Tolloid cleavage activates latent GDF8 by priming the pro‐complex for dissociation

Abstract

Growth differentiation factor 8 (GDF8)/myostatin is a latent TGF-β family member that potently inhibits skeletal muscle growth. Here, we compared the conformation and dynamics of precursor, latent, and Tolloid-cleaved GDF8 pro-complexes to understand structural mechanisms underlying latency and activation of GDF8. Negative stain electron microscopy (EM) of precursor and latent pro-complexes reveals a V-shaped conformation that is unaltered by furin cleavage and sharply contrasts with the ring-like, cross-armed conformation of latent TGF-β1. Surprisingly, Tolloid-cleaved GDF8 does not immediately dissociate, but in EM exhibits structural heterogeneity consistent with partial dissociation. Hydrogen-deuterium exchange was not affected by furin cleavage. In contrast, Tolloid cleavage, in the absence of prodomain-growth factor dissociation, increased exchange in regions that correspond in pro-TGF-β1 to the α1-helix, latency lasso, and β1-strand in the prodomain and to the β6'- and β7'-strands in the growth factor. Thus, these regions are important in maintaining GDF8 latency. Our results show that Tolloid cleavage activates latent GDF8 by destabilizing specific prodomain-growth factor interfaces and primes the growth factor for release from the prodomain.

Structure of the human myostatin precursor and determinants of growth factor latency

“…Thomas R Cotton et al Structure of human pro-myostatin The EMBO Journal features identified for pro-myostatin are conserved within the sequence of pro-GDF11, and as a result, we predict it will have a highly similar overall structure. This prediction is supported by the work of Pepinsky et al (2017) who recently demonstrated by negative-stain electron microscopy that latent pro-GDF11 adopts a V-shaped topology, very similar to that of our pro-myostatin structures and consistent with the EM analysis of pro-myostatin by Le et al (2018). We propose a model for the synthesis and activation of myostatin, based on the structures and data presented here and in other studies (Fig 8).…”

“…The open-armed conformation observed crystallographically (in two distinct crystal forms) also exists in solution, as shown by SAXS, and interestingly, the overall conformation (and associated particle dimensions) does not seem to change significantly upon cleavage of the pro-domain. These findings are corroborated further by Le et al (2018), who were able to clearly resolve the distinctive V-shape of promyostatin (and the furin-cleaved complex), using negative-stain electron microscopy (EM).…”

“…The arm domain is free to dissociate once the covalent linkage to the forearms is severed, which would then allow the helix-loop-helix epitope to dissociate as well ( Fig 8G and H). Increased rates of hydrogen/deuterium exchange at pro:mature interaction sites following TLD cleavage as shown by Le et al (2018) demonstrate increased lability of the shoulder and forearm following TLD cleavage, priming the complex for dissociation. Analysis of closely related GDF11 has shown that the N-terminal part of the pro-domain with a1 and a2 helices can remain associated with the mature GF, promoting its solubility while not affecting bioactivity, consistent with a stepwise dissociation model (Pepinsky et al, 2017).…”

Molecular characterization of latent GDF8 reveals mechanisms of activation

“…Taken together, our mutational analysis of the GDF8 prodomain is highly consistent with the structure of the prodomain:GDF8 complex and also consistent with previous truncation analysis (41,(44)(45)(46). Our results are also consistent with unpublished results from the laboratory of Tim Springer who performed a rigorous hydrogen-deuterium exchange followed by MS to map the interactions of the prodomain with the mature in solution (47).…”

Tolloid cleavage activates latent GDF8 by priming the pro-complex for dissociation

“…The results provide important insights into the structure and mechanism of activation of GDF8. Our results and those of two other groups were concurrently submitted to BioRxiv (Cotton, Fischer et al, 2017, Le, Iacob et al, 2017, Walker, McCoy et al, 2017b. After submission, we were able to read one another's manuscripts and learn of remarkable concordance.…”