Mutations in Hsp40 co-chaperone change the canonical interdomain interactions stimulating LGMDD1 myopathy.

Limb-girdle muscular dystrophy D1 (LGMDD1) is a rare, dominantly inherited neuromuscular protein-misfolding chaperonopathy caused by mutations in the Hsp40 co-chaperone DNAJB6, primarily in the glycine-phenylalanine (GF) or J-domains. Currently, no treatments are available, and a challenge in understanding the disease is identifying a specific client protein for DNAJB6 in skeletal muscle. DNAJB6 has homology to the yeast DNAJ family member, Sis1. Our previous research indicated that LGMDD1 GF domain mutants in Sis1 exhibit substrate-specific effects, influenced by Hsp70 activity. Herein, we employed functional assays along with advanced molecular simulation studies to understand the regulatory interdomain interactions in disease-causing mutants of DNAJB6 that cause LGMDD1 myopathy. We found that disease-causing novel mutations in the J-domain mimic the chaperone's substrate-bound state, both directly by disrupting J-GF contacts and indirectly by destabilizing the J-CTD inhibitory linkage. Both routes converge on similar interdomain rearrangements, indicating a unified pathway, wherein this premature allosteric switch locks the chaperone in an inactive conformation, blocking productive interactions with substrates and Hsp70. These mechanistic insights enhance our understanding of LGMDD1 myopathy and facilitate the identification of potential treatment strategies for the future.