Structural basis for p50RhoGAP BCH domain-mediated regulation of Rho inactivation

Area of Science:

• Molecular biology
• Cell signaling
• Structural biology

Background:

• Spatiotemporal regulation of signaling pathways relies on scaffolding proteins.
• The BNIP-2 and Cdc42GAP Homology (BCH) domain is critical for regulating small GTPases involved in cell morphogenesis.
• The precise mechanism of RhoA inactivation by p50RhoGAP (ARHGAP1) was previously unknown.

Purpose of the Study:

• To elucidate the molecular mechanism of RhoA inactivation by the p50RhoGAP BCH domain.
• To determine the structural basis for the regulatory function of the p50RhoGAP BCH domain.

Main Methods:

• Crystal structure determination of the *Schizosaccharomyces pombe* p50RhoGAP BCH domain.
• Modeling of the human p50RhoGAP BCH domain.
• In vitro biochemical assays and cell line studies.

Main Results:

• The p50RhoGAP BCH domain forms an intertwined asymmetric dimer with a RhoA-binding loop and a lipid-binding pocket.
• The β5-strand of the BCH domain engages in an intermolecular β-sheet, autoinhibiting the adjacent GAP domain.
• Mutation of the β5-strand releases autoinhibition, activating p50RhoGAP for RhoA inactivation and promoting BCH domain self-association.

Conclusions:

• The BCH domain's dimeric structure and the β5-strand are critical for p50RhoGAP's autoinhibition and RhoA regulation.
• Structural insights reveal how BCH domain-containing proteins achieve spatiotemporal control of Rho activity.