Abstract
Myostatin (or growth/differentiation factor 8 (GDF8)) is a member of the transforming growth factor β superfamily of growth factors and negatively regulates skeletal muscle growth. Its dysregulation is implicated in muscle wasting diseases. SRK-015 is a clinical-stage mAb that prevents extracellular proteolytic activation of pro- and latent myostatin. Here we used integrated structural and biochemical approaches to elucidate the molecular mechanism of antibody-mediated neutralization of pro-myostatin activation. The crystal structure of pro-myostatin in complex with 29H4-16 Fab, a high-affinity variant of SRK-015, at 2.79 Å resolution revealed that the antibody binds to a conformational epitope in the arm region of the prodomain distant from the proteolytic cleavage sites. This epitope is highly sequence-divergent, having only limited similarity to other closely related members of the transforming growth factor β superfamily. Hydrogen/deuterium exchange MS experiments indicated that antibody binding induces conformational changes in pro- and latent myostatin that span the arm region, the loops contiguous to the protease cleavage sites, and the latency-associated structural elements. Moreover, negative-stain EM with full-length antibodies disclosed a stable, ring-like antigen–antibody structure in which the two Fab arms of a single antibody occupy the two arm regions of the prodomain in the pro- and latent myostatin homodimers, suggesting a 1:1 (antibody:myostatin homodimer) binding stoichiometry. These results suggest that SRK-015 binding stabilizes the latent conformation and limits the accessibility of protease cleavage sites within the prodomain. These findings shed light on approaches that specifically block the extracellular activation of growth factors by targeting their precursor forms.
Highlights
Myostatin (or growth/differentiation factor 8 (GDF8)) is a member of the transforming growth factor  superfamily of growth factors and negatively regulates skeletal muscle growth
Myostatin (growth/differentiation factor 8 (GDF8); gene name MSTN, gene ID 2260) is a member of the transforming growth factor  (TGF-)3 superfamily of signaling proteins that is primarily expressed in skeletal muscle, with low-level expression reported in adipose and cardiac tissues [1, 2]
SRK-015 was constructed on a human IgG4 framework containing the Adair hinge mutation (S228P) [43] to reduce chain swapping, which can be observed for native IgG4 antibodies [44]. 29H4-16 is a high-affinity variant of the parental 29H4 antibody and contains four amino acids changes within the complementarity-determining regions (CDRs) of both the light and heavy chains
Summary
SRK-015 and related analogs bind and prevent pro- and latent myostatin activation. A phage-based discovery campaign aimed at identification of inhibitory antibodies targeting myostatin precursors yielded a number of specific antibodies capable of preventing proteolytic activation [41]. The corresponding peptides containing the tolloid/furin protease cleavage sites in latent myostatin displayed no significant changes in H/D exchange profile upon binding to 29H4-(16/GL) Fabs (Fig. 3, B, D, and F) This suggests that the overall flexibility of these regions is not affected in the presence of the antibody fragments. The superposition of the cocrystal structure of pro-myostatin–29H4-16 Fab (Fig. 2A) into the representative 2D class averages for pro-myostatin–29H4-16 hIgG4 (Fig. 4C) and latent myostatin–SRK-015 (Fig. 4E) showed a strikingly similar overall structural organization, with the exception of the presence of an additional density assigned to the Fc region of the full length antibody Taken together, these data suggest the utility of an integrated structural approach to map out the details of the binding mechanism and stoichiometry as well as the mech-. Anism of action of SRK-015, a neutralizing antibody against pro- and latent myostatin
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