Title: Molecular characterization of latent GDF8 reveals mechanisms of activation Running title: Activation of latent GDF8

Growth/differentiation factor 8 (GDF8) or myostatin negatively regulates muscle mass. GDF8 is held in a latent state through interactions with its N-terminal prodomain, much like TGF-β. Using a combination of small angle X-ray scattering and mutagenesis, we characterized the interactions of GDF8 with its prodomain. Our results show that the prodomain:GDF8 complex can exist in a fully latent state and an activated or ‘triggered’ state where the prodomain remains in complex with the mature domain. However, these states are not reversible, indicating the latent GDF8 is ‘spring-loaded’. Structural analysis shows that the prodomain:GDF8 complex adopts an ‘open’ configuration, distinct from the latency state of TGF-β and more similar to the ‘open’ state of Activin A and BMP9 (non-latent complexes). We determined that GDF8 maintains similar features for latency, including the alpha-1 helix and fastener elements, and identified a series of mutations in the prodomain of GDF8 that alleviate latency, including I56E, which does not require activation by the protease Tolloid. In vivo, active GDF8 variants were potent negative regulators of muscle mass, compared to wild-type GDF8. Collectively, these results help characterize the latency and activation mechanisms of GDF8. Introduction One of the most thoroughly described negative regulators of skeletal muscle mass is the TGF-β superfamily ligand growth/differentiation factor 8 (GDF8), also known as myostatin (1, 2). Genetic disruption of GDF8 results in substantial skeletal muscle growth (1-7). Further, a significant increase in muscle fiber size is also observed when adult animals are treated with agents that bioneutralize GDF8 (reviewed in (8)). As such, targeted inhibition of GDF8 is currently being pursued for the treatment of skeletal muscle-related disorders and associated symptoms (9, 10). GDF8, like numerous TGF-β family members, is a disulfide-linked dimer that is synthesized as a precursor protein which requires cleavage by a furin-like protease to yield a N-terminal prodomain and a Cterminal mature, signaling domain (11). Interestingly, for a number of TGF-β ligands the role of the prodomain extends beyond ligand maturation and folding support (12, 13), remaining non-covalently associated with the mature ligand following secretion in either a low-affinity, non-inhibitory or high-affinity, inhibitory fashion (reviewed in (14)). For example, the prodomains of TGF-β1, TGF-β2, TGF-β3, GDF11, and GDF8 hold the mature ligand in a latent or inactive state mediated by a non-covalent, yet high affinity, ligand-specific interaction (11, 15-18) whereas mature Activin A and BMP9 remain associated with, but are not inhibited by, their prodomain (19, 20). Activation of TGF-β1 and TGF-β3 requires covalent interactions with the extracellular matrix and cellular contractile forces to release the mature ligand (21-23). In fact, resolution of the latent TGFβ1 crystal structure provided a molecular explanation for how latency is exerted by the prodomain via a coordinated interaction between the N-terminal alpha helix (alpha1), latency lasso, and fastener of the prodomain with type I and type II receptor epitopes of the mature domain (23). On the other hand, GDF8 activation requires a second cleavage event within the prodomain via proteases from the BMP1/Tolloid (TLD) family of metalloproteases (24). However, the molecular and structural details of the GDF8 latent state have yet to be determined. Based on sequence conservation and prior biochemical data describing the N-terminal portion of the GDF8 prodomain (15), it is plausible that the molecular interactions and overall structure of the GDF8 latent complex may be similar to that of TGF-β1. However, the prodomains of a number of TGF-β family members share similar sequence conservation, yet they do not regulate the mature ligand in the same fashion and also exhibit significant structural diversity (19, 20). Therefore, while one might expect that GDF8 and TGF-β1 would share certain elements for how the prodomain binds and confers latency, it is possible that significant structural and molecular differences in these interactions occur as they exhibit profoundly different mechanisms of activation. However, this comparison is hindered by a lack of understanding of the GDF8 latent complex at the molecular level. In this study, we utilized small angle X-ray scattering (SAXS) and mutagenesis to characterize the GDF8 latent complex. Interestingly, SAXS analysis reveals that the GDF8 latent complex adopts a more ‘open’ conformation, similar to the overall structure of the BMP9 and Activin A prodomain complexes, which are not latent. The ‘open’ conformation of the GDF8 latent complex is in stark contrast to the ‘closed’ conformation adopted by the TGF-β1 latent complex. Furthermore, we identify key residues in the GDF8 prodomain that are responsible for promoting latency indicating that GDF8 and TGF-β1 share similar features for latency including a latency lasso. We further show that certain mutations in the prodomain of GDF8 can reduce latency, peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/155614 doi: bioRxiv preprint first posted online Jun. 26, 2017;