Rab35/ACAP2 and Rab35/RUSC2 Complex Structures Reveal Molecular Basis for Effector Recognition by Rab35 GTPase

Area of Science:

• Molecular Biology
• Structural Biology
• Cellular Biology

Background:

• Rab35 is a crucial regulator of membrane trafficking involved in cellular functions.
• Understanding Rab35-effector interactions is vital for deciphering its role in human diseases.
• Existing knowledge on the molecular basis of Rab35-effector binding is limited.

Purpose of the Study:

• To elucidate the molecular mechanisms underlying Rab35 interactions with its effectors ACAP2 and RUSC2.
• To determine the high-resolution crystal structures of Rab35-effector complexes.
• To design Rab35 mutants with altered effector binding specificity.

Main Methods:

• X-ray crystallography to obtain high-resolution structures of Rab35/ACAP2 and Rab35/RUSC2 complexes.
• Structural analysis to identify key interaction interfaces and binding modes.
• Site-directed mutagenesis to create and test specific Rab35 mutants.

Main Results:

• Determined crystal structures of Rab35 bound to ACAP2 and RUSC2, revealing novel binding interfaces.
• Identified a unique binding mode between Rab35 and ACAP2's ankyrin repeat and C-terminal helix.
• Characterized RUSC2's Arg1015 as a "pseudo-arginine finger" stabilizing GTP-bound Rab35.
• Successfully designed Rab35 mutants that selectively disrupt ACAP2 and RUSC2 interactions without affecting other effector bindings.
• Provided structural basis for disease-associated mutations at Rab35-effector interfaces.

Conclusions:

• The study provides unprecedented atomic-level insights into Rab35-effector interactions.
• The findings reveal novel binding mechanisms and facilitate the design of specific Rab35 mutants.
• These structures offer a foundation for understanding Rab35's role in health and disease and for developing targeted therapeutics.