Crystal Structure of Rac1 in Complex with the Guanine
Nucleotide Exchange Region of Tiam1
David K. Worthylake*, Kent L. Rossman†, & John Sondek *†‡
*Department of Pharmacology, †Department of Biochemistry and Biophysics,
‡Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel
Hill, Chapel Hill, NC 27599 USA
‡Corresponding author
Manuscript W06555A
Supplementary Information
Supplementary Figure 1 Experimental electron density. The Tiam1 DH domain
(yellow), Rac1 (green) and switch regions (red) are superimposed on the experimental
electron density (white) generated using density-modified phases and contoured at 1. 1
anomalous differences and model-derived phases were used to generate difference
density (red) contoured at 6 and encompassing Met 1224 (at right). This figure was
made using SPOCK (http://quorum.tamu.edu/jon/spock/)
Supplementary Figure 2 Sequence alignments. a, The all helical secondary structure of
the DH domain of Tiam1 is indicated in yellow above its sequence and agrees well with
secondary structure elements of other DH domains1-3 highlighted in grey. Tiam1
residues in red italics bury more than 10 Å2 of solvent accessible surface area upon
complex formation with Rac1; areas buried are charted in inset. Highly conserved
regions among all DH domains are labeled CR1-3 and the majority of Tiam1 residues
that interact with Rac1 are localized to CR1 and CR3 and most likely represent a
common binding surface for all combinations of G-proteins with Dbl family members.
Interacting residues outside the conserved regions localize to sequences within or
adjacent to helices 4b, 5, 310-7a, and 9. Residues in the DH domain of Trio that
possess significantly broadened amide resonances upon Rac1 addition2 are in black
italics and agree well with a subset of the contacts observed in the Tiam1Rac1 complex.
Borders of expression constructs are indicated with small arrows, small dots indicate
every 10 residues, and lightly shaded italicized residues could not be localized upon
structure determination. The secondary structure for Trio was taken from Liu et al. while
others were calculated using DSSP4. Twenty-two non-redundant DH domains were
aligned using ClustalX5 followed by minimal manual adjustment and only DH domains
of known structure are shown. Consensus sequence generated using GCG 6 (plurality, 8;
threshold, 1; individual weights, 1). b, Residues in Rac1 that bury more than 10 Å2 upon
complex formation with Tiam1 are indicated in bold italics and nomenclature for
secondary structure elements derives from the structure of Ras7. Buried residues
highlighted red differ between Rac1 and Cdc42 and are therefore likely to be important
for dictating specificity between G-proteins and DH domains. Also highlighted are the
switch regions that undergo conserved, nucleotide-dependent conformational alteration in
G-proteins, as well as, the 21 amino acid insertion unique to Rho family G-proteins,
absent in Ras, and involved in some effector interactions. Sequences for 16 Rho family
members and Ras were aligned using ClustalX5 and the subset shown. Secondary
structure was calculated using DSSP4
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