Iacob, R. E. | Search bioRxiv

bioRxiv2018

Fusion surface structure, function, and dynamics of gamete fusogen HAP2

HAP2 is a class II gamete fusogen in many eukaryotic kingdoms. A crystal structure of Chlamydomonas HAP2 shows a trimeric fusion state. Domains D1, D2.1 and D2.2 line the 3-fold axis; D3 and a stem pack against the outer surface. Surprisingly, hydrogen-deuterium exchange shows that surfaces of D1, D2.2 and D3 closest to the 3-fold axis are more dynamic than exposed surfaces. Three fusion helices in the fusion loop of each monomer expose hydrophobic residues at the trimer apex that are splayed from the 3-fold axis, leaving a solvent-filled cavity between the fusion loops in each monomer. At the base of the long fusion loop, Arg185 docks in a carbonyl cage. Comparisons to other structures, dynamics, and the greater effect on Chlamydomonas gamete fusion of mutation of axis-proximal than axis-distal fusion helices suggest that the apical portion of each monomer could tilt toward the 3-fold axis with merger of the fusion helices into a common fusion surface.

biochemistry↗

bioRxiv2017

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

Growth differentiation factor 8 (GDF8)/Myostatin is a latent TGF{-}{beta} 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{-}{beta}1. 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-{beta}1 to the 1-helix, latency lasso, and {beta}1 strand in the prodomain and to the {beta}6-7 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.

biochemistry↗