PDBsum entry 1nty

Signaling protein

PDB id: 1nty

Contents

Protein chain

305 a.a. *

Waters ×354

* Residue conservation analysis

PDB id:

1nty

Name:

Signaling protein

Title:

Crystal structure of the first dh/ph domain of trio to 1.7 a

Structure:

Triple functional domain protein. Chain: a. Fragment: n-terminal dh/ph domains, residues 1225-1535. Synonym: ptprf interacting protein. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: trio. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693.

Resolution:

1.70Å R-factor: 0.195 R-free: 0.214

Authors:

K.R.Skowronek,Y.Zheng,N.Nassar

Key ref:

K.R.Skowronek et al. (2004). The C-terminal basic tail of RhoG assists the guanine nucleotide exchange factor trio in binding to phospholipids. J Biol Chem, 279, 37895-37907. PubMed id: 15199069 DOI: 10.1074/jbc.M312677200

Date:

30-Jan-03 Release date: 29-Jun-04

Protein chain

O75962  (TRIO_HUMAN) -  Triple functional domain protein from Homo sapiens

Seq: 3097 a.a.

Struc: 305 a.a.

Enzyme reactions

• Enzyme class: E.C.2.7.11.1 - non-specific serine/threonine protein kinase.
• Reaction:
  1. L-seryl-[protein] + ATP = O-phospho-L-seryl-[protein] + ADP + H⁺
  2. L-threonyl-[protein] + ATP = O-phospho-L-threonyl-[protein] + ADP + H⁺

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.M312677200

J Biol Chem 279:37895-37907 (2004)

PubMed id: 15199069

The C-terminal basic tail of RhoG assists the guanine nucleotide exchange factor trio in binding to phospholipids.

K.R.Skowronek, F.Guo, Y.Zheng, N.Nassar.

ABSTRACT

The multidomain protein Trio regulates among others neuronal outgrowth and axonal guidance in vertebrates and invertebrates. Trio contains two Dbl-homology/pleckstrin homology (DH/PH) tandem domains that activate several RhoGTPases. Here, we present the x-ray structure of the N-terminal DH/PH, hereafter TrioN, refined to 1.7-A resolution. We show that the relative orientations of the DH and PH domains of TrioN and free Dbs are similar. However, this relative orientation is dissimilar to Dbs in the Dbs/Cdc42 structure. In vitro nucleotide exchange experiments catalyzed by TrioN show that RhoG is approximately 3x more efficiently exchanged than Rac and support the conclusion that RhoG is likely the downstream target of TrioN. Residues 54 and 69, which are not conserved between the two GTPases, are responsible for this specificity. Dot-blot assay reveals that the TrioN-PH domain does not detectably bind phosphatidylinositol 3,4-bisphosphate, PtdIns(3,4)P(2), or other phospholipids. This finding is supported by our three-dimensional structure and affinity binding experiments. Interestingly, the presence of RhoG but not Rac or a C-terminal-truncated RhoG mutant allows TrioN to bind PtdIns(3,4)P(2) with a micromolar affinity constant. We conclude the variable C-terminal basic tail of RhoG specifically assists the recruitment of the TrioN-PH domain to specific membrane-bound phospholipids. Our data suggest a role for the phosphoinositide 3-kinase, PI 3-kinase, in modulating the Trio/RhoG signaling pathway.

Selected figure(s)

Figure 4.
FIG. 4. A, superposition of the PH domains of PLC- 1 (light gray) and TrioN (dark gray). The residues of PLC- 1 involved in the binding to the Ins(1,4,5)P[3] moiety are shown in blue ball-and-stick. The corresponding residues in TrioN are shown in pink ball-and-stick and labeled. B, superposition of the PH domains of Grp1 (light gray) and TrioN (dark gray). The residues of Grp1 involved in the binding of Ins(1,3,4,5)P[4] are shown in blue ball-and-stick. The corresponding residues in TrioN are shown in pink ball-and-stick and labeled. C, sequence alignment of the PH domains of TrioN, PLC- 1, and Grp1 deduced from the superposition of their three-dimensional structures done with O (47). Secondary structure elements deduced from the TrioN-PH domain structure are also shown. Residues that interact with the phosphates of the inositol ring at positions 3, 4, and 5 are shown in red, green, and blue, respectively.

Figure 5.
FIG. 5. TrioN binds strongly to phospholipids only in the presence of RhoG. His[6]-tagged bacterially purified TrioN was applied to strips spotted with various phospholipids (Echelon) and revealed with an anti-His or an anti-GST antibody as detailed under "Experimental Procedures." All strips were revealed under the same conditions. Each experiments was repeated at least three times of which one representative is shown. A, His[6]-TrioN at 0.5 µg/ml. B, His[6]-TrioN at 50 µg/ml. C, His[6]-TrioN/RhoG complex at 0.5 µg/ml. D, RhoG deletion mutant lacking the C-terminal basic residues, RhoG 182, in complex with His[6]-TrioN at 0.5 µg/ml. E, GST-RhoG at 0.5 µg/ml. F, His[6]-TrioN/Rac complex at 0.5 µg/ml. G, Rac deletion mutant lacking the C-terminal basic residues, Rac 184, in complex with His[6]-TrioN at 0.5 µg/ml. H, His[6]-Rac at 0.5 µg/ml. I, bacterially expressed PLC- 1-PH domain at 0.5 µg/ml. This strip serves as a positive control and shows that our experimental procedure is reliable. J, nomenclature of the different phospholipids spotted on the nitrocellulose strip. Comparison of C and I shows that the binding affinity of TrioN/RhoG to PtdIns(3,4)P[2], PA, or PtdIns is similar to PLC- 1 binding to PtdIns(4,5)P[2] ( 1 µM) (62).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 37895-37907) copyright 2004.

Figures were selected by an automated process.