Maturation of the functional mouse CRES amyloid from globular form

Abstract

The epididymal lumen contains a complex cystatin-rich nonpathological 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 problem 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, allowing 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 progressed from monomeric folded protein to an advanced amyloid. We show the CRES monomer has a typical cystatin fold that assembles into highly branched amyloid matrices, comparable to those in vivo, by forming β-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 β-strand and by traditional cystatin domain swapping. Our results provide key insight into our understanding of functional amyloid assembly by revealing 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.

Myosin Binding Protein-C Forms Amyloid-Like Aggregates In Vitro

“…Diffuse rings on the diffraction pattern have been explained and described [ 48 ] in studies of non-oriented and weakly oriented amyloid samples. Similar diffuse ring reflections have been shown for amyloid aggregates of many proteins [ 10 , 40 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 ].…”

Cross-seeding between the functional amyloidogenic CRES and CRES3 family members and their regulation of Aβ assembly

“…1 C ). This observation, however, was not unexpected based on prior CD, FTIR, X-ray crystallization, and NMR studies of the related CRES that also revealed an antiparallel β-sheet-rich structure ( 25 , 26 ).

Figure 1 CRES3 forms SDS-sensitive and SDS-resistant aggregates.

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“…3 B ), the outlying peaks in red boxes correspond to the conserved cystatin α-helix. The lack of helical secondary structure in the CRES3 spectrum suggests that the assembly of the CRES3 amyloid involves a transition of α-helix to β-strand, similar to conformational changes observed in the polymerization of other amyloids including CRES ( 26 ). We confirmed that this is a likely possibility by repeating this exercise using our β-sheet and turn only CRES3 model ( Fig.…”

“…Using PROMALS3D ( 28 ) and Modeller ( 29 ), we computed a CRES3 model based on our recently reported X-ray crystallographic structure of CRES ( Fig. 3 A ) ( 26 ). In the second model, we computed a 3D CRES3 structure using our proposed domain-swapped amyloid-core model based upon the complete U- 15 N, 13 C chemical shift assignments of CRES amyloid ( Fig.…”

“…In the second model, we computed a 3D CRES3 structure using our proposed domain-swapped amyloid-core model based upon the complete U- 15 N, 13 C chemical shift assignments of CRES amyloid ( Fig. 3 C ) ( 26 ). In the overlay of the 13 C– 13 C cross-peaks predicted from the CRES structure ( Fig.…”