A catalytic coiled coil: structural insights into the activation of the Rab GTPase Sec4p by Sec2p - PubMed (original) (raw)

A catalytic coiled coil: structural insights into the activation of the Rab GTPase Sec4p by Sec2p

Gang Dong et al. Mol Cell. 2007.

Abstract

Rab GTPases, the largest subgroup in the superfamily of Ras-like GTPases, play regulatory roles in multiple steps of intracellular vesicle trafficking. They are activated by guanine nucleotide exchange factors (GEFs), which catalyze the interconversion of the GDP-bound, or inactive, form of Rab to the GTP-bound, or active, form. Relatively little is known of the mechanisms by which GEFs activate Rabs. Here, we present the crystal structure of the GEF domain of Sec2p in complex with its Rab partner Sec4p. The Sec2p GEF domain is a 220 Angstroms long coiled coil, striking in its simplicity and in the use of the coiled-coil motif for catalysis. The structure suggests a mechanism whereby Sec2p induces extensive structural rearrangements in the Sec4p switch regions and phosphate-binding loop that are incompatible with nucleotide binding. We show that Sec2p is specific for Sec4p and that specificity determinants reside in the two switch regions of Sec4p.

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Figures

Figure 1

Structure of the Rab GTPase Sec4p in complex with the GEF domain of Sec2p. (A) A ribbons diagram. Sec4p is grey and Sec2p purple/lilac. Switches I (indigo) and II (orange) and the P-loop of Sec4p are labeled. (B) Sec4p as a Cα worm on the Sec2p surface. (C) Residues of Sec4p within 4 Å of Sec2p are labeled. Residues contacted in Sec2p are boxed and purple or lilac, depending to which Sec2p monomer they belong. (D) Residues of Sec2p within 4 Å of Sec4p are labeled. Contacted residues in Sec4p are boxed; residues in Switch I and II are indigo and orange, respectively.

Figure 2

The Sec4p binding surface and conservation. (A) Surface representation of Sec2p. Residues within 4 Å of Sec4p are purple. (B) Residues identical in Sec2p and the mammalian Rabin3/8 and GRAB proteins are purple; similar residues (R=K, D=E, I=L=V) are magenta. (C) Sequence alignment of the Sec2p GEF domain with Rabin3/8 (human isoform β1) and GRAB (Rattus norvegicus). Identical and similar residues are purple and magenta, respectively. The Rabin3 and Rabin8 sequences are almost identical. Residues in Sec2p within 4 Å of Sec4p are boxed.

Figure 3

Superpositions of the Cα backbones of nucleotide/Mg++-free Sec4p from the Sec2p/Sec4p complex (lilac) with nucleotide bound forms of Sec4p (purple) (Stroupe & Brunger, 2000). In the nucleotide-free Sec4p from the complex, switches I and II and the P-loop are labeled, and it is superimposed with: (A) the GDP-bound form. Yellow circles indicate the Cα positions of Phe45. (B) an additional GDP-bound form of Sec4p. (C) the GppNHp-bound form. The conformation of switch II in nucleotide-free Sec4p from the complex more closely resembles that of the GppNHp-bound form than that of the GDP-bound form. (D) Nucleotide-free Sec4p with the positions of GppNHP and Mg++ derived from the GppNHp-bound Sec4p structure indicated. Phe45 and Ile50 are indicated.

Figure 4

Importance of the Sec4p switch regions for GEF activity. (A) Sequences of the switch regions and P-loops of different Rabs. Sec4p residues within 4 Å of Sec2p in the complex are underlined. (B) Comparison of the release of [3H]GDP from 200 nM wild type Sec4p and from the Ile50Ala Sec4p mutant in the absence and presence of 100 nM Sec2p exchange domain. Sec2p catalyzed exchange is impaired for the mutant as compared to wild type Sec4p. Here Sec4p constructs are expressed as GST-fusion proteins. (C) Comparison of the release of [3H]GDP from 200 nM wild type Ypt1p and Ypt1p with switch I replaced by the Sec4p sequence, Ypt1-SWI(Sec4), in the absence or presence of 100 nM Sec2p exchange domain. While Sec2p does not act on Ypt1p, Ypt1-SWI(Sec4) is a substrate. (D) Comparison of the release of [3H]GDP from 200 nM wild type Sec4p and Sec4p with switch I replaced by the Ypt1p sequence, Sec4-SWI(Ypt1), in the absence or presence of 100 nM Sec2p exchange domain. Sec2p does not stimulate GDP exchange for Sec4-SWI(Ypt1). (E) Comparison of the release of [3H]GDP from 200 nM Sec4p with switch II replaced by the Rab6 sequence, Sec4p-SWII(Rab6), and Rab6 with a Sec4p switch Rab6-SWII(Sec4), in the absence or presence of 100 nM Sec2p exchange domain. Sec2p does not act on Sec4p-SWII(Rab6) or Rab6-SWII(Sec4). Sec4p and Rab6 constructs are expressed as GST-fusions. Error bars represent standard deviation.

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A catalytic coiled coil: structural insights into the activation of the Rab GTPase Sec4p by Sec2p - PubMed (original) (raw)