CMT 2B: Disease mutations in Rab7 result in unregulated nucleotide exchange and

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Hum Mol Genet. 2009 Dec 22

Disease mutations in Rab7 result in unregulated nucleotide exchange and

inappropriate activation.

McCray BA, Skordalakes E, JP.

Department of Developmental Neurobiology, St. Jude Children's Research Hospital,

Memphis, TN 38105, USA.

Rab GTPases are molecular switches that orchestrate vesicular trafficking,

maturation, and fusion by cycling between an active, GTP-bound form and an

inactive, GDP-bound form. The activity cycle is coupled to GTP hydrolysis and is

tightly controlled by regulatory proteins. Missense mutations of the GTPase Rab7

cause a dominantly inherited axonal degeneration known as Charcot-Marie-Tooth

type 2B through an unknown mechanism.

We present the 2.8 A crystal structure of GTP-bound L129F mutant Rab7 which

reveals normal conformations of the effector binding regions and catalytic site,

but an alteration to the nucleotide binding pocket that is predicted to alter

GTP binding. Through extensive biochemical analysis we demonstrate that

disease-associated mutations in Rab7 do not lead to an intrinsic GTPase defect,

but permit unregulated nucleotide exchange leading to both excessive activation

and hydrolysis-independent inactivation. Consistent with augmented activity,

mutant Rab7 shows significantly enhanced interaction with a subset of effector

proteins. In addition, dynamic imaging demonstrates that mutant Rab7 is

abnormally retained on target membranes.

However, we show that increased activation of mutant Rab7 is counterbalanced by

unregulated, GTP hydrolysis-independent membrane cycling. Notably, disease

mutations are able to rescue the membrane cycling of a GTPase deficient mutant.

Thus, we demonstrate that disease mutations uncouple Rab7 from the spatial and

temporal control normally imposed by regulatory proteins and cause disease not

by a gain of novel toxic function, but by misregulation of native Rab7 activity.