Abstract
Despite the passage of ∼30 years since the complete primary sequence of the intermediate filament (IF) protein vimentin was reported, the structure remains unknown for both an individual protomer and the assembled filament. In this report, we present data describing the structure of vimentin linker 1 (L1) and rod 1B. Electron paramagnetic resonance spectra collected from samples bearing site-directed spin labels demonstrate that L1 is not a flexible segment between coiled-coils (CCs) but instead forms a rigid, tightly packed structure. An x-ray crystal structure of a construct containing L1 and rod 1B shows that it forms a tetramer comprising two equivalent parallel CC dimers that interact with one another in the form of a symmetrical anti-parallel dimer. Remarkably, the parallel CC dimers are themselves asymmetrical, which enables them to tetramerize rather than undergoing higher order oligomerization. This functionally vital asymmetry in the CC structure, encoded in the primary sequence of rod 1B, provides a striking example of evolutionary exploitation of the structural plasticity of proteins. EPR and crystallographic data consistently suggest that a very short region within L1 represents a minor local distortion in what is likely to be a continuous CC from the end of rod 1A through the entirety of rod 1B. The concordance of this structural model with previously published cross-linking and spectral data supports the conclusion that the crystallographic oligomer represents a native biological structure.
Highlights
The complete structure is not known for any intermediate filament (IF) protein
We present SDSL-EPR and crystallographic analyses of protein constructs containing the end of linker 1 (L1), the beginning of rod 1B, and most, if not all, of rod 1B
We present SDSL-EPR spectroscopy data establishing linker 1 (L1) as a very short discontinuity between the parallel ␣-helical CC structures formed by rod 1A and rod 1B on either side
Summary
The complete structure is not known for any intermediate filament (IF) protein. Results: Linker 1 and rod 1B in human vimentin were characterized using electron paramagnetic resonance spectroscopy and x-ray crystallography. We present SDSL-EPR and crystallographic analyses of protein constructs containing the end of L1, the beginning of rod 1B, and most, if not all, of rod 1B These data demonstrate that the non-CC region of L1 is likely to be shorter than predicted [29], whereas rod 1A and 1B form one continuous left-handed CC structure with the L1 segment representing only short perturbation. Our crystal structure demonstrates that rod 1B forms a parallel CC, which dimerizes with itself in an anti-parallel geometry to form a tetrameric ␣-helical bundle that is likely to be a key feature of higher order vimentin oligomerization and IF structure in vivo This interaction is centered near residue 191, as predicted by our prior EPR studies
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