the binding of ATP34 and the pseudokinase RYK, which does not bind nucleotide, also has a phenylalanine at this position35. In contrast, the presence of a phenylalanine in this position in LegK4 seems to stabilize the adenine ring rather than preventing nucleotide binding. The two hydroxyls of the ribose moiety are hydrogen bonded to the Asp152 side chain and the Gly199 carbonyl. The α- and β-phosphates interact with the invariant Lys104 and the Gln89, respectively (Fig. 4a). The γ-phosphate does not make direct interaction with the protein. The invariant Asp213, from the magnesium-binding loop, chelates the primary activating Mg2+ ion that directly binds the β-phosphate, and bridges the γ-phosphate via a water molecule. The secondary Mg2+ ion interacts with the Asn200 side chain and bridges the α- and β-phosphates (Fig. 4a).


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)

Nucleotide binding to LegK41–445.(a) Cartoon representation of superimposed active sites of apo-LegK41–445 (gold) and AMP-PNP•LegK41–445 (blue). Side chains of important residues are shown as ball and stick and coloured as the cartoon (carbon), red (oxygen) and blue (nitrogen). (b) Structural superimposition of apo-LegK41–445.(gold) and AMP-PNP•LegK41–445 illustrating the movement of the cap domain and the N-lobe. (c) Comparison of the architectures of the C- (yellow surface) and R-spines (red surface) in the apo (upper panels) and nucleotide bound states (lower panels) of LegK41–445 and mouse PKA mutant R194A (pdb code 4DFY).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Nucleotide binding to LegK41–445.(a) Cartoon representation of superimposed active sites of apo-LegK41–445 (gold) and AMP-PNP•LegK41–445 (blue). Side chains of important residues are shown as ball and stick and coloured as the cartoon (carbon), red (oxygen) and blue (nitrogen). (b) Structural superimposition of apo-LegK41–445.(gold) and AMP-PNP•LegK41–445 illustrating the movement of the cap domain and the N-lobe. (c) Comparison of the architectures of the C- (yellow surface) and R-spines (red surface) in the apo (upper panels) and nucleotide bound states (lower panels) of LegK41–445 and mouse PKA mutant R194A (pdb code 4DFY).
Mentions: In the AMP-PNP•LegK41–445 structure, one AMP-PNP molecule and two Mg2+ ions were present per LegK41–445 monomer (Fig. 4a). The activation segment, including the catalytic loop, the magnesium binding loop, the activation loop and the P+1 loop, adopts almost the same conformation as in the apo form (overall rmsd = 0.4 Å) (Fig. 4a). The nucleotide binds to LegK41–445 in a canonical manner, between the N- and C-lobe, with the adenine moiety buried in a hydrophobic pocket formed by Phe102, Phe147, Ile148 and Val212. The N1 and N6 amino groups of the adenine are hydrogen bonded to the Ile148 amide and to the Phe147 carbonyl, respectively while the N3 and N7 amino groups interact with surrounding bridging water molecules. Interestingly, the adenine is stacked by the Phe102 side chain. In the vast majority of kinases, this position is occupied by an alanine as part of the consensus “VAIK” sequence. In the case of kinase suppressor of Ras (KSR) 1, mutation of this residue to phenylalanine ablates the binding of ATP34 and the pseudokinase RYK, which does not bind nucleotide, also has a phenylalanine at this position35. In contrast, the presence of a phenylalanine in this position in LegK4 seems to stabilize the adenine ring rather than preventing nucleotide binding. The two hydroxyls of the ribose moiety are hydrogen bonded to the Asp152 side chain and the Gly199 carbonyl. The α- and β-phosphates interact with the invariant Lys104 and the Gln89, respectively (Fig. 4a). The γ-phosphate does not make direct interaction with the protein. The invariant Asp213, from the magnesium-binding loop, chelates the primary activating Mg2+ ion that directly binds the β-phosphate, and bridges the γ-phosphate via a water molecule. The secondary Mg2+ ion interacts with the Asn200 side chain and bridges the α- and β-phosphates (Fig. 4a).

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