Maturation of the functional mouse CRES amyloid from globular form

The epididymal lumen contains a complex cystatin-rich nonpatho-logical amyloid matrix with putative roles in sperm maturation and sperm protection. Given our growing understanding for the biological function of this and other functional amyloids, the prob-lem still remains: how functional amyloids assemble including their initial transition to early oligomeric forms. To examine this, we developed a protocol for the purification of nondenatured mouse CRES, a component of the epididymal amyloid matrix, allow-ing us to examine its assembly to amyloid under conditions that may mimic those in vivo. Herein we use X-ray crystallography, solution-state NMR, and solid-state NMR to follow at the atomic level the assembly of the CRES amyloidogenic precursor as it pro-gressed from monomeric folded protein to an advanced amyloid. We show the CRES monomer has a typical cystatin fold that assem-bles into highly branched amyloid matrices, comparable to those in vivo, by forming beta-sheet assemblies that our data suggest occur via two distinct mechanisms: a unique conformational switch of a highly flexible disulfide-anchored loop to a rigid beta-strand and by traditional cystatin domain swapping. Our results provide key in-sight into our understanding of functional amyloid assembly by re-vealing the earliest structural transitions from monomer to oligomer and by showing that some functional amyloid structures may be built by multiple and distinctive assembly mechanisms.