PDBsum entry 1txd

Signaling protein

PDB id: 1txd

Contents

Protein chain

352 a.a. *

Waters ×91

* Residue conservation analysis

PDB id:

1txd

Name:

Signaling protein

Title:

Crystal structure of the dh/ph domains of leukemia-associated rhogef

Structure:

Rho guanine nucleotide exchange factor 12. Chain: a. Fragment: dh/ph domains. Synonym: leukemia-associated rhogef. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: arhgef12, larg, kiaa0382. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.13Å R-factor: 0.237 R-free: 0.274

Authors:

R.Kristelly,G.Gao,J.J.Tesmer

Key ref:

R.Kristelly et al. (2004). Structural determinants of RhoA binding and nucleotide exchange in leukemia-associated Rho guanine-nucleotide exchange factor. J Biol Chem, 279, 47352-47362. PubMed id: 15331592 DOI: 10.1074/jbc.M406056200

Date:

03-Jul-04 Release date: 21-Sep-04

Protein chain

Q9NZN5  (ARHGC_HUMAN) -  Rho guanine nucleotide exchange factor 12 from Homo sapiens

Seq: 1544 a.a.

Struc: 352 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DOI no: 10.1074/jbc.M406056200

J Biol Chem 279:47352-47362 (2004)

PubMed id: 15331592

Structural determinants of RhoA binding and nucleotide exchange in leukemia-associated Rho guanine-nucleotide exchange factor.

R.Kristelly, G.Gao, J.J.Tesmer.

ABSTRACT

Rho guanine-nucleotide exchange factors (RhoGEFs) activate Rho GTPases, and thereby regulate cytoskeletal structure, gene transcription, and cell migration. Leukemia-associated RhoGEF (LARG) belongs to a small subfamily of RhoGEFs that are RhoA-selective and directly activated by the Galpha12/13 family of heterotrimeric G proteins. Herein we describe the atomic structures of the catalytic Dbl homology (DH) and pleckstrin homology (PH) domains of LARG alone and in complex with RhoA. These structures demonstrate that the DH/PH domains of LARG can undergo a dramatic conformational change upon binding RhoA, wherein both the DH and PH domains directly engage RhoA. Through mutational analysis we show that full nucleotide exchange activity requires a novel N-terminal extension on the DH domain that is predicted to exist in a broader family of RhoGEFs that includes p115-RhoGEF, Lbc, Lfc, Net1, and Xpln, and identify regions within the LARG PH domain that contribute to its ability to facilitate nucleotide exchange in vitro. In crystals of the DH/PH-RhoA complex, the active site of RhoA adopts two distinct GDP-excluding conformations among the four unique complexes in the asymmetric unit. Similar changes were previously observed in structures of nucleotide-free Ras and Ef-Tu. A potential protein-docking site on the LARG PH domain is also evident and appears to be conserved throughout the Lbc subfamily of RhoGEFs.

Selected figure(s)

Figure 1.
FIG. 1. Atomic structures of the LARG DH/PH domains and their complex with RhoA. a, the uncomplexed LARG DH/PH domains. In all figures, the DH domain is colored orange and the PH domain is blue. The six helical segments of the DH domain (9) are labeled, as are the N1 and N2 helices of the novel N1/ N2 extension at the N terminus of the domain. The side chains of Trp-769, which packs in the hydrophobic core of the N1/ N2 extension, and Glu-1023, which appears to contribute to PH domain-assisted nucleotide exchange, are shown as stick models. Like other RhoGEF PH domains, the LARG PH domain has an N-terminal extension that begins with an -helix ( N), followed by a -strand ( N), and then a 3[10] helix. The loop connecting N and the 3[10] helix is highly variable and can contain long inserts (disordered in the uncomplexed LARG DH/PH structure). The 3 and 4 strands of RhoGEF PH domains are also longer, allowing them to form an additional small -sheet with the N strand. b, the LARG DH/PH-RhoA complex. With respect to panel a, The PH domain has swung 30° downward to engage RhoA (green). The side chains of residues that form a conserved, solvent-exposed hydrophobic patch on the PH domain are drawn as stick models in yellow. This patch forms similar 2-fold dimer interfaces in both the DH/PH and DH/PH-RhoA crystal structures. Based on the position of the C terminus of RhoA (which is geranylgeranylated in vivo), the putative phospholipid binding surface of the PH domain and the flatness and positive charge of the top surface of the complex, the plasma membrane is predicted to run along the top of the panel. The LARG N-3[10] loop becomes ordered upon the binding of RhoA and forms an additional helix ( Nb). The inset shows the tetramer observed in the asymmetric unit of the LARG DH/PH-RhoA crystals, with the DH/PH domains rendered as space-filling models and the RhoA chains as green tubes. c, comparison of the DH/PH domains of LARG, intersectin (PDB code 1KI1 [PDB] ) and Dbs (1LB1 [PDB] ). The structures were aligned by superposition of their GTPase substrates (not shown). The orientation of the LARG PH domain with respect to the DH domain is most similar to that of Dbs, whereas that of intersectin is rotated 18° away from the DH domain and does not contact the GTPase substrate (10). The 6/ N helix of Dbs is shorter than that of LARG, allowing its extended 3- 4 loop to engage its GTPase substrate (not shown) (10). The conformation of the N-terminal DH/PH domains of Trio is essentially the same as that of Dbs (6). LARG, Dbs, and the N-terminal DH/PH domains of Trio exhibit PH domain-assisted nucleotide exchange in vitro, whereas intersectin does not.

Figure 2.
FIG. 2. The N1/ N2 extension of LARG. a, structure of the N1/ N2 extension and its contacts with the switch 1 region of RhoA. Side chains that contribute to the small hydrophobic core of the extension are shown except for Gln789 from 1, whose side-chain packs against Trp-769. Several hydrogen bonds (dashed yellow lines) also likely stabilize the extension: the side chain of Glu-790 forms two backbone hydrogen bonds with the N terminus of N1, and a backbone carbonyl in the 2- 3 loop forms a hydrogen bond with the side chain of Trp-769. The side chain of Gln-789 (not shown) also forms two backbone hydrogen bonds with the 2- 3 loop. b, sequence alignment of the N1/ N2 extensions from Lbc subfamily RhoGEFs, and comparison with the N-terminal extension of Vav. Although they form distinct structures, the N1/ N2 extension of LARG and an analogous N-terminal extension of Vav both appear to modulate RhoGEF activity (43). With respect to LARG, intersectin has a two-residue deletion and several amino acid substitutions in 1 (a glycine substituted for Glu-790 and arginine substituted for Ile-786 in LARG) that could potentially abrogate formation of an equivalent extension. In the crystal structure of the intersectin-Cdc42 complex (42), only four of the residues corresponding to the N-terminal extension (residues 1229-1232) were included in the recombinant protein. Residues that contribute to the hydrophobic core of the N1/ N2 extension are colored orange, and those equivalent to LARG-Glu-790 are green. The number preceding each sequence is the amino acid number of the starting residue. GenBankTM GI numbers of sequences used for the alignment are: LARG, 34395525; PDZ-RhoGEF, 34395516; p115RhoGEF, 34395524; GEF-H1/Lfc, 6919894; Lbc, 6016482; intersectin, 20141591; Net1, 16307475; Xpln, 9506401; Vav, 13124807.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 47352-47362) copyright 2004.

Figures were selected by the author.