A complete allosteric map of a GTPase switch in its native cellular network

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Signaling

Background:

• Allosteric regulation is crucial for protein function in cellular networks.
• The extent of allosteric regulation sites on proteins (few vs. many) remains an open question.
• GTPases act as molecular switches controlling cellular signaling via conformational changes.

Purpose of the Study:

• To investigate the distribution and nature of allosteric regulatory sites on GTPases at a residue level.
• To determine if GTPase regulation is confined to active sites or distributed throughout the protein structure.
• To create a functional map of regulatory sites for targeting GTPases.

Main Methods:

• Deep mutagenesis was employed to assay mutations in the GTPase Gsp1/Ran within its native biological network.
• Over 4,315 mutations were analyzed to identify those affecting protein function.
• Kinetic analysis was used to characterize the allosteric coupling of distal regulatory sites to the active site.

Main Results:

• 28% of assayed mutations in Gsp1/Ran exhibited significant gain-of-function responses.
• Gain-of-function mutations were identified at 60 positions, with 20 located outside the canonical active site regions.
• Distal regulatory sites were found to be allosterically coupled to the GTPase active site.

Conclusions:

• The GTPase switch mechanism demonstrates broad sensitivity to cellular allosteric regulation.
• Allosteric regulation of GTPases is not limited to a few canonical sites but occurs at numerous distributed positions.
• This research provides a functional map for understanding and targeting GTPases involved in essential biological processes.