PDBsum entry: 1iou

Membrane protein

PDB id: 1iou

Contents

Protein chain

140 a.a. *

* Residue conservation analysis

PDB id:

1iou

Name:

Membrane protein

Title:

Solution structure of ykt6p (1-140)

Structure:

Ykt6p. Chain: a. Fragment: residues 1-140. Synonym: synaptobrevin homolog 1. Engineered: yes

Source:

Saccharomyces cerevisiae. Baker's yeast. Organism_taxid: 4932. Expressed in: escherichia coli. Expression_system_taxid: 562

NMR struc:

20 models

Authors:

H.Tochio,M.M.K.Tsui,D.K.Banfield,M.Zhang

Key ref:

H.Tochio et al. (2001). An autoinhibitory mechanism for nonsyntaxin SNARE proteins revealed by the structure of Ykt6p. Science, 293, 698-702. PubMed id: 11474112 DOI: 10.1126/science.1062950

Date:

09-Apr-01 Release date: 06-May-03

Protein chain

P36015  (YKT6_YEAST) -  Synaptobrevin homolog YKT6

Seq: 200 a.a.

Struc: 140 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Gene Ontology (GO) functional annotation 

• Cellular component: integral to membrane (1 term)
• Biological process: transport (2 terms)

DOI no: 10.1126/science.1062950

Science 293:698-702 (2001)

PubMed id: 11474112

An autoinhibitory mechanism for nonsyntaxin SNARE proteins revealed by the structure of Ykt6p.

H.Tochio, M.M.Tsui, D.K.Banfield, M.Zhang.

ABSTRACT

Ykt6p is a nonsyntaxin SNARE implicated in multiple intracellular membrane trafficking steps. Here we present the structure of the NH2-terminal domain of Ykt6p (Ykt6pN, residues 1 to 140). The structure of Ykt6pN differed entirely from that of syntaxin and resembled the overall fold of the actin regulatory protein, profilin. Like some syntaxins, Ykt6p adopted a folded back conformation in which Ykt6pN bound to its COOH-terminal core domain. The NH2-terminal domain plays an important biological role in the function of Ykt6p, which in vitro studies revealed to include influencing the kinetics and proper assembly of SNARE complexes.

Selected figure(s)

Figure 2.
Fig. 2. Interaction of the core with the NH[2]-terminal domain of Ykt6p. (A) The surface exposed hydrophobic surface of Ykt6pN. Lys and Arg are in blue; Asp and Glu are in red; Phe, Val, Leu, Ile, Tyr, Ala, and Trp are in yellow; all other amino acids are shown in white. The conserved hydrophobic amino acid residues are labeled. The orientation of the molecule is as shown in (B). (B) Summary of 1H and 15N combined chemical shift changes in the NH[2]-domain of Ykt6p induced by the core domain. Because of the poor spectral quality of full-length Ykt6p, the shift perturbation was extracted from the 1H,15N- HSQC spectra of Phe42Glu-Ykt6pN and full-length, Phe42Glu-Ykt6p. (C) Representative regions of 1H,15N-HSQC spectra of Ykt6pN (blue), Ykt6pNC (green), and the full-length Ykt6p (red), showing the core domain binding-induced chemical shift changes in Ykt6pN. Inclusion of the intact core domain leads to the disappearance of a number of resonances for amino acids in III. In each case the concentration of the proteins used for NMR was <0.3 mM. The NMR spectra of Ykt6pN did not vary at a concentration range of 0.1 to 1.0 mM indicating that the protein chemical shift changes observed were not likely to be a result of nonspecific aggregation. (D) Amino acid sequence alignments of Ykt6p and its homologues. Completely conserved amino acid residues are highlighted in red, and highly conserved residues in green. The secondary structure of Ykt6pN is shown above the alignment. The heptad repeats region of the core domain is labeled as in (39). The isoprenylation motif of the protein is indicated by a dashed open box. The NCBI accession numbers of the proteins are Saccharomyces cerevisiae (NP012725), Candida albicans (CAA21982), Schizosaccharomyces pombe (CAA18664), mouse (NP062635), rat (AAD09152), human (NP006546), Xenopus leavis (AAC32182), Drosophila melanogastor (AAF46294), Caenorhabditis elegans (AAD31930), Nicotiana tabacum (AAD00116), and Arabidopsis thaliana (AAD00112).

Figure 3.
Fig. 3. The NH[2]-terminal domain of Ykt6p sequesters its core domain and is important for function. (A) Gel-filtration analysis showing that both Ykt6pN and Ykt6p exist as monomers in solution. The elution volumes of molecular mass standards are indicated on the top of the panel. The column buffer was identical to that used in NMR experiments and the concentration of the proteins was in the range of 0.02 to 0.15 mM. (B) Viability of yeast strains expressing mutant forms of Ykt6p. SARY158 cells [ykt6 pYKT6, see (14)] containing various ykt6 mutants were patched onto media containing 5-FOA. Lack of growth on 5-FOA indicates that the particular mutant cannot suffice as the sole source of Ykt6p. The plus symbols (+) indicate the relative growth rates of SARY158 cells expressing ykt6 mutant proteins as their sole source of Ykt6 protein where +++ is wild type, and - is no growth. (C) The NH[2]-domain of Ykt6p interacts with its core domain in the two-hybrid system. Core domain, NH[2]-domain, and full-length Ykt6p in bait vector were tested against wild-type NH[2]-domain, mutant (Phe42Glu) NH[2]-domain, and full-length wild-type Ykt6p or Phe42Glu-Ykt6p in prey vector. Empty prey vector was used as a control. (D) Phe42Glu-Ykt6p accelerates SNARE-complex formation in vitro. GST-Vti1p was incubated with (His)[6]-Sed5p (25 µg) and (His)[6]-Tlg1p (8 µg) together with either Ykt6p or Phe42Glu-Ykt6p (25 µg) at 4°C for 15 min, 30 min, 60 min, 120 min, or 22 hours (lanes 1 to 5 and 6 to 10, respectively). (E) Substitution of Ala for Phe at position 42 in the N-domain of Ykt6p results in defects in protein trafficking (20). ykt6^Phe42Ala cells accumulate p1 carboxypeptidase Y (CPY) at 12°C and miss-sort p2 CPY at 30°C. mCPY (mature form), p1 CPY (endoplasmic reticulum-modified form), p2 CPY (Golgi-modified form). (F) Phe42Ala-ykt6p is stable. Phe42Ala-ykt6 and wild-type (YKT6) yeast strains were grown at 12°C or 30°C to an optical density OD[660] of 0.6 and 1.5 OD[660] equivalents of total yeast protein were assayed by immunodetection with antibodies against Ykt6p.

The above figures are reprinted by permission from the AAAs: Science (2001, 293, 698-702) copyright 2001.

Figures were selected by an automated process.