Runaway domain swapping in amyloid-like fibrils of T7 endonuclease I.
Amyloid fibrils are associated with >20 fatal human disorders, including Alzheimer's, Parkinson's, and prion diseases. Knowledge of how soluble proteins assemble into amyloid fibrils remains elusive despite its potential usefulness for developing diagnostics and therapeutics. In at least some fibrils, runaway domain swapping has been proposed as a possible mechanism for fibril formation. In runaway domain swapping, each protein molecule swaps a domain into the complementary domain of the adjacent molecule along the fibril. Here we show that T7 endonuclease I, a naturally domain-swapped dimeric protein, can form amyloid-like fibrils. Using protein engineering, we designed a double-cysteine mutant that forms amyloid-like fibrils in which molecules of T7 endonuclease I are linked by intermolecular disulfide bonds. Because the disulfide bonds are designed to form only at the domain-swapped dimer interface, the resulting covalently linked fibrils show that T7 endonuclease I forms fibrils by a runaway domain swap. In addition, we show that the disulfide mutant exists in two conformations, only one of which is able to form fibrils. We also find that domain-swapped dimers, if locked in a close-ended dimeric form, are unable to form fibrils. Our study provides strong evidence for runaway domain swapping in the formation of an amyloid-like fibril and, consequently, a molecular explanation for specificity and stability of fibrils. In addition, our results suggest that inhibition of fibril formation for domain-swapped proteins may be achieved by stabilizing domain-swapped dimers.
Guo
Zhefeng
Z
Howard Hughes Medical Institute, University of California, Los Angeles-Department of Energy Institute for Genomics and Proteomics, Molecular Biology Institute, Box 951570, University of California, Los Angeles, CA 90095-1570, USA.
Eisenberg
David
D
D000682:Amyloid / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D003850:Deoxyribonuclease I / Q000378:metabolism*; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D019636:Neurodegenerative Diseases / Q000473:pathology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D000682:Amyloid; D004220:Disulfides; D003850:Deoxyribonuclease I; D003545:Cysteine