Crystal structure of tetrameric human Rabin8 GEF domain

Melanie Vetter; Niels Boegholm; Anni Christensen; Sagar Bhogaraju; Marie B Andersen; Anna Lorentzen; Esben Lorentzen

doi:10.1002/prot.25455

Crystal structure of tetrameric human Rabin8 GEF domain

Melanie Vetter, Niels Boegholm, Anni Christensen, Sagar Bhogaraju, Marie B Andersen, Anna Lorentzen, Esben Lorentzen

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Abstract

Rab GTPases and their effectors, activators and guanine nucleotide exchange factors (GEFs) are essential for vesicular transport. Rab8 and its GEF Rabin8 function in formation of the cilium organelle important for developmental signaling and sensory reception. Here, we show by size exclusion chromatography and analytical ultracentrifugation that Rabin8 exists in equilibrium between dimers and tetramers. The crystal structure of tetrameric Rabin8 GEF domain reveals an occluded Rab8 binding site suggesting that this oligomer is enzymatically inactive, a notion we verify experimentally using Rabin8/Rab8 GEF assays. We outline a procedure for the purification of active dimeric Rabin8 GEF‐domain for in vitro activity assays.

Original language	English
Journal	Proteins
Volume	86
Issue	4
Pages (from-to)	405-413
Number of pages	9
ISSN	0887-3585
DOIs	https://doi.org/10.1002/prot.25455
Publication status	Published - 10 Jan 2018

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title = "Crystal structure of tetrameric human Rabin8 GEF domain",

abstract = "Rab GTPases and their effectors, activators and guanine nucleotide exchange factors (GEFs) are essential for vesicular transport. Rab8 and its GEF Rabin8 function in formation of the cilium organelle important for developmental signaling and sensory reception. Here, we show by size exclusion chromatography and analytical ultracentrifugation that Rabin8 exists in equilibrium between dimers and tetramers. The crystal structure of tetrameric Rabin8 GEF domain reveals an occluded Rab8 binding site suggesting that this oligomer is enzymatically inactive, a notion we verify experimentally using Rabin8/Rab8 GEF assays. We outline a procedure for the purification of active dimeric Rabin8 GEF‐domain for in vitro activity assays.",

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T1 - Crystal structure of tetrameric human Rabin8 GEF domain

AU - Vetter, Melanie

AU - Boegholm, Niels

AU - Christensen, Anni

AU - Bhogaraju, Sagar

AU - Andersen, Marie B

AU - Lorentzen, Anna

AU - Lorentzen, Esben

PY - 2018/1/10

Y1 - 2018/1/10

N2 - Rab GTPases and their effectors, activators and guanine nucleotide exchange factors (GEFs) are essential for vesicular transport. Rab8 and its GEF Rabin8 function in formation of the cilium organelle important for developmental signaling and sensory reception. Here, we show by size exclusion chromatography and analytical ultracentrifugation that Rabin8 exists in equilibrium between dimers and tetramers. The crystal structure of tetrameric Rabin8 GEF domain reveals an occluded Rab8 binding site suggesting that this oligomer is enzymatically inactive, a notion we verify experimentally using Rabin8/Rab8 GEF assays. We outline a procedure for the purification of active dimeric Rabin8 GEF‐domain for in vitro activity assays.

AB - Rab GTPases and their effectors, activators and guanine nucleotide exchange factors (GEFs) are essential for vesicular transport. Rab8 and its GEF Rabin8 function in formation of the cilium organelle important for developmental signaling and sensory reception. Here, we show by size exclusion chromatography and analytical ultracentrifugation that Rabin8 exists in equilibrium between dimers and tetramers. The crystal structure of tetrameric Rabin8 GEF domain reveals an occluded Rab8 binding site suggesting that this oligomer is enzymatically inactive, a notion we verify experimentally using Rabin8/Rab8 GEF assays. We outline a procedure for the purification of active dimeric Rabin8 GEF‐domain for in vitro activity assays.

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DO - 10.1002/prot.25455

M3 - Journal article

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VL - 86

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JO - Proteins

JF - Proteins

IS - 4

ER -

Crystal structure of tetrameric human Rabin8 GEF domain

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