Murray Stewart

Classical Nuclear Localization Signals: Definition, Function, and Interaction with Importin α

The best understood system for the transport of macromolecules between the cytoplasm and the nucleus is the classical nuclear import pathway. In this pathway, a protein containing a classical basic nuclear localization signal (NLS) is imported by a heterodimeric import receptor consisting of the β-karyopherin importin β, which mediates interactions with the nuclear pore complex, and the adaptor protein importin α, which directly binds the classical NLS. Here we review recent studies that have advanced our understanding of this pathway and also take a bioinformatics approach to analyze the likely prevalence of this system in vivo. Finally, we describe how a predicted NLS within a protein of interest can be confirmed experimentally to be functionally important.

Molecular mechanism of the nuclear protein import cycle

The nuclear import of proteins through nuclear pore complexes (NPCs) illustrates how a complex biological function can be generated by a spatially and temporally organized cycle of interactions between cargoes, carriers and the Ran GTPase. Recent work has given considerable insight into this process, especially about how interactions are coordinated and the basis for the molecular recognition that underlies the process. Although considerable progress has been made in identifying and characterizing the molecular interactions in the soluble phase that drive the nuclear protein import cycle, understanding the precise mechanism of translocation through NPCs remains a major challenge.

Tropomyosin coiled-coil interactions: evidence for an unstaggered structure.

Stereochemical arguments based on models of the tropomyosin coiled-coil favour an unstaggered symmetrical form, since this allows the best packing of the hydrophobic groups on the inner face where the two helices interlock. The distribution of polar groups along one side of the helix also shows correlations between positive and negative charges which favour a symmetrical structure stabilized by salt bridges between the helices. The models also show that two symmetrical molecules can join end-to-end by an external overlap of the nonpolar zones at the termini, giving an effective length of 275 amino acids, which fits 14 repeats of Parrys (1974) 19 1/2-residue periodicity. If tropomyosin molecules join in this way without discontinuity of twist, and bind equivalently to all the troponin molecules on the actin helix, the supercoil must take n half-turns in a molecular length, where n must be even for a staggered structure. An unstaggered structure could make seven half-turns relative to the actin helix and present a similar binding surface to all seven actins along its length. Because of the compensating twist of the actin helix the tropomyosin molecule would itself make only six half-turns and have a pitch close to 137 A.

NTF2 mediates nuclear import of Ran

Importin β family transport receptors shuttle between the nucleus and the cytoplasm and mediate transport of macromolecules through nuclear pore complexes (NPCs). The interactions between these receptors and their cargoes are regulated by binding RanGTP; all receptors probably exit the nucleus complexed with RanGTP, and so should deplete RanGTP continuously from the nucleus. We describe here the development of an in vitro system to study how nuclear Ran is replenished. Nuclear import of Ran does not rely on simple diffusion as Rans small size would permit, but instead is stimulated by soluble transport factors. This facilitated import is specific for cytoplasmic RanGDP and employs nuclear transport factor 2 (NTF2) as the actual carrier. NTF2 binds RanGDP initially to NPCs and probably also mediates translocation of the NTF2–RanGDP complex to the nuclear side of the NPCs. A direct NTF2–RanGDP interaction is crucial for this process, since point mutations that disturb the RanGDP–NTF2 interaction also interfere with Ran import. The subsequent nuclear accumulation of Ran also requires GTP, but not GTP hydrolysis. The release of Ran from NTF2 into the nucleus, and thus the directionality of Ran import, probably involves nucleotide exchange to generate RanGTP, for which NTF2 has no detectable affinity, followed by binding of the RanGTP to an importin β family transport receptor.

Structural Basis for the Interaction between FxFG Nucleoporin Repeats and Importin-β in Nuclear Trafficking

We describe the crystal structure of a complex between importin-beta residues 1-442 (Ib442) and five FxFG nucleoporin repeats from Nsp1p. Nucleoporin FxFG cores bind on the convex face of Ib442 to a primary site between the A helices of HEAT repeats 5 and 6, and to a secondary site between HEAT repeats 6 and 7. Mutations at importin-beta Ile178 in the primary FxFG binding site reduce both binding and nuclear protein import, providing direct evidence for the functional significance of the importin-beta-FxFG interaction. The FxFG binding sites on importin-beta do not overlap with the RanGTP binding site. Instead, RanGTP may release importin-beta from FxFG nucleoporins by generating a conformational change that alters the structure of the FxFG binding site.

The 14-fold periodicity in α-tropomyosin and the interaction with actin

A Fourier analysis of the distributions of different types of amino acids in the sequence of tropomyosin shows strong 14th-order peaks in the profiles of both negatively charged and non-polar amino acids, with a period of 1923 residues and an overall repeat length of 275 ± 2 amino acids, which is shorter than the sequence length of 284 amino acids. Both peaks are statistically significant and confirm Parrys work (1974, 1975b). The regularities are analysed in terms of an assumed supercoil structure in which two α-helices lie parallel and in register to form a supercoil with a pitch of 137 A. These molecules are then assumed to overlap end-to-end by eight to nine amino acids so that the periodicity is continuous along an extended filament of linked tropomyosin molecules. The periodic features are stronger in the outer surface of the molecule away from the core of the supercoil. The sequence divides into 14 bands which each have a narrow zone of net positive charge and a broader negatively charged zone. Overlapping every positive zone is a hydrophobic zone which always has at least one non-polar group on the outer surface. Anomalies in the charge distribution are found near the molecular ends and close to Cys190. These are attributed to the end-to-end overlap site and the troponin binding site. In the thin filament the 137 A pitch supercoil would make seven half-twists relative to the twisted actin helix along a 385 A length, so that a pair of adjacent bands would be oriented equivalently with respect to a pair of actins 28 A apart. We therefore suggest that the bands (each containing one zone of each type) should be divided alternately into two series, α and β. Every pair of bands is 3913 residues long and each of the seven pairs corresponds with one segment of the 42-residue gene duplication repeat observed previously in the sequence. The disparity between the periods of 42 and 3913 is overcome by deletions and insertions. The 3913-residue periodicity is not simply a consequence of the supercoil structure or gene duplication but is probably a result of adaptation to the spatial periodicity of the actin helix in muscle. Although the α and β bands are alike in general, they differ systematically in detail and the α bands are more regular than the β. We propose that the seven α and seven β bands are alternative sets of sites which bind equivalently to complementary groups of sites on seven actins in the “relaxed” and “active” states of muscle, respectively. In each band the negative zone probably attaches to actin by magnesium bridges and the hydrophobic zone by direct contacts with the narrow outer edge of the supercoil. Since the supercoil twists 90 ° relative to actin on passing between adjacent α and β bands, a quarter rotation of the whole tropomyosin molecule would detach one set of seven sites and attach the other, allowing a highly co-operative switch mechanism.

Structural basis for the assembly of a nuclear export complex

The nuclear import and export of macromolecular cargoes through nuclear pore complexes is mediated primarily by carriers such as importin-β. Importins carry cargoes into the nucleus, whereas exportins carry cargoes to the cytoplasm. Transport is orchestrated by nuclear RanGTP, which dissociates cargoes from importins, but conversely is required for cargo binding to exportins. Here we present the 2.0 Å crystal structure of the nuclear export complex formed by exportin Cse1p complexed with its cargo (Kap60p) and RanGTP, thereby providing a structural framework for understanding nuclear protein export and the different functions of RanGTP in export and import. In the complex, Cse1p coils around both RanGTP and Kap60p, stabilizing the RanGTP-state and clamping the Kap60p importin-β-binding domain, ensuring that only cargo-free Kap60p is exported. Mutagenesis indicated that conformational changes in exportins couple cargo binding to high affinity for RanGTP, generating a spring-loaded molecule to facilitate disassembly of the export complex following GTP hydrolysis in the cytoplasm.

Structural basis for nuclear import complex dissociation by RanGTP.

Nuclear protein import is mediated mainly by the transport factor importin-β that binds cytoplasmic cargo, most often via the importin-α adaptor, and then transports it through nuclear pore complexes. This active transport is driven by disassembly of the import complex by nuclear RanGTP. The switch I and II loops of Ran change conformation with nucleotide state, and regulate its interactions with nuclear trafficking components. Importin-β consists of 19 HEAT repeats that are based on a pair of antiparallel α-helices (referred to as the A- and B-helices). The HEAT repeats stack to yield two C-shaped arches, linked together to form a helicoidal molecule that has considerable conformational flexibility. Here we present the structure of full-length yeast importin-β (Kap95p or karyopherin-β) complexed with RanGTP, which provides a basis for understanding the crucial cargo-release step of nuclear import. We identify a key interaction site where the RanGTP switch I loop binds to the carboxy-terminal arch of Kap95p. This interaction produces a change in helicoidal pitch that locks Kap95p in a conformation that cannot bind importin-α or cargo. We suggest an allosteric mechanism for nuclear import complex disassembly by RanGTP.

Structural basis for the interaction between NTF2 and nucleoporin FxFG repeats

Interactions with nucleoporins containing FxFG‐repeat cores are crucial for the nuclear import of RanGDP mediated by nuclear transport factor 2 (NTF2). We describe here the 1.9 Å resolution crystal structure of yeast NTF2‐N77Y bound to a FxFG‐nucleoporin core, which provides a basis for understanding this interaction and its role in nuclear trafficking. The two identical FxFG binding sites on the dimeric molecule are formed by residues from each chain of NTF2. Engineered mutants at the interaction interface reduce the binding of NTF2 to nuclear pores and cause reduced growth rates and Ran mislocalization when substituted for the wild‐type protein in yeast. Comparison with the crystal structure of FG‐nucleoporin cores bound to importin‐β and TAP/p15 identified a number of common features of their binding sites. The structure of the binding interfaces on these transport factors provides a rationale for the specificity of their interactions with nucleoporins that, combined with their weak binding constants, facilitates rapid translocation through NPCs during nuclear trafficking.

INTERMEDIATE FILAMENT STRUCTURE AND ASSEMBLY

Intermediate filaments are constructed from two-chain alpha-helical coiled-coil molecules arranged on an imperfect helical lattice. Filament structure and assembly can be influenced at several different structural levels, including molecular structure, oligomer formation and filament nucleation and elongation. Consequently, it can sometimes be difficult to interpret mutagenesis data unequivocally, although regions near the amino and carboxyl termini of the rod domain of the molecule are known to be important for the production of native filaments. Imperfections in molecular packing may be important in filament assembly and dynamics.