A crystallographic view of interactions between Dbs and Cdc42: PH domain-assisted guanine nucleotide exchange - PubMed (original) (raw)
A crystallographic view of interactions between Dbs and Cdc42: PH domain-assisted guanine nucleotide exchange
Kent L Rossman et al. EMBO J. 2002.
Abstract
Dbl-related oncoproteins are guanine nucleotide exchange factors (GEFs) specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. While it is known that the DH domain is the principal catalytic subunit, recent biochemical data indicate that for some Dbl-family proteins, such as Dbs and Trio, PH domains may cooperate with their associated DH domains in promoting guanine nucleotide exchange of Rho GTPases. In order to gain an understanding of the involvement of these PH domains in guanine nucleotide exchange, we have determined the crystal structure of a DH/PH fragment from Dbs in complex with Cdc42. The complex features the PH domain in a unique conformation distinct from the PH domains in the related structures of Sos1 and Tiam1.Rac1. Consequently, the Dbs PH domain participates with the DH domain in binding Cdc42, primarily through a set of interactions involving switch 2 of the GTPase. Comparative sequence analysis suggests that a subset of Dbl-family proteins will utilize their PH domains similarly to Dbs.
Figures
Fig. 1. Structure of the Dbs·Cdc42 complex. (A) A schematic representation of the putative signaling domains and their associated sequence ranges within full-length Dbs (murine, DDBJ/EMBL/GenBank accession No. AAB33461), as predicted by the protein sequence analysis program SMART (Schultz et al., 1998). Also indicated is the fragment of Dbs used for crystallization of the Dbs·Cdc42 complex (residues 623–967). In addition to the DH and PH domains, Dbs also possesses a domain homologous to the Saccharomyces cerevisiae phosphatidylinositol transfer protein Sec14p (SEC14), two spectrin repeats (SPEC) and Src homology 3 domain (SH3). (B) A ribbon diagram of the Dbs·Cdc42 complex shows that the DH domain (yellow) of Dbs primarily engages the switch regions (red) of Cdc42 (green). The PH domain (blue) also interacts with the GTPase by means of a conformation unique from that of Sos1 and Tiam1·Rac1. The disordered β1/β2 loop within the PH domain is gray. (C) The Dbs·Cdc42 complex is shown rotated 90° about the vertical axis relative to its orientation in (B). (D) The experimental electron density (white) contoured at 1.2σ in the vicinity of the vacant nucleotide binding site. Bound waters are magenta.
Fig. 2. Sequence alignments of Dbl-family exchange factors and Rho GTPases. (A) Sequences for Dbs, Tiam1 and Sos1 were aligned using Clustal_X v1.8 (Thompson et al., 1997), and manually altered to further align secondary structure elements. The consensus sequence was generated from a Dbl-family alignment of 47 non-redundant sequences and conserved regions (CR) are boxed. Crystallographically determined helices (yellow) and β-strands (green) of Dbs are shown above the alignment, and similar secondary structure elements of Tiam1 and Sos1 are shaded. Red italicized amino acids indicate direct contacts with bound GTPases (Dbs·Cdc42 or Tiam1·Rac1). PH domain residues thought to be involved in binding phospholipids are indicated by blue italics (Koshiba et al., 1997; see Figure 8A). Small arrows indicate construct borders, lightened italicized residues are disordered in the structures, small dots indicate 10-residue spans and numbers indicate amino acid positions within the full-length proteins. DDBJ/EMBL/GenBank accession Nos are: Dbs, AAB33461; Tiam1, Q60610; Sos1, A37488. (B) Sequences for 16 Rho GTPases and Ras were aligned using Clustal_X v1.8 (Thompson et al., 1997) and those for Cdc42 and Rac1 are shown. Residues in Cdc42 and Rac1 that bury >10 Å2 upon complex formation with Dbs or Tiam1, respectively, are indicated in red italics, and nomenclature for secondary structure elements are derived from the structure of Ras (Pai et al., 1990). In blue italics are buried residues that differ between Cdc42 and Rac1 and are probably important for dictating specificity between GTPases and DH domains (Worthylake et al., 2000; Karnoub et al., 2001). Also highlighted (underscore boxes) are the switches that undergo conserved, nucleotide-dependent conformational alteration in GTPases, as well as the 21 amino acid insertion unique to Rho GTPases. The buried accessible surface areas for residues losing >10 Å2 upon complex formation within (C) the Dbs DH/PH domain or (D) Cdc42 were determined and plotted for each residue number.
Fig. 3. Comparison of the relative orientations between DH and PH domains from Dbs, Tiam1 and Sos1. The DH and PH domains from the structures of Dbs·Cdc42 (top), Tiam1·Rac1 (bottom left) and Sos1 (bottom right) were aligned by least squares superposition of the DH domains conserved regions 1–3. DH domains are colored yellow and PH domains are blue. To aid visualization, the C-terminal helix of each PH domain (αC) is colored from blue to red (N- to C-terminus, respectively) and the αC helical axes are represented by black dashed lines. The yellow dashed line in Sos1 indicates the disordered residues in the DH domain. Whether the PH domains of Tiam1 and Sos1 can adopt Dbs-like conformations to enhance exchange, e.g. at cell membranes or through the actions of accessory proteins, is unclear.
Fig. 4. Interactions between the PH domain of Dbs and Cdc42. Stereo view of the PH domain (blue) participating with α6 of the DH domain (yellow) to bind switch 2 (red) and α3b (green) of Cdc42. Dashed lines indicate hydrogen bonds (<3.3Å).
Fig. 5. Biochemical analysis of PH domain-mediated interactions with Cdc42. Substitutions within the DH/PH fragment of Dbs (K885A, Y889F, H814A) remove specific interactions with Cdc42 and differentially affect exchange activity on (A) Cdc42 or (B) RhoA. For comparison, also shown is the wild-type (wt) DH/PH fragment, as well as the isolated DH domain (DH) and no exchange factor (none). (C) Substitutions in Cdc42 (R66A, H103A) at sites in contact with the Dbs PH domain exhibit minor effects on Dbs-stimulated guanine nucleotide exchange compared with wild-type Cdc42.
Fig. 6. Comparison of native and Dbs(Y889F)·Cdc42 structures. (A) 2_F_o – _F_c density contoured at 1.2σ on the final coordinates of the native complex and (B) Dbs(Y889F)·Cdc42 in the vicinity of Tyr889. For illustrative purposes, the wild-type coordinates including Tyr889 were refined using the Dbs(Y889F)·Cdc42 data. _F_o – _F_c difference density (green) contoured at –6.0σ surrounds the position of the inappropriate tyrosine hydroxyl oxygen atom.
Fig. 7. Sequence alignment of Dbs-like Dbl-family members. A multiple sequence alignment of non-redundant Dbl-family members was used to identify sequences containing identity to residues in Dbs important for PH domain enhancement of nucleotide exchange. Only the regions corresponding to α6 of the DH domain and β1 and β4 of the PH domain of Dbs are shown. Identical residues are shown boxed in black; similar conserved residues are boxed blue. Highlighted in red are the highly conserved residues corresponding to His814, Gln834 and Tyr889 of Dbs. Trio/N and Unc-73/N refer to the N-terminal DH and PH domains within these proteins. M.m., Mus musculus; H.s., Homo sapiens; D.m., Drosophila melanogaster; C.e., Caenorhabditis elegans.
Fig. 8. The structure of Dbs·Cdc42 suggests a model for membrane engagement. (A) The structures of the Grp1 and Dapp1 PH domains bound to Ins(1,3,4,5)P4 (RCSB accession Nos 1FGY and 1FAO, respectively) were superimposed upon the Dbs·Cdc42 crystal structure coordinates to identify residues within the Dbs PH domain likely to participate in phospholipid binding. Shown are the Ins(1,3,4,5)P4 (magenta and green) from the Grp1 structure and a subset of Dbs residues (Arg861, Lys874, Lys892, Gln893 and Tyr924) (light brown) at positions common to Dapp1 or Grp1 that are utilized for inositol phosphate recognition. (B) The electrostatic potential (contoured from –2 kT to +2 kT) for the Dbs·Cdc42 complex. Regions covered by blue and red mesh indicate positive and negative electrostatic potential, respectively. (C) The probable orientation of Dbs·Cdc42 (colored as in Figure 1) at negatively charged membranes (silver) as suggested by the highly polarized electrostatic potential, and known sites of membrane engagement by Cdc42 (geranylgeranyl) and PH domains [phosphatidylinositol (4,5) bisphosphate].
References
- Aghazadeh B., Zhu,K., Kubiseski,T.J., Liu,G.A., Pawson,T., Zheng,Y. and Rosen,M.K. (1998) Structure and mutagenesis of the Dbl homology domain. Nature Struct. Biol., 5, 1098–1107. - PubMed
- Aghazadeh B., Lowry,W.E., Huang,X.Y. and Rosen,M.K. (2000) Structural basis for relief of autoinhibition of the Dbl homology domain of proto-oncogene Vav by tyrosine phosphorylation. Cell, 102, 625–633. - PubMed
- Aravind L., Neuwald,A.F. and Ponting,C.P. (1999) Sec14p-like domains in NF1 and Dbl-like proteins indicate lipid regulation of Ras and Rho signaling. Curr. Biol., 9, R195–R197. - PubMed
- Boriack-Sjodin P.A., Margarit,S.M., Bar-Sagi,D. and Kuriyan,J. (1998) The structural basis of the activation of Ras by Sos. Nature, 394, 337–343. - PubMed
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