Intermediate filament structure: the bottom-up approach

Intermediate filaments (IFs) result from a key cytoskeletal protein class in metazoan cells, but currently there is no consensus as to their three-dimensional architecture. IF proteins form elongated dimers based on the coiled-coil structure within their central ‘rod’ domain. Here we focus on the atomic structure of this elementary dimer, elucidated using X-ray crystallography on multiple fragments and electron paramagnetic resonance experiments on spin-labelled vimentin samples. In line with conserved sequence features, the rod of all IF proteins is composed of three coiled-coil segments containing heptad and hendecad repeats and interconnected by linkers. In addition, the next assembly intermediate beyond the dimer, the tetramer, could be modelled. The impact of these structural results towards understanding the assembly mechanism is discussed.

Introduction

Intermediate filaments (IFs) have a clear-cut importance for the functioning of metazoan cells. However, at present there is no consensus with regard to the architecture and assembly mechanism of these nanofilaments. In fact, the 10 nm wide IFs have proven to be much more challenging for structural studies than microtubules and F-actin, for both of which a complete 3D description, including atomic resolution data on their respective building blocks and the symmetry of the assembled filament, has long been available [1, 2].

All IF proteins, classified into five main families (‘types’, see http://www.interfil.org), have a distinct tripartite organization. In their primary structure, one can discern a highly α-helical central domain (‘rod’), flanked by the N-terminal (‘head’) and C-terminal (‘tail’) domains highly variable in sequence and length and typically containing little secondary structure [3]. The rod domain forms an α-helical coiled coil, one of the principal motifs of protein architecture (Box 1), composed of two parallel in register chains. Most IF proteins form homodimers, although preferential heterodimers such as keratins as well as further heterotypic assemblies in vivo are also observed [4]. It is the coiled-coil rod that is responsible for the overall elongated shape of the elementary dimer, which measures 45–48 nm in cytoplasmic IF proteins and 50–52 nm in nuclear lamins [5, 6].

Even though the assembly of IFs in their natural habitat — either the cytoplasm or the nucleus — occurs in the presence of many other protein factors, various IF types could also be reproduced in vitro by manipulating with solution parameters. Thus IFs qualify as self-assembling systems [7]. There are several assembly principles that are valid for all IFs. First, the axes of the dimers are aligned roughly in the filament direction; second, dimers associate laterally via several distinct modes [8]; and finally, longitudinal growth of the filament depends on the end-to-end interaction of dimers. The exact assembly pathway varies for different IF types [4, 9]. For instance, vimentin is found in a low molarity neutral buffer in the form of tetramers resulting from two half-staggered antiparallel dimers (so-called A₁₁ tetramers). Subsequent increase of ionic strength yields higher lateral assembly intermediates such as octamers and the so-called unit-length filaments (ULFs) typically containing 4 octamers, followed by longitudinal assembly that eventually results in native-like filaments [4, 10].

Here we will focus on the considerable advances that have been made in the recent years towards the atomic structure of the elementary IF dimer, contributing to a better understanding of both the IF architecture and assembly mechanism.