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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

Structural flexibility of the cap domain.Cartoon view of the interface between the cap domain (pink) and the N-lobe (green) of AMP-PNP•LegK41–445 (a) and apo-LegK41–445 (b). Side chains of principal residues participating in the interface in chain A2 are depicted as ball and sticks with carbon atoms coloured as the cartoon, oxygen in red and nitrogen in blue. (c) Structures of mouse NIK (pdb code 4G3C) and human MARK1 (pdb code 2HAK) kinases represented in the same orientation as LegK4 in (a) with the N-lobe coloured in green and the NIK N-terminal extension coloured in purple and MARK1 UBA domain coloured in magenta.
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f2: Structural flexibility of the cap domain.Cartoon view of the interface between the cap domain (pink) and the N-lobe (green) of AMP-PNP•LegK41–445 (a) and apo-LegK41–445 (b). Side chains of principal residues participating in the interface in chain A2 are depicted as ball and sticks with carbon atoms coloured as the cartoon, oxygen in red and nitrogen in blue. (c) Structures of mouse NIK (pdb code 4G3C) and human MARK1 (pdb code 2HAK) kinases represented in the same orientation as LegK4 in (a) with the N-lobe coloured in green and the NIK N-terminal extension coloured in purple and MARK1 UBA domain coloured in magenta.

Mentions: The short amino terminal domain, named “cap” hereafter is well defined in chain A1, in which it consists of four small α-helices (α1 to α4) and a β-strand. βN1 runs antiparallel to the N-lobe strands β1 and β4 resulting in an extended six-stranded open β-barrel. The interactions between the cap domain and the N-lobe are extensive, burying a total of 970 Å2. A hydrophobic patch formed by α2 Leu22, Leu23 and Val26 and α3 Ile32 and Ile36 interacts with N-lobe residues Ile59, Phe61 (β1), Ile72 (β2), Tyr93 and Leu95 (β3). The interface also involves several hydrogen bonds including salt bridges between Asp56 and Arg46 and between Lys2 and Asp96 (Fig. 2a). Comparison of the cap domain in chains A1, A2, B1 and B2 reveals that parts of the cap domain are disordered as well as the contacting N-lobe elements (Fig. 2a,b). While no obvious homologue structure of this motif could be found using the DALI server, PDBefold26 identified several ubiquitin-associated domains (UBA) as remote structural homologues. UBA domains have been described in human AMPK-related kinases, among which are members of the MAP/microtubule affinity-regulating kinases family (MARK)2728 (Fig. 2c). In this context, these short helical domains are C-terminal to the kinase domain and can regulate kinase activities through an interaction with the kinase N-lobe at β4 and the β2-β3 loop. Interestingly the cap domain is also similar to the N-terminal extension of the nuclear factor κB-inducing kinase (NIK). Like in LegK4, the extension of NIK also extends the N-lobe β-sheet. The extension of NIK also contains two extra helices, which stabilizes helix αC in the active orientation and maintains the kinase domain in the catalytically competent conformation29 (Fig. 2c). Although there are superficial similarities between the cap domain of LegK4 and UBA domains of MARK family kinases and the N-terminal extension of NIK, they bind to different faces of the N-lobe (Fig. 2a–c).


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)

Structural flexibility of the cap domain.Cartoon view of the interface between the cap domain (pink) and the N-lobe (green) of AMP-PNP•LegK41–445 (a) and apo-LegK41–445 (b). Side chains of principal residues participating in the interface in chain A2 are depicted as ball and sticks with carbon atoms coloured as the cartoon, oxygen in red and nitrogen in blue. (c) Structures of mouse NIK (pdb code 4G3C) and human MARK1 (pdb code 2HAK) kinases represented in the same orientation as LegK4 in (a) with the N-lobe coloured in green and the NIK N-terminal extension coloured in purple and MARK1 UBA domain coloured in magenta.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Structural flexibility of the cap domain.Cartoon view of the interface between the cap domain (pink) and the N-lobe (green) of AMP-PNP•LegK41–445 (a) and apo-LegK41–445 (b). Side chains of principal residues participating in the interface in chain A2 are depicted as ball and sticks with carbon atoms coloured as the cartoon, oxygen in red and nitrogen in blue. (c) Structures of mouse NIK (pdb code 4G3C) and human MARK1 (pdb code 2HAK) kinases represented in the same orientation as LegK4 in (a) with the N-lobe coloured in green and the NIK N-terminal extension coloured in purple and MARK1 UBA domain coloured in magenta.
Mentions: The short amino terminal domain, named “cap” hereafter is well defined in chain A1, in which it consists of four small α-helices (α1 to α4) and a β-strand. βN1 runs antiparallel to the N-lobe strands β1 and β4 resulting in an extended six-stranded open β-barrel. The interactions between the cap domain and the N-lobe are extensive, burying a total of 970 Å2. A hydrophobic patch formed by α2 Leu22, Leu23 and Val26 and α3 Ile32 and Ile36 interacts with N-lobe residues Ile59, Phe61 (β1), Ile72 (β2), Tyr93 and Leu95 (β3). The interface also involves several hydrogen bonds including salt bridges between Asp56 and Arg46 and between Lys2 and Asp96 (Fig. 2a). Comparison of the cap domain in chains A1, A2, B1 and B2 reveals that parts of the cap domain are disordered as well as the contacting N-lobe elements (Fig. 2a,b). While no obvious homologue structure of this motif could be found using the DALI server, PDBefold26 identified several ubiquitin-associated domains (UBA) as remote structural homologues. UBA domains have been described in human AMPK-related kinases, among which are members of the MAP/microtubule affinity-regulating kinases family (MARK)2728 (Fig. 2c). In this context, these short helical domains are C-terminal to the kinase domain and can regulate kinase activities through an interaction with the kinase N-lobe at β4 and the β2-β3 loop. Interestingly the cap domain is also similar to the N-terminal extension of the nuclear factor κB-inducing kinase (NIK). Like in LegK4, the extension of NIK also extends the N-lobe β-sheet. The extension of NIK also contains two extra helices, which stabilizes helix αC in the active orientation and maintains the kinase domain in the catalytically competent conformation29 (Fig. 2c). Although there are superficial similarities between the cap domain of LegK4 and UBA domains of MARK family kinases and the N-terminal extension of NIK, they bind to different faces of the N-lobe (Fig. 2a–c).

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