Monitoring intermediate filament assembly by small-angle x-ray scattering reveals the molecular architecture of assembly intermediates

Abstract

Intermediate filaments (IFs), along with microtubules, microfilaments, and associated cross-bridging proteins, constitute the cytoskeleton of metazoan cells. While crystallographic data on the dimer representing the elementary IF "building block" have recently become available, little structural detail is known about both the mature IF architecture and its assembly pathway. Here, we have applied solution small-angle x-ray scattering to investigate the in vitro assembly of a 53-kDa human IF protein vimentin at pH 8.4 by systematically varying the ionic strength conditions, and complemented these experiments by electron microscopy and analytical ultracentrifugation. While a vimentin solution in 5 mM Tris.HCl (pH 8.4) contains predominantly tetramers, addition of 20 mM NaCl induces further lateral assembly evidenced by the shift of the sedimentation coefficient and yields a distinct octameric intermediate. Four octamers eventually associate into unit-length filaments (ULFs) that anneal longitudinally. Based on the small-angle x-ray scattering experiments supplemented by crystallographic data and additional structural constraints, 3D molecular models of the vimentin tetramer, octamer, and ULF were constructed. Within each of the three oligomers, the adjacent dimers are aligned exclusively in an approximately half-staggered antiparallel A(11) mode with a distance of 3.2-3.4 nm between their axes. The ULF appears to be a dynamic and a relatively loosely packed structure with a roughly even mass distribution over its cross-section.

Fig. 1.

Vimentin assembly monitored by SAXS and AUC. (A) Experimental SAXS curves for the K139C vimentin mutant at: 4.2 mg/ml in 5 mM Tris·HCl (pH 8.4) (blue diamonds) overlaid with the calculated scattering from the final tetramer model (blue line), 1.6 mg/ml in the same buffer in the presence of 20 mM NaCl (red squares) overlaid with the calculated scattering from the final octamer model (red line), and 4 mg/ml in the same buffer in the presence of 75 mM NaCl (black circles) overlaid with the calculated scattering from the final ULF model (black line). (B) Corresponding cross-sectional distance distributions p_cr(r). (C) Distribution of sedimentation coefficients as determined from AUC velocity runs at 17°C with a K139C vimentin solution at 2 mg/ml in 5 mM Tris·HCl (pH 8.4) (black squares) and 4 mg/ml in 5 mM Tris·HCl (pH 8.4) in the presence of 12 mM NaCl (red squares).

Fig. 2.

Electron micrographs of negatively stained vimentin samples. (A and B) WT assembled at ≈5 mg/ml in 5 mM Tris·HCl buffer (pH 8.4) in the presence of 50 and 100 mM NaCl, respectively. (C) K139C mutant at ≈5 mg/ml in 5 mM Tris·HCl (pH 8.4) with 100 mM NaCl. (D) K139C mutant assembled for 2 s under standard conditions (0.1 mg/ml protein, 25 mM Tris·HCl, pH 7.5, and 50 mM NaCl). (Scale bar: 100 nm.)

Fig. 3.

Vimentin tetramer and octamer. (A) Side view of the tetramer as a ribbon diagram overlapped with a semitransparent surface (yellow). (B) Cross-sectional view at the position indicated by triangles in A. (C) Side view of the octamer. (D) Cross-sectional view of the octamer.

Fig. 4.

Vimentin ULF. (A) Cross-section of the initial model featuring a 4-fold symmetrical arrangement of octamers. (B) Refined model with a flattened cross-section. (C) Calculated scattering from the initial symmetrical model (blue diamonds), flattened model (red squares), and the final randomized model (green triangles) overlaid with the experimental data for the K139C mutant vimentin variant at 4 mg/ml in 5 mM Tris·HCl buffer (pH 8.4) with 75 mM NaCl (black circles). (D) Corresponding p_cr(r) functions. (E) Side view of the final randomized model. (F) Cross-section of the position indicated by triangles in E in the final randomized model.