Abstract
The Rho GDP-dissociation inhibitors (GDIs) negatively regulate Rho-family GTPases. The inhibitory activity of GDI derives both from an ability to bind the carboxy-terminal isoprene of Rho family members and extract them from membranes, and from inhibition of GTPase cycling between the GTP- and GDP-bound states. Here we demonstrate that these binding and inhibitory functions of rhoGDI can be attributed to two structurally distinct regions of the protein. A carboxy-terminal folded domain of relative molecular mass 16,000 (M[r] 16K) binds strongly to the Rho-family member Cdc42, yet has little effect on the rate of nucleotide dissociation from the GTPase. The solution structure of this domain shows a beta-sandwich motif with a narrow hydrophobic cleft that binds isoprenes, and an exposed surface that interacts with the protein portion of Cdc42. The amino-terminal region of rhoGDI is unstructured in the absence of target and contributes little to binding, but is necessary to inhibit nucleotide dissociation from Cdc42. These results lead to a model of rhoGDI function in which the carboxy-terminal binding domain targets the amino-terminal inhibitory region to GTPases, resulting in membrane extraction and inhibition of nucleotide cycling.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Cattle
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Cell Cycle Proteins / chemistry
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Cell Cycle Proteins / metabolism*
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GTP Phosphohydrolases / metabolism*
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GTP-Binding Proteins / chemistry*
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GTP-Binding Proteins / metabolism*
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Guanine Nucleotide Dissociation Inhibitors*
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Guanosine Diphosphate / analogs & derivatives
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Guanosine Diphosphate / metabolism*
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Humans
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Hydrogen Bonding
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Magnetic Resonance Spectroscopy
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Models, Molecular
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Molecular Sequence Data
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Molecular Weight
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Protein Binding
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Protein Conformation
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Protein Folding
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Protein Prenylation
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Protein Structure, Secondary
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Protein Structure, Tertiary
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Sequence Alignment
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Thermodynamics
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cdc42 GTP-Binding Protein, Saccharomyces cerevisiae
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ortho-Aminobenzoates / metabolism
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rho Guanine Nucleotide Dissociation Inhibitor alpha
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rho-Specific Guanine Nucleotide Dissociation Inhibitors
Substances
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ARHGDIA protein, human
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Cell Cycle Proteins
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Guanine Nucleotide Dissociation Inhibitors
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ortho-Aminobenzoates
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rho Guanine Nucleotide Dissociation Inhibitor alpha
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rho-Specific Guanine Nucleotide Dissociation Inhibitors
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3'-(methylanthraniloyl)-2'-deoxy-guanosine diphosphate
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Guanosine Diphosphate
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GTP Phosphohydrolases
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GTP-Binding Proteins
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cdc42 GTP-Binding Protein, Saccharomyces cerevisiae