Sergei Strelkov

Coiled coils: a highly versatile protein folding motif

The alpha-helical coiled coil is one of the principal subunit oligomerization motifs in proteins. Its most characteristic feature is a heptad repeat pattern of primarily apolar residues that constitute the oligomer interface. Despite its simplicity, it is a highly versatile folding motif: coiled-coil-containing proteins exhibit a broad range of different functions related to the specific design of their coiled-coil domains. The architecture of a particular coiled-coil domain determines its oligomerization state, rigidity and ability to function as a molecular recognition system. Much progress has been made towards understanding the factors that determine coiled-coil formation and stability. Here we discuss this highly versatile protein folding and oligomerization motif with regard to its structural architecture and how this is related to its biological functions.

Conserved Segments 1A and 2B of the Intermediate Filament Dimer: Their Atomic Structures and Role in Filament Assembly.

Intermediate filaments (IFs) are key components of the cytoskeleton in higher eukaryotic cells. The elementary IF ‘building block’ is an elongated coiled‐coil dimer consisting of four consecutive α‐helical segments. The segments 1A and 2B include highly conserved sequences and are critically involved in IF assembly. Based on the crystal structures of three human vimentin fragments at 1.4–2.3 Å resolution (PDB entries 1gk4, 1gk6 and 1gk7), we have established the molecular organization of these two segments. The fragment corresponding to segment 1A forms a single, amphipatic α‐helix, which is compatible with a coiled‐coil geometry. While this segment might yield a coiled coil within an isolated dimer, monomeric 1A helices are likely to play a role in specific dimer–dimer interactions during IF assembly. The 2B segment reveals a double‐stranded coiled coil, which unwinds near residue Phe351 to accommodate a ‘stutter’. A fragment containing the last seven heptads of 2B interferes heavily with IF assembly and also transforms mature vimentin filaments into a new kind of structure. These results provide the first insight into the architecture and functioning of IFs at the atomic level.

Intermediate filaments: primary determinants of cell architecture and plasticity

Intermediate filaments (IFs) are major constituents of the cytoskeleton and nuclear boundary in animal cells. They are of prime importance for the functional organization of structural elements. Depending on the cell type, morphologically similar but biochemically distinct proteins form highly viscoelastic filament networks with multiple nanomechanical functions. Besides their primary role in cell plasticity and their established function as cellular stress absorbers, recently discovered gene defects have elucidated that structural alterations of IFs can affect their involvement both in signaling and in controlling gene regulatory networks. Here, we highlight the basic structural and functional properties of IFs and derive a concept of how mutations may affect cellular architecture and thereby tissue construction and physiology.

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

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 coeficient 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 A11 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.

Intermediate filament protein structure determination.

Publisher Summary X-ray crystallography is one of the few experimental methods that makes possible to study intermediate filament (IF) structure at atomic resolution; however, the prerequisite for a crystallographic analysis is the ability to produce macroscopic, well-ordered crystals. This chapter focuses on the existing experience with x-ray crystallography. It also discusses another diffraction method—small angle x-ray scattering (SAXS)–which is used to investigate IF structure at higher assembly levels than the dimer. It outlines the approaches that bring together data on the IF protein structure obtained by various methods, aiming at constructing the three-dimensional model of complete IFs—a goal that has not been achieved yet. Although, the structural resolution provided by SAXS is less than that of x-ray crystallography, the advantage of SAXS is that it works on protein solutions and, hence, does not depend on the availability of crystals. Moreover, SAXS allows the monitoring of the consequent steps of IF assembly by varying the solution conditions and provides additional information on the assembly pathway. The crystallization of stable complexes formed by various IF fragments is a difficult task; however, this should provide the essential atomic detail on the four dimer–dimer contact types occurring in mature IFs. Both x-ray crystallography and SAXS have a large future potential toward getting more structural insight into the interactions of the elementary IF dimers during assembly.