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The structural basis of Arfaptin-mediated cross-talk between Rac and Arf signalling pathways

Nature volume 411, pages 215–219 (2001)Cite this article

Abstract

Small G proteins are GTP-dependent molecular switches that regulate numerous cellular functions1. They can be classified into homologous subfamilies that are broadly associated with specific biological processes. Cross-talk between small G-protein families has an important role in signalling, but the mechanism by which it occurs is poorly understood2. The coordinated action of Arf and Rho family GTPases is required to regulate many cellular processes including lipid signalling3, cell motility4 and Golgi function5. Arfaptin is a ubiquitously expressed protein implicated in mediating cross-talk between Rac (a member of the Rho family) and Arf small GTPases. Here we show that Arfaptin binds specifically to GTP-bound Arf1 and Arf6, but binds to Rac·GTP and Rac·GDP with similar affinities. The X-ray structure of Arfaptin reveals an elongated, crescent-shaped dimer of three-helix coiled-coils. Structures of Arfaptin with Rac bound to either GDP or the slowly hydrolysable analogue GMPPNP show that the switch regions adopt similar conformations in both complexes. Our data highlight fundamental differences between the molecular mechanisms of Rho and Ras family signalling, and suggest a model of Arfaptin-mediated synergy between the Arf and Rho family signalling pathways.

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Figure 1: Sequence homology and structure of Arfaptin.
Figure 2: Arfaptin binding and competition studies.
Figure 3: Structure of the Rac–Arfaptin complex.
Figure 4: Switch I conformations of Rac in complexes with Arfaptin.

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Acknowledgements

We acknowledge S. Price for assistance with protein purification; G. Dodson and A. Lane for critically reading the manuscript; T. Magee for helpful discussions; D. Jones for expression clones of Arf; the staff of SRS Daresbury and ESRF Grenoble, particularly G. Leonard, for beamtime allocation and assistance with X-ray data collection; and J. F. Eccleston for assistance with fluorescence experiments.

Author information

Author notes

  1. C. Tarricone

    Present address: European Institute of Oncology, Via Ripamonti 435, 20141, Milan, Italy

  2. C. Tarricone, B. Xiao and N. Justin: These authors contributed equally to this work

  3. S. J. Gamblin: Correspondence and requests for materials should be addressed to S.J.G.

Authors and Affiliations

  1. Division of Protein Structure, National Institute for Medical Research, Mill Hill, NW7 1AA, London, UK

    C. Tarricone, B. Xiao, N. Justin, P. A. Walker, K. Rittinger, S. J. Gamblin & S. J. Smerdon

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  1. C. Tarricone
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Corresponding author

Correspondence to S. J. Gamblin.

Additional information

Coordinates have been deposited in the Brookhaven Database under accession codes 1I49 (Arfaptin), 1I4D (Arfaptin–RacGDP monoclinic form), 1I4L (Arfaptin–RacGDP tetragonal form) and 1I4T (Arfaptin–RacGMPPNP).

Supplementary information

Table 1. X-ray data collection, phasing and refinement

Arfaptin

Space-group: P3221 a=b=146.3Å c=82.7Å

Table 1
Full size table
Table 2
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Table 3
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Table 4
Full size table

Arfaptin/Rac.GDP

Space group: P21 a=63.6Å, b=47.5Å, c=125.3Å, b=97.5°

Table 5
Full size table

Space group: P41 a=b=112.7Å, c=68.2Å

Table 6
Full size table

Arfaptin/RacQ61L.GMPPNP

Space group: P41 a=b=112.7Å, c=68Å

Table 7
Full size table

1 Nobs/Nunique

2 Rsym = Sj|<I> - Ij|/S<I> where Ij is the intensity of the jth reflection and <I> is the average intensity.

3 Rcryst = Shkl |Fobs - Fcalc|/ Shkll Fobs

4 Rfree - as for Rcryst but calculated on 5% of the data excluded from the refinement calculation.

Figure 2.

(GIF 18.7 KB)

a. Isothermal titration calorimetry measurements of Rac1 binding to Arfaptin. The upper panel shows the raw data of an ITC experiment performed at 22 °C. The lower panel shows the integrated heat changes, corrected for the heat of dilution (the binding reaction and the heat of dilution have different signs), and the fitted curve based on a single site model. For each type of experiment titrations were performed as follows: GTPase into Arfaptin and Arfaptin into GTPase in order to confirm the stoichiometry of interaction. The figure shows the titration of Arfaptin (0.3mM dimer) into Rac1.GMPPNP at 0.05mM which gives Kd = 4.5m M and a binding ratio of 2.0 (Arfaptin monomer/Rac).

b. Arf1 competition for Rac binding to Arfaptin monitored by fluorescence anisotropy from Rac.mant-nucleotide. The figure shows titration of Arf1.GMPPNP (<) and Arf1.GDP (•) into Arfaptin/Rac1.mant-GDP at 40m M. The error bars for the Arf1.GMPPNP titration represent the standard error of three measurements. The half-way point of the signal change corresponds approximately to the concentration of the mixture in the cuvette as would be expected for competing species with similar affinities for Arfaptin. Titrations using Rac.GDP (non-mant) also gave a reduction in anisotropy with similar start and end points.

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Tarricone, C., Xiao, B., Justin, N. et al. The structural basis of Arfaptin-mediated cross-talk between Rac and Arf signalling pathways. Nature 411, 215–219 (2001). https://doi.org/10.1038/35075620

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