Structural basis of high-affinity nuclear localization signal interactions with importin-α.

Classical nuclear localization signals (cNLSs), comprising one (monopartite cNLSs) or two clusters of basic residues connected by a 10-12 residue linker (bipartite cNLSs), are recognized by the nuclear import factor importin-α. The cNLSs bind along a concave groove on importin-α; however, specificity determinants of cNLSs remain poorly understood. We present a structural and interaction analysis study of importin-α binding to both designed and naturally occurring high-affinity cNLS-like sequences; the peptide inhibitors Bimax1 and Bimax2, and cNLS peptides of cap-binding protein 80. Our data suggest that cNLSs and cNLS-like sequences can achieve high affinity through maximizing interactions at the importin-α minor site, and by taking advantage of multiple linker region interactions. Our study defines an extended set of binding cavities on the importin-α surface, and also expands on recent observations that longer linker sequences are allowed, and that long-range electrostatic complementarity can contribute to cNLS-binding affinity. Altogether, our study explains the molecular and structural basis of the results of a number of recent studies, including systematic mutagenesis and peptide library approaches, and provides an improved level of understanding on the specificity determinants of a cNLS. Our results have implications for identifying cNLSs in novel proteins.

Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights.

Phosphorylation adjacent to nuclear localization signals (NLSs) is involved in the regulation of nucleocytoplasmic transport. The nuclear isoform of human dUTPase, an enzyme that is essential for genomic integrity, has been shown to be phosphorylated on a serine residue (Ser11) in the vicinity of its nuclear localization signal; however, the effect of this phosphorylation is not yet known. To investigate this issue, an integrated set of structural, molecular and cell biological methods were employed. It is shown that NLS-adjacent phosphorylation of dUTPase occurs during the M phase of the cell cycle. Comparison of the cellular distribution of wild-type dUTPase with those of hyperphosphorylation- and hypophosphorylation-mimicking mutants suggests that phosphorylation at Ser11 leads to the exclusion of dUTPase from the nucleus. Isothermal titration microcalorimetry and additional independent biophysical techniques show that the interaction between dUTPase and importin-α, the karyopherin molecule responsible for `classical' NLS binding, is weakened significantly in the case of the S11E hyperphosphorylation-mimicking mutant. The structures of the importin-α-wild-type and the importin-α-hyperphosphorylation-mimicking dUTPase NLS complexes provide structural insights into the molecular details of this regulation. The data indicate that the post-translational modification of dUTPase during the cell cycle may modulate the nuclear availability of this enzyme.

Ligand-induced conformational changes within a hexameric Acyl-CoA thioesterase.

Acyl-coenzyme A (acyl-CoA) thioesterases play a crucial role in the metabolism of activated fatty acids, coenzyme A, and other metabolic precursor molecules including arachidonic acid and palmitic acid. These enzymes hydrolyze coenzyme A from acyl-CoA esters to mediate a range of cellular functions including ß-oxidation, lipid biosynthesis, and signal transduction. Here, we present the crystal structure of a hexameric hot-dog domain-containing acyl-CoA thioesterase from Bacillus halodurans in the apo-form and provide structural and comparative analyses to the coenzyme A-bound form to identify key conformational changes induced upon ligand binding. We observed dramatic ligand-induced changes at both the hot-dog dimer and the trimer-of-dimer interfaces; the dimer interfaces in the apo-structure differ by over 20% and decrease to about half the size in the ligand-bound state. We also assessed the specificity of the enzyme against a range of fatty acyl-CoA substrates and have identified a preference for short-chain fatty acyl-CoAs. Coenzyme A was shown both to negatively regulate enzyme activity, representing a direct inhibitory feedback, and consistent with the structural data, to destabilize the quaternary structure of the enzyme. Coenzyme A-induced conformational changes in the C-terminal helices of enzyme were assessed through mutational analysis and shown to play a role in regulating enzyme activity. The conformational changes are likely to be conserved from bacteria through to humans and provide a greater understanding, particularly at a structural level, of thioesterase function and regulation.

Molecular basis for specificity of nuclear import and prediction of nuclear localization.

Although proteins are translated on cytoplasmic ribosomes, many of these proteins play essential roles in the nucleus, mediating key cellular processes including but not limited to DNA replication and repair as well as transcription and RNA processing. Thus, understanding how these critical nuclear proteins are accurately targeted to the nucleus is of paramount importance in biology. Interaction and structural studies in the recent years have jointly revealed some general rules on the specificity determinants of the recognition of nuclear targeting signals by their specific receptors, at least for two nuclear import pathways: (i) the classical pathway, which involves the classical nuclear localization sequences (cNLSs) and the receptors importin-α/karyopherin-α and importin-ß/karyopherin-ß1; and (ii) the karyopherin-ß2 pathway, which employs the proline-tyrosine (PY)-NLSs and the receptor transportin-1/karyopherin-ß2. The understanding of specificity rules allows the prediction of protein nuclear localization. We review the current understanding of the molecular determinants of the specificity of nuclear import, focusing on the importin-α•cargo recognition, as well as the currently available databases and predictive tools relevant to nuclear localization. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.

Importin-beta is a GDP-to-GTP exchange factor of Ran: implications for the mechanism of nuclear import.

Ran-GTP interacts strongly with importin-beta, and this interaction promotes the release of the importin-alpha-nuclear localization signal cargo from importin-beta. Ran-GDP also interacts with importin-beta, but this interaction is 4 orders of magnitude weaker than the Ran-GTP.importin-beta interaction. Here we use the yeast complement of nuclear import proteins to show that the interaction between Ran-GDP and importin-beta promotes the dissociation of GDP from Ran. The release of GDP from the Ran-GDP-importin-beta complex stabilizes the complex, which cannot be dissociated by importin-alpha. Although Ran has a higher affinity for GDP compared with GTP, Ran in complex with importin-beta has a higher affinity for GTP. This feature is responsible for the generation of Ran-GTP from Ran-GDP by importin-beta. Ran-binding protein-1 (RanBP1) activates this reaction by forming a trimeric complex with Ran-GDP and importin-beta. Importin-alpha inhibits the GDP exchange reaction by sequestering importin-beta, whereas RanBP1 restores the GDP nucleotide exchange by importin-beta by forming a tetrameric complex with importin-beta, Ran, and importin-alpha. The exchange is also inhibited by nuclear-transport factor-2 (NTF2). We suggest a mechanism for nuclear import, additional to the established RCC1 (Ran-guanine exchange factor)-dependent pathway that incorporates these results.

Crystallization of the flexible nuclear import receptor importin-beta in the unliganded state.

The transport of macromolecules across the nuclear envelope is an essential eukaryotic process that enables proteins such as transcription factors, polymerases and histones to gain access to the genetic material contained within the nucleus. Importin-beta plays a central role in the nucleocytoplasmic transport process, mediating nuclear import through a range of interactions with cytoplasmic, nuclear and nuclear pore proteins such as importin-alpha, Ran, nucleoporins and various cargo molecules. The unliganded form of the full-length yeast importin-beta has been expressed and crystallized. The crystals were obtained by vapour diffusion at pH 6.5 and 290 K. The crystals belonged to space group P2(1) (unit-cell parameters a = 58.17, b = 127.25, c = 68.52 A, beta = 102.23). One molecule is expected in the asymmetric unit. The crystals diffracted to 2.4 A resolution using a laboratory X-ray source and were suitable for crystal structure determination.

Crystallography and protein-protein interactions: biological interfaces and crystal contacts.

Crystallography is commonly used for studying the structures of protein-protein complexes. However, a crystal structure does not define a unique protein-protein interface, and distinguishing a 'biological interface' from 'crystal contacts' is often not straightforward. A number of computational approaches exist for distinguishing them, but their error rate is high, emphasizing the need to obtain further data on the biological interface using complementary structural and functional approaches. In addition to reviewing the computational and experimental approaches for addressing this problem, we highlight two relevant examples. The first example from our laboratory involves the structure of acyl-CoA thioesterase 7, where each domain of this two-domain protein was crystallized separately, but both yielded a non-functional assembly. The structure of the full-length protein was uncovered using a combination of complementary approaches including chemical cross-linking, analytical ultracentrifugation and mutagenesis. The second example involves the platelet glycoprotein Ibalpha-thrombin complex. Two groups reported the crystal structures of this complex, but all the interacting interfaces differed between the two structures. Our computational analysis did not fully resolve the reasons for the discrepancies, but provided interesting insights into the system. This review highlights the need to complement crystallographic studies with complementary experimental and computational approaches.

Quantitative structural analysis of importin-ß flexibility: paradigm for solenoid protein structures.

The structure of solenoid proteins facilitates a higher degree of flexibility than most folded proteins. In importin-ß, a nuclear import factor built from 19 tandem HEAT repeats, flexibility plays a crucial role in allowing interactions with a range of different partners. We present a comprehensive analysis of importin-ß flexibility based on a number of different approaches. We determined the crystal structure of unliganded Saccharomyces cerevisiae importin-ß (Kap95) to allow a quantitative comparison with importin-ß bound to different partners. Complementary mutagenesis, small angle X-ray scattering and molecular dynamics studies suggest that the protein samples several conformations in solution. The analyses suggest the flexibility of the solenoid is generated by cumulative small movements along its length. Importin-ß illustrates how solenoid proteins can orchestrate protein interactions in many cellular pathways.

Kap95p binding induces the switch loops of RanGDP to adopt the GTP-bound conformation: implications for nuclear import complex assembly dynamics.

The asymmetric distribution of the nucleotide-bound state of Ran across the nuclear envelope is crucial for determining the directionality of nuclear transport. In the nucleus, Ran is primarily in the guanosine 5'-triphosphate (GTP)-bound state, whereas in the cytoplasm, Ran is primarily guanosine 5'-diphosphate (GDP)-bound. Conformational changes within the Ran switch I and switch II loops are thought to modulate its affinity for importin-beta. Here, we show that RanGDP and importin-beta form a stable complex with a micromolar dissociation constant. This complex can be dissociated by importin-beta binding partners such as importin-alpha. Surprisingly, the crystal structure of the Kap95p-RanGDP complex shows that Kap95p induces the switch I and II regions of RanGDP to adopt a conformation that resembles that of the GTP-bound form. The structure of the complex provides insights into the structural basis for the gradation of affinities regulating nuclear protein transport.

Structural basis for recruitment of tandem hotdog domains in acyl-CoA thioesterase 7 and its role in inflammation.

Acyl-CoA thioesterases (Acots) catalyze the hydrolysis of fatty acyl-CoA to free fatty acid and CoA and thereby regulate lipid metabolism and cellular signaling. We present a comprehensive structural and functional characterization of mouse acyl-CoA thioesterase 7 (Acot7). Whereas prokaryotic homologues possess a single thioesterase domain, mammalian Acot7 contains a pair of domains in tandem. We determined the crystal structures of both the N- and C-terminal domains of the mouse enzyme, and inferred the structure of the full-length enzyme using a combination of chemical cross-linking, mass spectrometry, and molecular modeling. The quaternary arrangement in Acot7 features a trimer of hotdog fold dimers. Both domains of Acot7 are required for activity, but only one of two possible active sites in the dimer is functional. Asn-24 and Asp-213 (from N- and C-domains, respectively) were identified as the catalytic residues through site-directed mutagenesis. An enzyme with higher activity than wild-type Acot7 was obtained by mutating the residues in the nonfunctional active site. Recombinant Acot7 was shown to have the highest activity toward arachidonoyl-CoA, suggesting a function in eicosanoid metabolism. In line with the proposal, Acot7 was shown to be highly expressed in macrophages and up-regulated by lipopolysaccharide. Overexpression of Acot7 in a macrophage cell line modified the production of prostaglandins D2 and E2. Together, the results link the molecular and cellular functions of Acot7 and identify the enzyme as a candidate drug target in inflammatory disease.