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. 2010 Jun 24;5(6):e11291.
doi: 10.1371/journal.pone.0011291.

The minimal autoinhibited unit of the guanine nucleotide exchange factor intersectin

K Farid Ahmad  1 , Wendell A Lim
Affiliations

The minimal autoinhibited unit of the guanine nucleotide exchange factor intersectin

K Farid Ahmad et al. PLoS One. .

Abstract

Intersectin-1L is a member of the Dbl homology (DH) domain guanine nucleotide exchange factors (GEF) which control Rho-family GTPase signaling. Intersectin-1L is a GEF that is specific for Cdc42. It plays an important role in endocytosis, and is regulated by several partners including the actin regulator N-WASP. Intact intersectin-1L shows low Cdc42 exchange activity, although the isolated catalytic DH domain shows high activity. This finding suggests that the molecule is autoinhibited. To investigate the mechanism of autoinhibition we have constructed a series of domain deletions. We find that the five SH3 domains of intersectin are important for autoinhibition, with the fifth domain (SH3(E)) being sufficient for the bulk of the autoinhibitory effect. This SH3 domain appears to primarily interact with the DH domain. We have determined the crystal structure of the SH3(E)-DH domain construct, which shows a domain swapped arrangement in which the SH3 from one monomer interacts with the DH domain of the other monomer. Analytical ultracentrifugation and gel filtration, however, show that under biochemical concentrations, the construct is fully monomeric. Thus we propose that the actual autoinhibited structure contains the related intramolecular SH3(E)-DH interaction. We propose a model in which this intramolecular interaction may block or distort the GTPase binding region of the DH domain.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The minimal autoinhibited unit of intersectin-1L is comprised of the SH3(E)-DH domain fragment (1141–1435).
a) Schematic of intersectin 1L domain structure, with regulatory (SH3 domains) and output domains (DH-PH) highlighted. b) In vitro fluorescence assay showing loading of mant-GDP onto GTPase Cdc42 by DH-PH domain-containing fragments of intersectin 1L. Fragments containing SH3(E) domain exhibit approximately 3-fold repression compared to the DH-PH domain. c) In vitro fluorescence assay comparing loading of mant-GDP onto GTPase Cdc42 by intersectin(SH3(E)-DH) versus intersectin(DH). SH3(E)-DH is repressed approximately 2.5-fold.
Figure 2
Figure 2. Observed secondary structure of intersectin-1L SH3(E)-DH (1141–1435).
a) Sequence alignment of murine intersectin SH3 domains A through E. The observed secondary structure of SH3(E) is indicated above the sequence. The numbering is according to murine intersectin-1L. Black and gray backgrounds are used to indicate identical and/or conserved residues found in at least 50% of the proteins at a given position. b) Sequence alignment of selected DH domains and the observed secondary structure of the intersectin-1L DH domain. Residues from intersectin-1L are numbered. Highly conserved regions among all DH domains are labeled in red as CR1-3.
Figure 3
Figure 3. Ribbon diagram of the intersectin-1L SH3(E)-DH (1141–1435) monomer.
The SH3(E) domain does not interact with the DH domain in the same chain. The linker joining the SH3(E) domain and the DH domain (1204–1225) is colored in purple. The secondary structure elements of the SH3(E) domain and the DH domain are indicated.
Figure 4
Figure 4. Superposition of intersectin-1L DH domains.
The two “repressed” murine intersectin-1L DH domain monomers (blue, and red) and “active” human intersectin-1L DH domain (green). The least-squares superposition of these three chains result in an average pairwise rmsd value of less than 1.0 Å.
Figure 5
Figure 5. Structure of the intersectin-1L SH3(E)-DH domain homodimer.
a) Ribbon diagram of the intersectin-1L SH3(E)-DH domain homodimer. One monomer is colored red, the other blue. The SH3(E) domain of the blue monomer was not present in electron density maps and is modeled here in a transparent blue. b) Schematic diagram of the intersectin-1L SH3(E)-DH domain homodimer, illustrating the terms related to 3D domain swapping. For simplicity, only elements pertaining to one chain are labeled. The closed interface is the interface between the SH3(E) domain and the DH domain. It exists in both the monomer and the domain-swapped dimer. The open interface is the interface between DH domain monomers. It exists only in the domain-swapped dimer, but not in the monomer. The hinge loop connects the SH3(E) domain and the DH domain. It adopts different conformations in the monomer and the domain-swapped dimer. c) Schematic diagram of possible domain organization in SH3E-DH domain monomer. The SH3(E) domain (the swapped domain) forms an intramolecular interaction the DH, and in a manner identical to that of the SH3(E)-DH domain intermolecular interaction in the dimer.
Figure 6
Figure 6. Parallel display of the intersectin-1L DH domain homodimer and intersectin-1L DH domain-Cdc42 complex.
The DH domain in red is in the same orientation in both structures. The DH domain of the second monomer of the intersectin DH domain homodimer is in blue. The Cdc42 molecule in the DH domain-Cdc42 complex is green. The binding site for Cdc42 on the DH domain is occluded by DH-DH homodimer formation.
Figure 7
Figure 7. Intersectin(SH3(E)-DH) is monomeric.
a) Elution profile of Intersectin(SH3(E)-DH) from a Superdex-75 size-exclusion column. Intersectin(SH3(E)-DH) elutes as a single peak between the elution volumes of ovalbumin (44 kDa) and chymotrypsinogen (25 kDa). b) Analytical ultracentrifugation (AUC) analysis of intersectin(SH3(E)-DH). The top curve is at 14,000 rpm and the bottom at 20,000 rpm. Shown below are the residuals for fits to the data. The data fit to an effective molecular weight (σ) of 1.5. c) Predicted σ values of different oligomeric states of intersectin(SH3(E)-DH). A σ value of 1.5 correlates to a molecular weight of 32 kDa, based on AUC analysis of the 12.9 kDa protein profillin (σ = 0.6092).
Figure 8
Figure 8. Surface representation of intersectin-1L DH domain – SH3(E) domain interaction.
The DH domain from one monomer is in red, and the SH3(E) domain from the other monomer is in blue. The SH3(E) domain binds to the DH domain on the side of the molecule opposite the Cdc42 binding site (labeled). The polyproline type II helix binding groove on the SH3E domain (purple) does not overlap with the DH domain- SH3(E) domain interface.
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References

    1. Rossman KL, Der CJ, Sondek J. GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol. 2005;6:167–180. - PubMed
    1. Jaffe AB, Hall A. Rho GTPases: biochemistry and biology. Annu Rev Cell Dev Biol. 2005;21:247–269. - PubMed
    1. Hoffman GR, Cerione RA. Signaling to the Rho GTPases: networking with the DH domain. FEBS Lett. 2002;513:85–91. - PubMed
    1. Van Aelst L, D'Souza-Schorey C. Rho GTPases and signaling networks. Genes Dev. 1997;11:2295–2322. - PubMed
    1. Mackay DJ, Hall A. Rho GTPases. J Biol Chem. 1998;273:20685–20688. - PubMed

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