PDBsum entry: 1lf8

Signaling protein

PDB id: 1lf8

Contents

Protein chains

151 a.a. *

159 a.a. *

Ligands

SEP-ASP-GLU-ASP-LEU-LEU-HIS-ILE ×4

Waters ×300

* Residue conservation analysis

PDB id:

1lf8

Name:

Signaling protein

Title:

Complex of gga3-vhs domain and ci-mpr c-terminal phosphopeptide

Structure:

Adp-ribosylation factor binding protein gga3. Chain: a, b, c, d. Fragment: vhs domain (residues 1-166). Synonym: golgi-localized, gamma ear-containing, arf- binding protein 3. Engineered: yes. Cation-independent mannose-6-phosphate receptor. Chain: e, f, g, h. Fragment: c-terminus (residues 2480-2491).

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008. Synthetic: yes. Other_details: the peptide was chemically synthesized. The sequence of the peptide is naturally found in homo sapiens (human).

Biol. unit:

Octamer (from PQS)

Resolution:

2.30Å R-factor: 0.211 R-free: 0.255

Authors:

Y.Kato,S.Misra,R.Puertollano,J.H.Hurley,J.S.Bonifacino

Key ref:

Y.Kato et al. (2002). Phosphoregulation of sorting signal-VHS domain interactions by a direct electrostatic mechanism. Nat Struct Biol, 9, 532-536. PubMed id: 12032548 DOI: 10.1038/nsb0698-422

Date:

10-Apr-02 Release date: 26-Jun-02

Protein chains

Q9NZ52  (GGA3_HUMAN) -  ADP-ribosylation factor-binding protein GGA3

Seq: 723 a.a.

Struc: 151 a.a.

Protein chains

Q9NZ52  (GGA3_HUMAN) -  ADP-ribosylation factor-binding protein GGA3

Seq: 723 a.a.

Struc: 159 a.a.

 Gene Ontology (GO) functional annotation 

• Biological process: intracellular protein transport (1 term)

DOI no: 10.1038/nsb0698-422

Nat Struct Biol 9:532-536 (2002)

PubMed id: 12032548

Phosphoregulation of sorting signal-VHS domain interactions by a direct electrostatic mechanism.

Y.Kato, S.Misra, R.Puertollano, J.H.Hurley, J.S.Bonifacino.

ABSTRACT

Phosphorylation of the cytosolic tails of transmembrane receptors can regulate their intracellular trafficking. The structural basis for such regulation, however, has not been explained in most cases. The cytosolic tail of the cation-independent mannose 6-phosphate receptor contains a serine residue within an acidic-cluster dileucine signal that is important for the function of the receptor in the biosynthetic sorting of lysosomal hydrolases. We show here that phosphorylation of this Ser enhances interactions of the signal with its recognition module, the VHS domain of the GGA proteins. Crystallographic analyses demonstrate that the phosphoserine residue interacts electrostatically with two basic residues on the VHS domain of GGA3, thus providing an additional point of attachment of the acidic-cluster dileucine signal to its recognition module.

Selected figure(s)

Figure 3.
Figure 3. Phosphorylation enhances binding of the CI-MPR tail to the GGA3 VHS domain in vitro. a, Recombinant His[6]−GGA3-VHS was tested for interactions with GST fusion proteins bearing the cytosolic tail sequences indicated on top. Some of the GST fusion protein samples were subjected to phosphorylation by recombinant CKII in the absence or presence of heparin, as indicated in the figure. The GST fusion proteins were collected by incubation with glutathione−Sepharose, and the bound His[6]−GGA3-VHS was resolved by SDS-PAGE and detected by immunoblotting with anti-His[6]. Notice the increased binding of His[6]−GGA3-VHS to the GST−CI-MPR tail construct upon treatment with CKII (lane 2). b, Isothermal titration calorimetry measuring the affinities of unphosphorylated CI-MPR (CI) and phosphorylated CI-MPR (pCI) peptides for GGA1-VHS and GGA3-VHS domains. Inset: Differential heat released when 1 mM pCI-MPR peptide is injected into 50 M GGA1-VHS. The trace is shown after subtraction of data from injection of pCI-MPR into a buffer blank. c, K[d] and n (stoichiometry) for the different peptide−VHS domain interactions expressed as the mean s.d.

Figure 4.
Figure 4. Structure of the GGA3 VHS domain bound to phosphorylated CI-MPR peptide. a, Overall architecture of the complex. The helices of the VHS domain are numbered. The peptide (CPK bonds) is bound between helices 6 and 8 of the VHS domain. The phosphoserine (pSer (-1)) and the two critical Leu residues (Leu 3 and Leu 4) of the peptide and the VHS residues that interact with the phosphoserine (Lys 86 and Arg 88) are labeled. b, Surface electrostatics of the VHS domain with the bound CI-MPR phosphopeptide. The key pSer (-1), Asp 0, Leu 3 and Leu 4 residues and the C-terminus of the phosphopeptide are labeled. Saturated red and blue areas are at -10kT and +10kT, respectively. c, Stereo view of the phosphoserine-binding site. The phosphopeptide residues have gray carbon bonds, and residues from the VHS domain are colored green The electron density for the phosphopeptide residues is shown as a SIGMAA-weighted 2mF[o] - dF[c] omit map (phosphopeptide omitted, map contoured at 0.9 ). Interactions between the side chains of Lys 86 and Arg 88 and the phosphoserine are shown as dashed lines. d, Superimposition of the first five residues of the CI-MPR peptide and residues 86−88 of the VHS domain in the phosphopeptide complex and the native peptide complex. The phosphopeptide and VHS domain residues from the phosphopeptide complex structure are colored as in (c). In the unphosphorylated peptide complex, the peptide and the VHS domain residues are colored magenta and cyan, respectively.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Biol (2002, 9, 532-536) copyright 2002.

Figures were selected by the author.