CMT2N-causing aminoacylation domain mutants enable Nrp1 interaction with AlaRS

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• Aminoacyl-tRNA synthetases (aaRS) are the largest protein family linked to Charcot-Marie-Tooth disease (CMT) through dominant mutations.
• Charcot-Marie-Tooth disease subtype 2N (CMT2N) is associated with mutations in AlaRS, with some located in the aminoacylation domain, initially suggesting a tRNA-charging defect.

Purpose of the Study:

• To investigate the pathogenic mechanisms of CMT2N-associated AlaRS mutations.
• To determine if tRNA-charging ability is affected by CMT2N mutations.
• To explore the interaction of mutant AlaRS with neuropilin 1 (Nrp1).

Main Methods:

• In vitro biochemical assays to assess aminoacylation activity.
• Analysis of patient cell samples.
• X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange (HDX), switchSENSE hydrodynamic diameter, and protease digestion to study protein structure and dynamics.
• Investigating interactions between AlaRS and Nrp1.

Main Results:

• The R329H AlaRS mutation, common in CMT2N, does not affect aminoacylation in vitro or in patient cells.
• Mutant AlaRS proteins, unlike wild-type, interact with Nrp1, an interaction confirmed in patient samples.
• Structural analysis reveals mutation-induced loosening of the aminoacylation domain correlating with Nrp1 interaction.
• Mutations in different AlaRS domains induce distinct structural changes and pathogenic mechanisms.

Conclusions:

• Aminoacylation per se is not the relevant pathology in CMT2N.
• Aberrant interaction with Nrp1 is a key pathogenic mechanism for certain CMT2N mutations.
• Nrp1 is broadly implicated in CMT pathogenesis across tRNA synthetase family members.
• Different mutations within the same protein can lead to neuropathy via distinct molecular pathways.