Crystal structure of a GDP-bound <i>Bombyx mori</i> RabX6

Rab GTPases are molecular switches that control intracellular vesicular transport by cycling between GDP- and GTP-bound states. Insects encode an insect-specific subset, RabX; Bombyx mori RabX6 (BmRabX6) has been implicated in testis development and neuropeptide secretion, but its structure and mechanism were unknown. Here we report the 3.1 Å crystal structure of BmRabX6 in complex with GDP and Mg2+ (PDB: 9VLB), the first structure of an insect-specific Rab GTPase. BmRabX6 adopts the canonical small GTPase fold with conserved P-loop and Switch I/II, and displays a GDP-binding mode similar to vertebrate Rabs. Two features distinguish BmRabX6. First, the catalytic glutamine required for GTP hydrolysis in typical Rabs is naturally replaced by methionine (Met69) and oriented away from the nucleotide, consistent with obligate GAP-assisted hydrolysis. Second, one residue of the hydrophobic effector-binding triad is histidine (His47), suggesting a potential shift toward a hydrophilic interface-mediated interaction distinct from canonical Rab-effector recognition. AlphaFold3-based complex modeling further identified BmH9J2P5 as a prioritized GTPase-activating protein (GAP) candidate interacting with BmRabX6. These adaptations suggest that BmRabX6 preserves core nucleotide cycling while employing divergent regulatory chemistry tuned to insect physiology. Our structure provides a framework for testing GAP dependence and effector specificity of RabX6 in reproductive and neuronal tissues and illustrates how strategic amino-acid substitutions diversify Rab function.