Related Experiment Video
Updated: Aug 4, 2026
10:37
Comparing the Affinity of GTPase-binding Proteins using Competition Assays
Published on: October 8, 2015
Structure-based mutagenesis reveals distinct functions for Ras switch 1 and switch 2 in Sos-catalyzed guanine
B E Hall1, S S Yang, P A Boriack-Sjodin
1Department of Molecular Genetics and Microbiology and the Graduate Programs in Molecular Pharmacology and Molecular and Cellular Biology, State University of New York at Stony Brook, New York 11794-5222, USA.
The Journal of Biological Chemistry
|May 3, 2001
Summary
Site-directed mutagenesis reveals how the guanine nucleotide exchange factor Sos activates Ras GTPases. Sos binding to Ras switch 2 anchors it, while interactions with switch 1 disrupt nucleotide binding, promoting GDP dissociation.
Area of Science:
- Molecular Biology
- Cell Signaling
- Protein Structure-Function Relationships
Background:
- Ras GTPases are critical binary switches regulating cell growth and differentiation.
- The guanine nucleotide exchange factor Sos activates Ras in response to external signals.
- Previous structural studies revealed Sos induces conformational changes in Ras switch 1 and switch 2 regions.
Purpose of the Study:
- To investigate the functional significance of Sos-induced conformational changes in Ras.
- To elucidate the specific roles of Ras switch 1 and switch 2 interactions with Sos in Ras activation.
Main Methods:
- Site-directed mutagenesis of key residues in Ras and Sos.
- Analysis of Ras-Sos binding affinity and catalytic function.
- Structure-function analysis based on previous crystallographic data.
Main Results:
- Mutagenesis of Ras switch 2-Sos interface residues showed limited impact on binding, with Tyr64 being crucial.
- Mutations in Ras switch 2 residues interacting with the nucleotide-binding site did not affect Sos catalytic function.
- Mutations in Ras switch 1 residues disrupted Sos binding and increased nucleotide dissociation rates.
Conclusions:
- Sos interaction with Ras switch 2 primarily mediates stable anchoring to the guanine nucleotide exchange factor.
- Sos interaction with Ras switch 1 is critical for disrupting the nucleotide-binding site and facilitating GDP release.
- Distinct functional roles of switch 1 and switch 2 interactions explain Sos-mediated Ras activation.
Related Concept Videos
02:14GTPases and their Regulation
Guanine nucleotide-binding proteins (G-proteins), also known as GTPases, are a superfamily of proteins that regulate many cellular processes, such as cell signaling, vesicular transport, and the regulation of cell shape and motility. Mutation or dysfunction of these proteins can lead to disease. There are around 40,000 known G-proteins that can broadly be classified into two groups ‒ small G-proteins consisting of a single domain and large multi-domain G-proteins.
Large G-proteins, also known...
Large G-proteins, also known...
01:14Rab Proteins
Rab proteins constitute the largest family of monomeric GTPases, of which 70 members are present in humans. Rab proteins and their effectors regulate consecutive stages of vesicle transport such as vesicle transport, docking, and fusion to the correct recipient membrane.
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...
02:38The Ras Gene
The Ras-gene-encoded proteins are regulators of signaling pathways controlling cell proliferation, differentiation, or cell survival. The Ras-gene family in humans constitutes three primary members—the HRas, NRas, and KRas. These genes code for four functionally distinct yet closely related proteins—the HRas, NRas, KRas4A, and KRas4B. The involvement of mutant Ras genes in human cancer was first discovered in 1982 and is among the most common causes of human tumorigenesis.
Ras is a superfamily...
Ras is a superfamily...
01:25Rab Cascades
Rab GTPases act in a regulated cascade during membrane fusion, helping the lipid bilayers mix. The Rab family of proteins are active when bound to GTP, and inactive when bound to GDP. Hence, they act as guanine nucleotide-dependent molecular switches. Rab-GTP recognizes and binds to long or short-range tethering proteins to capture the target vesicle. These tethers coordinate with SNAREs on the vesicle and the target membrane to assemble the trans SNARE complex that locks the mixing bilayers.
01:24Small GTPases - Ras and Rho
Ras and Rho are small monomeric GTPases that act downstream of receptor tyrosine kinase (RTK) and regulate various cellular processes. These GTPases switch between active and inactive states by binding to guanine nucleotides.
Three regulatory proteins control their activity:
Three regulatory proteins control their activity:
02:38The Ras Gene
The Ras-gene-encoded proteins are regulators of signaling pathways controlling cell proliferation, differentiation, or cell survival. The Ras-gene family in humans constitutes three primary members—the HRas, NRas, and KRas. These genes code for four functionally distinct yet closely related proteins—the HRas, NRas, KRas4A, and KRas4B. The involvement of mutant Ras genes in human cancer was first discovered in 1982 and is among the most common causes of human tumorigenesis.
Ras is a superfamily...
Ras is a superfamily...