PDBsum entry 2v8s

Protein transport

PDB id: 2v8s

Contents

Protein chains

137 a.a. *

93 a.a. *

Ligands

GOL ×5

Waters ×75

* Residue conservation analysis

PDB id:

2v8s

Name:

Protein transport

Title:

Vti1b habc domain - epsinr enth domain complex

Structure:

Clathrin interactor 1. Chain: e. Fragment: enth, residues 20-166. Synonym: epsinr, epsin-4, epsin-related protein, enthoprotin, clathrin-interacting protein localized in the trans-golgi region, clint. Engineered: yes. Vesicle transport through interaction with t-snares homolog 1b.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

2.22Å R-factor: 0.201 R-free: 0.284

Authors:

D.J.Owen,A.J.Mccoy,B.M.Collins,S.E.Miller

Key ref:

S.E.Miller et al. (2007). A SNARE-adaptor interaction is a new mode of cargo recognition in clathrin-coated vesicles. Nature, 450, 570-574. PubMed id: 18033301 DOI: 10.1038/nature06353

Date:

14-Aug-07 Release date: 27-Nov-07

Protein chain

Q14677  (EPN4_HUMAN) -  Clathrin interactor 1 from Homo sapiens

Seq: 625 a.a.

Struc: 137 a.a.

Protein chain

Q9UEU0  (VTI1B_HUMAN) -  Vesicle transport through interaction with t-SNAREs homolog 1B from Homo sapiens

Seq: 232 a.a.

Struc: 93 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chains E, V: E.C.?

DOI no: 10.1038/nature06353

Nature 450:570-574 (2007)

PubMed id: 18033301

A SNARE-adaptor interaction is a new mode of cargo recognition in clathrin-coated vesicles.

S.E.Miller, B.M.Collins, A.J.McCoy, M.S.Robinson, D.J.Owen.

ABSTRACT

Soluble NSF attachment protein receptors (SNAREs) are type II transmembrane proteins that have critical roles in providing the specificity and energy for transport-vesicle fusion and must therefore be correctly partitioned between vesicle and organelle membranes. Like all other cargo, SNAREs need to be sorted into the forming vesicles by direct interaction with components of the vesicles' coats. Here we characterize the molecular details governing the sorting of a SNARE into clathrin-coated vesicles, namely the direct recognition of the three-helical bundle H(abc) domain of the mouse SNARE Vti1b by the human clathrin adaptor epsinR (EPNR, also known as CLINT1). Structures of each domain and of their complex show that this interaction (dissociation constant 22 muM) is mediated by surface patches composed of approximately 15 residues each, the topographies of which are dependent on each domain's overall fold. Disruption of the interface with point mutations abolishes the interaction in vitro and causes Vti1b to become relocalized to late endosomes and lysosomes. This new class of highly specific, surface-surface interaction between the clathrin coat component and the cargo is distinct from the widely observed binding of short, linear cargo motifs by the assembly polypeptide (AP) complex and GGA adaptors and is therefore not vulnerable to competition from standard motif-containing cargoes for incorporation into clathrin-coated vesicles. We propose that conceptually similar but mechanistically different interactions will direct the post-Golgi trafficking of many SNAREs.

Selected figure(s)

Figure 1.
Figure 1: Mapping the binding sites on the Vti1b H[abc] domain and the EPNR ENTH Delta-alpha-0 domain on their isolated structures. a, b, Ribbon diagram showing the three-helix bundle (H[abc] domain) of uncomplexed Vti1b (a, light green) and of uncomplexed EPNR ENTH 0 (b, pale pink). Surface views are shown in the same orientations. Mutated residues on both representations are coloured green if they affected binding to EPNR, pink if they affected binding to Vti1b H[abc] and grey-blue if there was no effect (surface views only). c–f, Pull-down experiments detecting the binding of EPNR ENTH–Myc constructs to GST–Vti1b by western blotting for the Myc tag. c, The effect of mutations in GST–Vti1b on their binding of wild-type EPNR ENTH–Myc. d, e, The effect of point (d) and helix-deletion (e) mutations in EPNR ENTH–Myc on their binding to wild-type GST–Vti1b. f, The effect of the charge-swap mutations EPNR(R146E) and Vti1b(E23R) introduced on the basis of the complex structure.

Figure 2.
Figure 2: The EPNR ENTH Delta-alpha-0–Vti1b H[abc] domain complex. a, The complex is shown, with Vti1b coloured dark green to pale green (N to C) and EPNR coloured pale pink to dark pink (N to C). Enlarged views are shown of key residues in the binding interface. The putative lipid binding helix 0 of EPNR is shown in grey. The proposed orientation of the remaining portions of Vti1b and EPNR are indicated by dotted lines. Charge-swap mutations are boxed. b, Surface representations of the complex, with each rotated by 90 degrees to show the 'footprint' of interaction coloured green on Vti1b H[abc] and pink on EPNR ENTH. C ribbons and side chains participating in the interaction are shown through the different surfaces. Mutated residues that have been demonstrated to affect binding are boxed. c, Structure-based sequence alignment of the H[abc] domains of mouse Vti1b, mouse Vti1a and yeast Vti1 (with conserved residues boxed in green), and of human EPNR ENTH domain with yeast Ent3 (with conserved residues shown in pink). Residues in which mutation abolishes binding between Vti1b and EPNR are marked with an asterisk. Residues that have significant roles in the binding interface are indicated by a triangle.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2007, 450, 570-574) copyright 2007.

Figures were selected by an automated process.