DNA modulates structural transitions and oligomerization kinetics of the functional amyloid CRES

Functional amyloids play critical roles in diverse physiological processes; however, the molecular mechanisms regulating their assembly remain unknown. The mouse epididymal lumen contains a functional amyloid- and nucleic acid-rich extracellular matrix with roles in host defense and sperm maturation. Cystatin-related epididymal spermatogenic (CRES), a reproductive cystatin and key component of the mammalian epididymal amyloid matrix, assembles into structurally heterogeneous amyloid forms to support the various roles of this extracellular matrix. Here, we show that CRES binds double-stranded DNA with sub-micromolar affinity in a sequence independent manner and that DNA binding accelerates amyloid formation by increasing local protein concentration and promoting specific oligomerization pathways. Using NMR spectroscopy, site-directed mutagenesis, and biophysical analyses, we find that DNA interacts primarily with the CRES loop region, thereby occluding one assembly mechanism and redirecting oligomerization through a pathway involving the L1 loop. These DNA-mediated changes in assembly kinetics and pathway selection suggest a regulatory mechanism for achieving structural and functional diversity in non-pathological amyloid systems. Our findings provide a molecular framework for understanding nucleic acid-guided amyloidogenesis and highlight how functional amyloids may exploit multiple assembly routes to fulfill their physiological roles.