Characterization of the linker 2 region in human vimentin using site-directed spin labeling and electron paramagnetic resonance.

Intermediate filaments (IFs1) are one of the three major classes of cytoskeletal structures in metazoan cells. Though more than 60 IF genes have been defined in the human genome, most IFs are assembled from just 1 or 2 IF proteins. Which specific IF protein(s) is/are expressed varies by cell type and the state of development/differentiation (1–4). Though there is considerable diversity in the IF family in both size and primary sequence, the vast majority of IF proteins show the conservation of several features: (1) they are localized to and/or can assemble into 8–11 nm IFs, and (2) they exhibit a common predicted domain structure. The predicted domain structure consists of (a) head and tail domains that vary widely in size and primary sequence and show no obvious predicted secondary structure and (b) a central rod domain that is approximately 310 amino acids in length. This rod domain is predicted to consist of extended runs of an α helix containing a heptad repeat pattern, which predicts the formation of coiled-coils (coil domains) separated by short regions called linkers that are not predicted to be α-helical, nor do they show a heptad repeat pattern (1–3, 5–16). However, depending on how parameters are set in programs that make such predictions, the strength of the coiled-coil prediction can vary quite dramatically (Figure 1) (17). Importantly, no direct test of structure has ever been made in these linker regions. FIGURE 1 Vimentin Schematic. A traditional schematic of the vimentin molecule is shown, with coil domains flanking the noncoil or linker region. Three variants of Coils version 2.1 (http://www.ch.embnet.org/software/COILS) predictions used to generate such models ... No intact IF protein or IF has been crystallized, thus a high-resolution structural map of neither the IF nor the IF protein has been determined. However, a variety of approaches, both theoretical and experimental have contributed to a model of IF structure and assembly. Strelkov and co-workers, for example, have been successful at the crystallization of fragments of the homopolymeric protein vimentin (18, 19). From the solution of these crystals’ structures has come the first detailed pictures of 2 regions of IF coiled-coil structure. The higher order structure of IF proteins within a filament and within assembly intermediates has been approached with cross-linking methodology. From both vimentin and keratin cross-link studies have emerged data that suggest a set of overlapping structures that are found in assembled filaments (20–23). We have employed site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) to characterize the full range of IF structure, yielding data on secondary, tertiary, and quaternary structures, and identifying sites where proteins are in close contact with one another in intact filaments (24–26). Collectively, these data support a model of the IF as beginning with the formation of an in-parallel and in-register dimer, where the central rod domain forms, at least in part, a coiled-coil dimer. Dimers then assemble into antiparallel tetramers with rod domains 1 associated (A11 interaction), followed by a species with rod domains 2 associated in an antiparallel interaction (A22 interaction). Cross-linking and SDSL–EPR have both contributed to establishing relationships between adjacent dimers, providing data on the relative orientation and registry of adjacent dimers. Most data that have been generated address the structure of the rod domain and its assembly into a coiled-coil dimer, and the relationship between adjacent rod domains in higher order structures. Essentially, nothing is known about the structure of the linker domains, and very little data has been generated that addresses the structure of either head or tail domains. In this article, we use SDSL-EPR to describe a series of spin-labeled mutants in the linker region of human vimentin (residues 281–304). EPR analysis of these spin-labeled mutants indicates that this region lacks coiled-coil structure, but also shows that several sites in this linker region are positioned extremely close to one another in intact filaments and that the polypeptide backbone in this region is quite rigid. Of considerable interest is evidence that the region from 283–291 assembles into its final structure much earlier than the coiled-coil domains within rod 1B and 2B that we have examined and is highly resistant to thermal denaturation.