Abstract:BackgroundGrowth/differentiation factor 8 (GDF8) and GDF11 are two highly similar members of the transforming growth factor β (TGFβ) family. While GDF8 has been recognized as a negative regulator of muscle growth and differentiation, there are conflicting studies on the function of GDF11 and whether GDF11 has beneficial effects on age-related dysfunction. To address whether GDF8 and GDF11 are functionally identical, we compared their signaling and structural properties.ResultsHere we show that, despite their h… Show more
“…The individual mature GF protomers also overlay well with the structure of myostatin bound to follistatin 288 (PDB: 3HH2, Cα RMSD: 0.63 Å, 65 atoms), but exhibit a shift in inter‐protomer angle (Fig C; Cash et al , ). This observation is consistent with that of Walker et al , who recently showed that the mature myostatin GF dimer crystallises with radically different inter‐protomer angles in apo and FST‐288 bound states (Fig C; Walker et al , 2017a). Conformational plasticity is similarly well documented for activin A, which has inter‐protomer angles ranging from 50° in complex with type II receptor ecto‐domain (PDB: 1NYS), to 108° when bound to FST‐315 (PDB: 2P6A; Wang et al , ).…”
Myostatin, a key regulator of muscle mass in vertebrates, is biosynthesised as a latent precursor in muscle and is activated by sequential proteolysis of the pro-domain. To investigate the molecular mechanism by which pro-myostatin remains latent, we have determined the structure of unprocessed pro-myostatin and analysed the properties of the protein in its different forms. Crystal structures and SAXS analyses show that pro-myostatin adopts an open, V-shaped structure with a domain-swapped arrangement. The pro-mature complex, after cleavage of the furin site, has significantly reduced activity compared with the mature growth factor and persists as a stable complex that is resistant to the natural antagonist follistatin. The latency appears to be conferred by a number of distinct features that collectively stabilise the interaction of the pro-domains with the mature growth factor, enabling a regulated stepwise activation process, distinct from the prototypical pro-TGF-β1. These results provide a basis for understanding the effect of missense mutations in pro-myostatin and pave the way for the design of novel myostatin inhibitors.
“…The individual mature GF protomers also overlay well with the structure of myostatin bound to follistatin 288 (PDB: 3HH2, Cα RMSD: 0.63 Å, 65 atoms), but exhibit a shift in inter‐protomer angle (Fig C; Cash et al , ). This observation is consistent with that of Walker et al , who recently showed that the mature myostatin GF dimer crystallises with radically different inter‐protomer angles in apo and FST‐288 bound states (Fig C; Walker et al , 2017a). Conformational plasticity is similarly well documented for activin A, which has inter‐protomer angles ranging from 50° in complex with type II receptor ecto‐domain (PDB: 1NYS), to 108° when bound to FST‐315 (PDB: 2P6A; Wang et al , ).…”
Myostatin, a key regulator of muscle mass in vertebrates, is biosynthesised as a latent precursor in muscle and is activated by sequential proteolysis of the pro-domain. To investigate the molecular mechanism by which pro-myostatin remains latent, we have determined the structure of unprocessed pro-myostatin and analysed the properties of the protein in its different forms. Crystal structures and SAXS analyses show that pro-myostatin adopts an open, V-shaped structure with a domain-swapped arrangement. The pro-mature complex, after cleavage of the furin site, has significantly reduced activity compared with the mature growth factor and persists as a stable complex that is resistant to the natural antagonist follistatin. The latency appears to be conferred by a number of distinct features that collectively stabilise the interaction of the pro-domains with the mature growth factor, enabling a regulated stepwise activation process, distinct from the prototypical pro-TGF-β1. These results provide a basis for understanding the effect of missense mutations in pro-myostatin and pave the way for the design of novel myostatin inhibitors.
“…The growth factor dimer adopts a closed conformation in pro‐TGF‐β1 (pdb: 3RJR) (K) (Shi et al , ) and pro‐BMP9 (pdb: 4YCG) (L) (Mi et al , ), and an open conformation in pro‐GDF8 (pdb: 5NTU). N–PCrystal structures of the GDF8 growth factor in different conformations. The apo form (pdb: 5JI1) (Walker et al , ) adopts an open conformation (N). In contrast, the GDF8 growth factor adopts a closed conformation when in complex with the antagonist FSL3 (O) (pdb: 3sek) (Cash et al , ) and yet another conformation when bound to Fab (P) (pdb: 5f3b) (Apgar et al , ).Source data are available online for this figure.…”
Section: Resultsmentioning
confidence: 99%
“…If the GF in the pro‐activin A complex had a closed conformation, as seen in some activin GF complex structures with inhibitors and receptors, the pro‐complex would have to assume a markedly more obtuse V‐angle (Hinck et al , ; Wang et al , ). Notably, the apo GDF8 GF dimer structure adopts an open GF conformation (Fig N; Walker et al , ) with a more acute V‐angle than in the pro‐complex (Fig M), whereas crystal structures of the GDF8 GF in complex with antagonists reveal a closed conformation and a complex with Fab reveals yet another conformation (Fig O and P; Apgar et al , ; Hinck et al , ). Our HDX results showed no changes in GDF8 GF regions that alter in conformation between the open and closed GF conformations, including the GF α3′‐helix (Hinck et al , ), and thus provided no evidence for a change in overall GF conformation associated with TLL2 cleavage.…”
Section: Discussionmentioning
confidence: 99%
“…N-P Crystal structures of the GDF8 growth factor in different conformations. The apo form (pdb: 5JI1) (Walker et al, 2017a) adopts an open conformation (N). In contrast, the GDF8 growth factor adopts a closed conformation when in complex with the antagonist FSL3 (O) (pdb: 3sek) (Cash et al, 2012) and yet another conformation when bound to Fab (P) (pdb: 5f3b) (Apgar et al, 2016).…”
mentioning
confidence: 99%
“…In particular, sequences that correspond to the a1-helix, latency lasso (including the 6-residue latency helix insertion), a1-helix, fastener, and b1-strand in the prodomain and the b6 0 -and 7 0strands in the GF of GDF8 are strongly conserved in GDF11 (Hinck et al, 2016). Although GF factor structures of GDF8 and 11 vary in conformation, follistatin 288-bound structures of both are remarkably alike (RMSD = 0.66 Å ; Cash et al, 2009;Apgar et al, 2016;Padyana et al, 2016;Walker et al, 2017a), suggesting that interaction with the same binding partner imposes similar structural constraints on the GDF8 and 11 GFs. These observations combined with conservation of overall domain architecture and secondary structure in the family (Shi et al, 2011;Mi et al, 2015;Hinck et al, 2016;Wang et al, 2016;Cotton et al, 2018) suggest that latent GDF11 forms similar prodomain-GF interfaces.…”
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.
Cleft lip with or without cleft palate (CL/P) is generally viewed as a complex trait with multiple genetic and environmental contributions. In 70% of cases, CL/P presents as an isolated feature and/or deemed nonsyndromic. In the remaining 30%, CL/P is associated with multisystem phenotypes or clinically recognizable syndromes, many with a monogenic basis. Here we report the identification, via exome sequencing, of likely pathogenic variants in two genes that encode interacting proteins previously only linked to orofacial clefting in mouse models. A variant in GDF11 (encoding growth differentiation factor 11), predicting a p.(Arg298Gln) substitution at the Furin protease cleavage site, was identified in one family that segregated with CL/P and both rib and vertebral hypersegmentation, mirroring that seen in Gdf11 knockout mice. In the second family in which CL/P was the only phenotype, a mutation in FST (encoding the GDF11 antagonist, Follistatin) was identified that is predicted to result in a p.(Cys56Tyr) substitution in the region that binds GDF11. Functional assays demonstrated a significant impact of the specific mutated amino acids on FST and GDF11 function and, together with embryonic expression data, provide strong evidence for the importance of GDF11 and Follistatin in the regulation of human orofacial development.
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