Norbert Geisler

Neurofilament architecture combines structural principles of intermediate filaments with carboxy-terminal extensions increasing in size between triplet proteins.

Mammalian neurofilament triplet proteins (68 K, 160 K and 200 K) have been correlated by a biochemical, immunological and protein chemical study. The 160 K and 200 K triplet proteins are intermediate filament proteins in their own right, since they reveal the alpha‐helical coiled‐coil rod domain analyzed in detail for the 68 K protein. Triplet proteins display two distinct arrays. Their amino‐terminal region built analogously to non‐neuronal intermediate filament proteins should allow a co‐polymerization process via the interaction of coiled‐coil domains. The extra mass of all triplet proteins is allocated to carboxy‐terminally located extensions of increasing size and unique amino acid sequences. These may provide highly charged scaffolds suitable for interactions with other neuronal components. Such a domain of 68 K reveals, in sequence analysis, 47 glutamic acids within 106 residues. The epitope recognized by a monoclonal antibody reacting probably with all intermediate filament proteins has been mapped. It is located within the last 20 residues of the rods, where six distinct intermediate filament proteins point to a consensus sequence.

Self-assembly in vitro of the 68,000 molecular weight component of the mammalian neurofilament triplet proteins into intermediate-sized filaments☆

The mammalian neurofilament triplet proteins (210, 160 and 68 × 103 Mr proteins) are resolved by anion exchange chromatography in the presence of urea. Upon dialysis against physiological buffers at 37 °C only the 68 × 103 Mr protein shows self-assembly into morphologically normal intermediate-sized filaments. Addition of 210 × 103 Mr protein to 68 × 103 Mr protein leads to shorter filaments, which upon embedding reveal a rough surface and whisker-like protrusions that are not present on the smooth surface of filaments assembled from 68 × 103 Mr protein alone. Certain emerging principles of neurofilament structure are discussed, emphasizing a possible relation between neurofilaments and other intermediate-sized filaments.

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.

Proteinchemical characterization of three structurally distinct domains along the protofilament unit of desmin 10 nm filaments

Limited chymotryptic cleavage of soluble chicken gizzard desmin protofilaments allows the characterization of three structurally distinct domains. A surface-exposed very basic amino-terminal region (the headpiece) with an amino acid sequence excluding alpha-helical organization (7.5 kd) is separated from the perhaps globular carboxy-terminal 48 residues (the tailpiece) by a distinctly different middle domain of approximately 330 residues. This 38 kd domain is very rich in alpha-helix (at least 83%), and electron microscopy reveals a thin rod with a length of 500 +/- 50 A. Amino acid sequence data also show that the rod domain is interrupted by a nonhelical portion. An alpha-helical array is able to form a coiled-coil spanning the carboxy-terminal half of the 38 kd domain. The alpha-type diffraction pattern of 10 nm filaments arises from a coiled-coil conformation displayed through most but not all of the middle domain of the protofilaments.

SDS—PAGE strongly overestimates the molecular masses of the neurofilament proteins

Direct molecular mass determination of the three porcine neurofilament proteins (H, M and L) was performed in 6 M guanidine—HCl using analytical gel filtration and sedimentation equilibrium centrifugation. The results show that SDS—PAGE strongly overestimates the values of the ‘higher molecular mass’ components H and M. This discrepancy stems from the carboxyterminal extensions known to have unusual amino acid composition.

Intermediate filament forming ability of desmin derivatives lacking either the amino-terminal 67 or the carboxy-terminal 27 residues☆

Amino acid sequence data and results from limited proteolytic digestion have been used to define the three-domain structure of intermediate filament proteins. A centrally located highly alpha-helical domain of about 310 residues well-conserved in sequence principles and length is flanked by the highly variable sequences of the non-alpha-helical headpiece and tailpiece. A direct involvement in filament formation of one or both terminal domains was previously proposed for desmin since chymotryptic removal of head and tailpiece provided a derivative unable to form filaments. In order to evaluate directly the importance of these regions we have prepared desmin derivatives lacking either the amino-terminal 67 (T-desmin) or carboxy-terminal 27 residues (L-desmin). Whereas the latter derivative is fully polymerization-competent the fragment lacking only the basic and arginine-rich headpiece cannot form filaments on its own and remains in a protofilamentous stage. These structures of T-desmin are not incorporated into filaments when mixed with protofilaments of desmin. If, however, the two proteins are mixed in 7 M-urea subsequent dialysis provides morphologically normal filaments containing T-desmin. The results suggest that at least certain hybrid protofilaments containing less than four headpieces are accepted in the filament. The removal of the 27 carboxy-terminal residues in L-desmin, although not interfering with filament formation, leads to a change in surface since filaments show lateral aggregation at 170 mM but not at 50 mM salt. The results are discussed in relation to current models of intermediate filament structure.

Reconstitution of intermediate-sized filaments from denatured monomeric vimentin.

Abstract Vimentin, a major cytoskeletal protein of many cells of mesenchymal tissues as well as of cultured cells of various kinds, is the constituent protein of a special class of intermediate-sized (7 to 11 nm) filaments. These are insoluble at high and low ionic strength over the physiologically relevant pH range (5·5 to 7·5). Vimentin from cytoskeletons of various cells (porcine eye lens tissue, mouse 3T3, rat RVF-SMC, and hamster BHK-21 cells) was purified and solubilized in urea or guanidinium hydrochloride. When solutions of denatured, monomeric vimentin were dialyzed against buffers of various ionic strengths containing 2-mercaptoethanol, vimentin renatured and long (up to 3·5 μm) intermediate-sized filaments were formed. The reconstituted filaments were indistinguishable from native vimentin filaments by electron microscopy and X-ray diffraction. Native and reconstituted intermediate-sized filaments containing other proteins (prekeratin, desmin) were examined in parallel. The high degree of molecular order in reconstituted filaments was demonstrated by meridional reflections at 0·15 and 0·51 nm and equatorial reflections at 0·98 and 6·3 nm, indicating coiled-coil arrangements of α-helices of vimentin. The results show that vimentin spontaneously refolds into α-helical arrangements and assembles, over a broad range of salt concentrations, into long intermediate-sized filaments. The process does not involve disulfide bond formation or the presence of other proteins. The similar reconstitution properties of various intermediate filament proteins suggest that such proteins, albeit different in polypeptide composition and amino acid sequence, contain regions with similar principles of sequence arrangement that define α-helical domains and are responsible for the assembly into morphologically similar intermediate-sized filaments.

Antiparallel orientation of the two double-stranded coiled-coils in the tetrameric protofilament unit of intermediate filaments☆

The chymotryptically excised middle domain of desmin slightly exceeds in length the structurally conserved alpha-helical middle region documented in all intermediate filament proteins by amino acid sequence data. This rod domain is a protofilament derivative with a tetrameric organization, thus indicating the presence of two double-stranded coiled-coil units. We now show by immunoelectron microscopy that Fab fragments of a desmin-specific monoclonal antibody mixed with the rod lead to dumb-bell-shaped structures. The tagging of both ends together with the length of the rod (48 nm) argues for an antiparallel orientation of the two coiled-coils without a major stagger. This information combined with the lateral 21 nm periodicity of the intermediate filament observed by us and others leads to a structural hypothesis similar to those entertained from X-ray data on wool alpha-keratins, although here an antiparallel tetrameric unit of some 60 to 66 nm is invoked, which has never been isolated. The structure that we discuss allows for the existence of both the particles, and the antibody experiment strongly supports the antiparallel orientation postulated in both approaches. The tube-like filament structure proposed for the intermediate filament agrees with recent mass per unit length measurements and allows for two minor classes of intermediate filaments with different values in this property as also found experimentally.

A periodic ultrastructure in intermediate filaments.

Abstract Intermediate sized filaments reconstituted in vitro from purified desmin, epidermal keratin and the M r 68,000 protein of neurofilaments were examined after high resolution metal shadowing. The filaments demonstrate a marked longitudinal periodicity of about 21 nm. This is the first procedure that allows detection of a periodic substructure in these filaments using the electron microscope.

Protein-chemical characterization of NF-H, the largest mammalian neurofilament component; intermediate filament-type sequences followed by a unique carboxy-terminal extension

NF‐H has the highest mol. wt. of the three mammalian neurofilament components (NF‐L, NF‐M, NF‐H). In spite of its unusually large mol. wt., estimated to be 200 K by gel electrophoresis, NF‐H contains sequences which identify it as an integral intermediate filament (IF) protein in its amino‐terminal region. We have isolated and partially characterized a basic, non‐α‐helical segment located at the amino‐terminal end with properties similar to headpieces of other non‐epithelial IF proteins. The highly α‐helical 40‐K fragment excised by chymotrypsin is now identified by the amino acid sequence of a 17‐K fragment. This sequence can be unambiguously aligned with the rod region of other IF proteins and covers about half of the presumptive coiled‐coil arrays. NF‐H and NF‐M show 45% sequence identity in this region. The extra mass of NF‐H in comparison with most other IF proteins arises from a carboxy‐terminal extension thought to be responsible for inter‐neurofilament cross‐bridges in axons. This autonomous domain has a unique amino acid composition characterized by a high content of proline, alanine and particularly of lysine and glutamic acid. The NF‐H tailpiece extension also carries a large number of serine phosphates, which are not evenly distributed, but are restricted to the amino‐terminal part. Having now delineated the intermediate filament‐type sequences for all three neurofilament proteins it seems very likely that the three components interact via coiled‐coil interactions. They all carry unique carboxy‐terminal extensions which increase in length from NF‐L to NF‐H and seem to extend from the filament wall.