Because of extraordinarily tight coiled-coil associations of type I and type II keratins, the composition and structure of keratin subunits has been difficult to determine. We report here the use of novel genetic and biochemical methods to explore the early stages of keratin filament assembly. Using bacterially expressed humans K5 and K14, we show that remarkably, these keratins behave as 1:1 complexes even in 9 M urea and in the presence of a reducing agent. Gel filtration chromatography and chemical cross-linking were used to identify heterodimers and heterotetramers as the most stable building blocks of keratin filament assembly. EM suggested that the dimer consists of a coiled-coil of K5 and K14 aligned in register and in parallel fashion, and the tetramer consists of two dimers in antiparallel fashion, without polarity. In 4 M urea, both end-to-end and lateral packing of tetramers occurred, leading to a variety of larger heteromeric complexes. The coexistence of multiple, higher-ordered associations under strongly denaturing conditions suggests that there may not be a serial sequence of events leading to the assembly of keratin intermediate filaments, but rather a number of associations may take place in parallel.

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