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Structural analyses of Legionella LepB reveal a new GAP fold that catalytically mimics eukaryotic RasGAP.

Yu Q, Hu L, Yao Q, Zhu Y, Dong N, Wang DC, Shao F - Cell Res. (2013)

Bottom Line: We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors.This conformationally rearranged Gln70 acts as the catalytic cis-glutamine, therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB.Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs, particularly those from bacterial pathogens.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.

ABSTRACT
Rab GTPases are emerging targets of diverse bacterial pathogens. Here, we perform biochemical and structural analyses of LepB, a Rab GTPase-activating protein (GAP) effector from Legionella pneumophila. We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors. Exhaustive mutation analyses identify Arg444 as the arginine finger, but no catalytically essential glutamine residues. Crystal structures of LepB313-618 alone and the GAP domain of Legionella drancourtii LepB in complex with Rab1-GDP-AlF3 support the catalytic role of Arg444, and also further reveal a 3D architecture and a GTPase-binding mode distinct from all known GAPs. Glu449, structurally equivalent to TBC RabGAP glutamine finger in apo-LepB, undergoes a drastic movement upon Rab1 binding, which induces Rab1 Gln70 side-chain flipping towards GDP-AlF3 through a strong ionic interaction. This conformationally rearranged Gln70 acts as the catalytic cis-glutamine, therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB. Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs, particularly those from bacterial pathogens.

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Crystal structure of LepB313-618 and comparison with the VirA/EspG-family bacterial RabGAP and the TBC domain of Gyp1p. (A) Overall structure of LepB313-618 in comparison with those of VirA (PDB ID: 4FMB) and Gyp1p (PDB ID: 2G77). Secondary structure elements are drawn with α-helices as coils, β-strands as flat arrows and loops as lines. LepB313-618, VirA and Gyp1p are colored blue, orange and green, respectively. (B) Superimposition of LepB313-618 structure onto those of VirA and Gyp1p using Arg444/Glu449 in LepB313-618, Arg188/Gln280 in VirA and Arg343/Gln378 in Gyp1p as the references. Shown on the right is a close-up view with marked reference residues in stick models. α-helices are shown as cylinders colored as that in A. (C) Structural comparison of Arg444/Glu449 in LepB313-618 with Arg343/Gln378 in Gyp1p. GDP and AlF3 in the Gyp1p-Rab1 complex structure are shown as yellow sticks. Mg2+ and the nucleophilic water are depicted as magenta and red sphere, respectively. Polar interactions between GDP-AlF3 and Arg343/Gln378 in Gyp1p are denoted by black dashed lines.
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fig3: Crystal structure of LepB313-618 and comparison with the VirA/EspG-family bacterial RabGAP and the TBC domain of Gyp1p. (A) Overall structure of LepB313-618 in comparison with those of VirA (PDB ID: 4FMB) and Gyp1p (PDB ID: 2G77). Secondary structure elements are drawn with α-helices as coils, β-strands as flat arrows and loops as lines. LepB313-618, VirA and Gyp1p are colored blue, orange and green, respectively. (B) Superimposition of LepB313-618 structure onto those of VirA and Gyp1p using Arg444/Glu449 in LepB313-618, Arg188/Gln280 in VirA and Arg343/Gln378 in Gyp1p as the references. Shown on the right is a close-up view with marked reference residues in stick models. α-helices are shown as cylinders colored as that in A. (C) Structural comparison of Arg444/Glu449 in LepB313-618 with Arg343/Gln378 in Gyp1p. GDP and AlF3 in the Gyp1p-Rab1 complex structure are shown as yellow sticks. Mg2+ and the nucleophilic water are depicted as magenta and red sphere, respectively. Polar interactions between GDP-AlF3 and Arg343/Gln378 in Gyp1p are denoted by black dashed lines.

Mentions: LepB GAP domain adopts a wide “V”-shape architecture, consisting of 10 α-helices and no β-strands (Figure 3A). The more elongated left arm of the “V” shape is mainly formed by a four-helix bundle (α2, α3, α5 and α6) packed by α1 and α4 on opposite sides. α1 at the top end of the left arm connects the GAP domain to the N-terminal domain (not shown in the picture) through a linking loop; α4 is situated in the valley bottom. The right arm is made up of four helices (α7-α10) organized into a more globular shape. α7 and α8, projecting perpendicular to the plane of the V shape, is located closer to the valley bottom; α9 and α10, parallel to each other, cross α7 and pack against α7/α8 from outside. Notably, the overall shape and structural architecture of LepB GAP domain shows no similarity to that of eukaryotic TBC domain (illustrated using Gyp1p TBC domain)20 (Figure 3A and 3B). LepB GAP domain is also structurally distinct from the VirA/EspG family of bacterial RabGAPs that adopt an α/β fold despite that they are also of prokaryotic origin and highly specific for Rab1. The structural divergence of LepB and the VirA/EspG family from eukaryotic TBC GAPs shall bring insights into the evolution of bacterial virulence activity.


Structural analyses of Legionella LepB reveal a new GAP fold that catalytically mimics eukaryotic RasGAP.

Yu Q, Hu L, Yao Q, Zhu Y, Dong N, Wang DC, Shao F - Cell Res. (2013)

Crystal structure of LepB313-618 and comparison with the VirA/EspG-family bacterial RabGAP and the TBC domain of Gyp1p. (A) Overall structure of LepB313-618 in comparison with those of VirA (PDB ID: 4FMB) and Gyp1p (PDB ID: 2G77). Secondary structure elements are drawn with α-helices as coils, β-strands as flat arrows and loops as lines. LepB313-618, VirA and Gyp1p are colored blue, orange and green, respectively. (B) Superimposition of LepB313-618 structure onto those of VirA and Gyp1p using Arg444/Glu449 in LepB313-618, Arg188/Gln280 in VirA and Arg343/Gln378 in Gyp1p as the references. Shown on the right is a close-up view with marked reference residues in stick models. α-helices are shown as cylinders colored as that in A. (C) Structural comparison of Arg444/Glu449 in LepB313-618 with Arg343/Gln378 in Gyp1p. GDP and AlF3 in the Gyp1p-Rab1 complex structure are shown as yellow sticks. Mg2+ and the nucleophilic water are depicted as magenta and red sphere, respectively. Polar interactions between GDP-AlF3 and Arg343/Gln378 in Gyp1p are denoted by black dashed lines.
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fig3: Crystal structure of LepB313-618 and comparison with the VirA/EspG-family bacterial RabGAP and the TBC domain of Gyp1p. (A) Overall structure of LepB313-618 in comparison with those of VirA (PDB ID: 4FMB) and Gyp1p (PDB ID: 2G77). Secondary structure elements are drawn with α-helices as coils, β-strands as flat arrows and loops as lines. LepB313-618, VirA and Gyp1p are colored blue, orange and green, respectively. (B) Superimposition of LepB313-618 structure onto those of VirA and Gyp1p using Arg444/Glu449 in LepB313-618, Arg188/Gln280 in VirA and Arg343/Gln378 in Gyp1p as the references. Shown on the right is a close-up view with marked reference residues in stick models. α-helices are shown as cylinders colored as that in A. (C) Structural comparison of Arg444/Glu449 in LepB313-618 with Arg343/Gln378 in Gyp1p. GDP and AlF3 in the Gyp1p-Rab1 complex structure are shown as yellow sticks. Mg2+ and the nucleophilic water are depicted as magenta and red sphere, respectively. Polar interactions between GDP-AlF3 and Arg343/Gln378 in Gyp1p are denoted by black dashed lines.
Mentions: LepB GAP domain adopts a wide “V”-shape architecture, consisting of 10 α-helices and no β-strands (Figure 3A). The more elongated left arm of the “V” shape is mainly formed by a four-helix bundle (α2, α3, α5 and α6) packed by α1 and α4 on opposite sides. α1 at the top end of the left arm connects the GAP domain to the N-terminal domain (not shown in the picture) through a linking loop; α4 is situated in the valley bottom. The right arm is made up of four helices (α7-α10) organized into a more globular shape. α7 and α8, projecting perpendicular to the plane of the V shape, is located closer to the valley bottom; α9 and α10, parallel to each other, cross α7 and pack against α7/α8 from outside. Notably, the overall shape and structural architecture of LepB GAP domain shows no similarity to that of eukaryotic TBC domain (illustrated using Gyp1p TBC domain)20 (Figure 3A and 3B). LepB GAP domain is also structurally distinct from the VirA/EspG family of bacterial RabGAPs that adopt an α/β fold despite that they are also of prokaryotic origin and highly specific for Rab1. The structural divergence of LepB and the VirA/EspG family from eukaryotic TBC GAPs shall bring insights into the evolution of bacterial virulence activity.

Bottom Line: We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors.This conformationally rearranged Gln70 acts as the catalytic cis-glutamine, therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB.Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs, particularly those from bacterial pathogens.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.

ABSTRACT
Rab GTPases are emerging targets of diverse bacterial pathogens. Here, we perform biochemical and structural analyses of LepB, a Rab GTPase-activating protein (GAP) effector from Legionella pneumophila. We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors. Exhaustive mutation analyses identify Arg444 as the arginine finger, but no catalytically essential glutamine residues. Crystal structures of LepB313-618 alone and the GAP domain of Legionella drancourtii LepB in complex with Rab1-GDP-AlF3 support the catalytic role of Arg444, and also further reveal a 3D architecture and a GTPase-binding mode distinct from all known GAPs. Glu449, structurally equivalent to TBC RabGAP glutamine finger in apo-LepB, undergoes a drastic movement upon Rab1 binding, which induces Rab1 Gln70 side-chain flipping towards GDP-AlF3 through a strong ionic interaction. This conformationally rearranged Gln70 acts as the catalytic cis-glutamine, therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB. Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs, particularly those from bacterial pathogens.

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