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-alpha, 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-alpha-wild-type and the importin-alpha-hyperphosphorylation-mimicking dUTPase NLS complexes provide structural insights into the molecular details of this regulation. The data indicate that the posttranslational modification of dUTPase during the cell cycle may modulate the nuclear availability of this enzyme

Structural and Functional Characterisation of Nuclear Transport Protein Complexes

In eukaryotic cells, the nucleus and cytoplasm are physically separated by the nuclear membrane, segregating nuclear DNA replication and RNA transcription from cytoplasmic protein synthesis. This compartmentalisation therefore requires an efficient mechanism to transport mRNA and proteins between the nucleus and cytoplasm. Bi-directional transport of these molecules occurs through large macromolecular channels, called nuclear pore complexes that traverse the nuclear envelope. Although small molecules can passively diffuse through the nuclear pore channels, proteins >40 kDa require an active transport mechanism for nuclear translocation. Nuclear protein import is a process that delivers protein cargo into the nucleus in a regulated and efficient manner. The majority of nuclear transport pathways are mediated by the β-karyopherin superfamily of proteins, while the small GTPase Ran imparts directionality on the transport cycle. Many aspects of the nuclear transport pathway have been characterised structurally and biochemically. Nonetheless, there are still various interactions that have been identified in past studies that are not adequately described within our current model of protein transport. The objective of this study was to structurally and functionally investigate a range of proteins and protein complexes within the nuclear transport cycle, for which their role in protein import was unclear, or have not been sufficiently structurally characterised. In particular, this study has gained new insights into the specificity determinants of the importin-α and nuclear localisation signal (NLS) interaction, through the structural characterization of the importin-α:high-affinity NLS interaction. Additionally, this work has extended our understanding of importin-β flexibility in both the unliganded state, and in complex with importin-α through the use of small-angle x-ray scattering techniques. Lastly, the structures of importin-β:Ran complexes with and without bound nucleotide were determined using x-ray crystallography. These results have shed light on the Ran GDP-to-GTP exchange activity of importin-β, and have revealed a novel conformation for the C-terminal helix of Ran when in complex with importin-β

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

Conditions are reported for the crystallization of the flexible nuclear import receptor importin-β. Preliminary X-ray diffraction data indicate that the structure can be solved using molecular replacement

Importin-β Is a GDP-to-GTP Exchange Factor of Ran: IMPLICATIONS FOR THE MECHANISM OF NUCLEAR IMPORT*

Ran-GTP interacts strongly with importin-β, and this interaction promotes the release of the importin-α-nuclear localization signal cargo from importin-β. Ran-GDP also interacts with importin-β, but this interaction is 4 orders of magnitude weaker than the Ran-GTP·importin-β interaction. Here we use the yeast complement of nuclear import proteins to show that the interaction between Ran-GDP and importin-β promotes the dissociation of GDP from Ran. The release of GDP from the Ran-GDP-importin-β complex stabilizes the complex, which cannot be dissociated by importin-α. Although Ran has a higher affinity for GDP compared with GTP, Ran in complex with importin-β has a higher affinity for GTP. This feature is responsible for the generation of Ran-GTP from Ran-GDP by importin-β. Ran-binding protein-1 (RanBP1) activates this reaction by forming a trimeric complex with Ran-GDP and importin-β. Importin-α inhibits the GDP exchange reaction by sequestering importin-β, whereas RanBP1 restores the GDP nucleotide exchange by importin-β by forming a tetrameric complex with importin-β, Ran, and importin-α. 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

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-alpha/karyopherin-alpha and importin-beta/karyopherin-beta 1; and (ii) the karyopherin-beta 2 pathway, which employs the proline-tyrosine (PY)-NLSs and the receptor transportin-1/karyopherin-beta 2. 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-alpha.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. (C) 2010 Elsevier B.V. All rights reserved

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-β. Here, we show that RanGDP and importin-β form a stable complex with a micromolar dissociation constant. This complex can be dissociated by importin-β binding partners such as importin-α. 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

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 Ibα–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. © The Authors Journal compilation © 2008 Biochemical Societ

Remdesivir and Mortality in Patients with COVID-19.

BACKGROUND: The impact of remdesivir (RDV) on COVID-19 mortality is controversial, and the mortality effect in sub-groups of baseline disease severity has been incompletely explored. The purpose of this study was to assess the association of RDV with mortality in patients with COVID-19. METHODS: In this retrospective cohort study we compared persons receiving RDV to persons receiving best supportive care (BSC). Patients hospitalized between 2/28/20 - 5/28/20 with laboratory confirmed SARS-CoV-2 infection were included when they developed COVID-19 pneumonia on chest radiography, and hypoxia requiring supplemental oxygen or SpO2 ≤ 94% on room air. The primary outcome was overall survival assessed with time-dependent Cox proportional-hazards regression and multivariable adjustment, including calendar time, baseline patient characteristics, corticosteroid use and effects for hospital. RESULTS: 1,138 patients were enrolled including 286 who received RDV, and 852 treated with BSC, 400 of whom received hydroxychloroquine. Corticosteroids were used in 20.4% of the cohort (12.6% in RDV and 23% in BSC). In persons receiving RDV compared to those receiving BSC the HR (95%CI) for death was 0.46 (0.31 - 0.69) in the univariate model, p CONCLUSION: Treatment with RDV was associated with lower mortality compared to BSC. These findings remain the same in the subgroup with baseline use of low-flow oxygen