Head and Rod 1 Interactions in Vimentin

Atya Aziz,John F Hess,Madhu S Budamagunta,Paul G Fitzgerald,John C Voss

doi:10.1074/jbc.m809029200

Atya Aziz, John F Hess + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m809029200

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Abstract

We have used site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) to identify residues 17 and 137 as sites of interaction between the head domain and rod domain 1A of the intermediate filament protein vimentin. This interaction was maximal when compared with the spin labels placed at up- and downstream positions in both head and rod regions, indicating that residues 17 and 137 were the closest point of interaction in this region. SDSL EPR characterization of residues 120-145, which includes the site of head contact with rod 1A, reveals that this region exhibits the heptad repeat pattern indicative of alpha-helical coiled-coil structure, but that this heptad repeat pattern begins to decay near residue 139, suggesting a transition out of coiled-coil structure. By monitoring the spectra of spin labels placed at the 17 and 137 residues during in vitro assembly, we show that 17-137 interaction occurs early in the assembly process. We also explored the effect of phosphorylation on the 17-137 interaction and found that phosphorylation-induced changes affected the head-head interaction (17-17) in the dimer, without significantly influencing the rod-rod (137-137) and head-rod (17-137) interactions in the dimer. These data provide the first direct evidence for, and location of, head-rod interactions in assembled intermediate filaments, as well as direct evidence of coiled-coil structure in rod 1A. Finally, the data identify changes in the structure in this region following in vitro phosphorylation.

Highlights

The intermediate filament (IF)2 protein family is one of the largest in the human genome, with more than 65 members
Structure prediction programs suggest the central ␣-helical rod domain can be subdivided into extended regions where the coiled-coil prediction is very strong, interrupted by short segments where this prediction weakens [11,12,13, 15] The head and tail domains that flank the central rod domain show great variation in both size and sequence and almost nothing is known of their structure or relationships in intact filaments
Very little experimental data are available for the structure of head region, and virtually no data on secondary structure are available to suggest any specific model of interaction between head and rod regions

Summary

EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis, Cloning, Overexpression, Purification, and Spin Labeling of Human Vimentin—Vimentin mutants were produced by site-directed mutagenesis, purified and spin-labeled as described in detail previously[36, 37, 40].3 In short, site-directed mutagenesis was used to introduce cysteine residues at specific sites in a slightly modified vimentin expression construct (generously provided by Roy Quinlan, University of Durham, Durham, UK) using the Stratagene QuikChange kit. Site-directed Mutagenesis, Cloning, Overexpression, Purification, and Spin Labeling of Human Vimentin—Vimentin mutants were produced by site-directed mutagenesis, purified and spin-labeled as described in detail previously[36, 37, 40].3. Mutant vimentin protein was produced by bacterial overexpression. Site-directed spin labeling was done by first treating the purified protein with 100 ␮M TCEP (Tris-(2-carboxyethyl) phosphine, hydrochloride; Molecular Probes, Eugene, OR) followed by spin labeling with 500 ␮M O-87500 ((1-oxyl-2,2,5,5-tetramethyl-_3-pyrroline-3methyl) methanethiosulfonate- d15[MTSL-d15]; (Toronto Research Chemicals, Toronto, Canada) to make the EPR-active protein having the nitroxide spin label on the targeted cysteine residue. The vimentin mutants were spin-labeled with 500 ␮M 3-malemidoproxyl (253375 Sigma Aldrich).

Kinase buffer

RESULTS

DISCUSSION