Proteins are translated in the cytoplasm, but many need to access the nucleus to perform their functions. Understanding how these nuclear proteins are transported through the nuclear envelope and how the import processes are regulated is therefore an important aspect of understanding cell function. Structural biology has played a key role in understanding the molecular events during the transport processes and their regulation, including the recognition of nuclear targeting signals by the corresponding receptors. Here, we review the structural basis of the principal nuclear import pathways and the molecular basis of their regulation. The pathways involve transport factors that are members of the β-karyopherin family, which can bind cargo directly (e.g., importin-β, transportin-1, transportin-3, importin-13) or through adaptor proteins (e.g., importin-α, snurportin-1, symportin-1), as well as unrelated transport factors such as Hikeshi, involved in the transport of heat-shock proteins, and NTF2, involved in the transport of RanGDP. Solenoid proteins feature prominently in these pathways. Nuclear transport factors recognize nuclear targeting signals on the cargo proteins, including the classical nuclear localization signals, recognized by the adaptor importin-α, and the PY nuclear localization signals, recognized by transportin-1. Post-translational modifications, particularly phosphorylation, constitute key regulatory mechanisms operating in these pathways.

Importin-␣ is the nuclear import receptor that recognizes the classic monopartite and bipartite nuclear localization sequences (cNLSs), which contain one or two clusters of basic amino acids, respectively. Different importin-␣ paralogs in a single organism are specific for distinct repertoires of cargos. Structural studies revealed that monopartite cNLSs and the C-terminal basic clusters of the bipartite cNLSs bind to the same site on importin-␣, termed the major cNLS-binding site. We used an oriented peptide library approach with five degenerate positions to probe the specificity of the major cNLS-binding site in importin-␣. We identified the sequences KKKRR, KKKRK, and KKRKK as the optimal sequences for binding to this site for mouse importin-␣2, human importin-␣1, and human importin-␣5, respectively. The crystal structure of mouse importin-␣2 with its optimal peptide confirmed the expected binding mode resembling the binding of simian virus 40 large tumor-antigen cNLS. Binding assays confirmed that the peptides containing these sequences bound to the corresponding proteins with low nanomolar affinities. Nuclear import assays showed that the sequences acted as functional cNLSs, with specificity for particular importin-␣s. This is the first time that structural information has been linked to an oriented peptide library screening approach for importin-␣; the results will contribute to understanding of the sequence determinants of cNLSs, and may help identify as yet unidentified cNLSs in novel proteins.Cellular compartments are the defining feature of a eukaryotic cell. The nucleus allows the separation of the genetic material and transcriptional machinery from the translational and metabolic processes in the cytoplasm. The nucleus is defined by a double membrane called the nuclear envelope, and the transport across this barrier occurs through large macromolecular assemblies called the nuclear pore complexes (1, 2). Transport of proteins through this pore is facilitated by nucleocytoplasmic transport factors termed karyopherins. These proteins recognize special sequences within the cargo proteins and selectively transport them in (importins) or out (exportins) of the nucleus.

Ku70 and Ku80 form a heterodimeric complex involved in multiple nuclear processes. This complex plays a key role in DNA repair due to its ability to bind DNA double-strand breaks and facilitate repair by the nonhomologous end-joining pathway. Ku70 and Ku80 have been proposed to contain bipartite and monopartite nuclear localization sequences (NLSs), respectively, that allow them to be translocated to the nucleus independently of each other via the classical importin-α (Impα)/importin-β-mediated nuclear import pathway. To determine the structural basis of the recognition of Ku70 and Ku80 proteins by Impα, we solved the crystal structures of the complexes of Impα with the peptides corresponding to the Ku70 and Ku80 NLSs. Our structural studies confirm the binding of the Ku80 NLS as a classical monopartite NLS but reveal an unexpected binding mode for Ku70 NLS with only one basic cluster bound to the receptor. Both Ku70 and Ku80 therefore contain monopartite NLSs, and sequences outside the basic cluster make favorable interactions with Impα, suggesting that this may be a general feature in monopartite NLSs. We show that the Ku70 NLS has a higher affinity for Impα than the Ku80 NLS, consistent with more extensive interactions in its N-terminal region. The prospect of nuclear import of Ku70 and Ku80 independently of each other provides a powerful regulatory mechanism for the function of the Ku70/Ku80 heterodimer and independent functions of the two proteins.

MLH1 and PMS2 proteins form the MutLα heterodimer, which plays a major role in DNA mismatch repair (MMR) in humans. Mutations in MMR-related proteins are associated with cancer, especially with colon cancer. The N-terminal region of MutLα comprises the N-termini of PMS2 and MLH1 and, similarly, the C-terminal region of MutLα is composed by the C-termini of PMS2 and MLH1, and the two are connected by linker region. The nuclear localization sequences (NLSs) necessary for the nuclear transport of the two proteins are found in this linker region. However, the exact NLS sequences have been controversial, with different sequences reported, particularly for MLH1. The individual components are not imported efficiently, presumably due to their C-termini masking their NLSs. In order to gain insights into the nuclear transport of these proteins, we solved the crystal structures of importin-α bound to peptides corresponding to the supposed NLSs of MLH1 and PMS2 and performed isothermal titration calorimetry to study their binding affinities. Both putative MLH1 and PMS2 NLSs can bind to importin-α as monopartite NLSs, which is in agreement with some previous studies. However, MLH1-NLS has the highest affinity measured by a natural NLS peptide, suggesting a major role of MLH1 protein in nuclear import compared to PMS2. Finally, the role of MLH1 and PMS2 in the nuclear transport of the MutLα heterodimer is discussed.

The crystal structure of an acidic phospholipase A2 isolated from Bothrops jararacussu venom (BthA‐I) chemically modified with p‐bromophenacyl bromide (BPB) has been determined at 1.85 Å resolution. The catalytic, platelet‐aggregation inhibition, anticoagulant and hypotensive activities of BthA‐I are abolished by ligand binding. Electron‐density maps permitted unambiguous identification of inhibitor covalently bound to His48 in the substrate‐binding cleft. The BthA‐I–BPB complex contains three structural regions that are modified after inhibitor binding: the Ca2+‐binding loop, β‐wing and C‐terminal regions. Comparison of BthA‐I–BPB with two other BPB‐inhibited PLA2 structures suggests that in the absence of Na+ ions at the Ca2+‐binding loop, this loop and other regions of the PLA2s undergo structural changes. The BthA‐I–BPB structure reveals a novel oligomeric conformation. This conformation is more energetically and conformationally stable than the native structure and the abolition of pharmacological activities by the ligand may be related to the oligomeric structural changes. A residue of the `pancreatic' loop (Lys69), which is usually attributed as providing the anticoagulant effect, is in the dimeric interface of BthA‐I–BPB, leading to a new hypothesis regarding the abolition of this activity by BPB.

Flap endonuclease 1 (FEN1) is a member of the nuclease family and is structurally conserved from bacteriophages to humans. This protein is involved in multiple DNA-processing pathways, including Okazaki fragment maturation, stalled replication-fork rescue, telomere maintenance, long-patch base-excision repair and apoptotic DNA fragmentation. FEN1 has three functional motifs that are responsible for its nuclease, PCNA-interaction and nuclear localization activities, respectively. It has been shown that the C-terminal nuclear localization sequence (NLS) facilitates nuclear localization of the enzyme during the S phase of the cell cycle and in response to DNA damage. To determine the structural basis of the recognition of FEN1 by the nuclear import receptor importin , the crystal structure of the complex of importin with a peptide corresponding to the FEN1 NLS was solved. Structural studies confirmed the binding of the FEN1 NLS as a classical bipartite NLS; however, in contrast to the previously proposed 354 KRKX 8 KKK 367 sequence, it is the 354 KRX 10 KK-AK 369 sequence that binds to importin . This result explains the incomplete inhibition of localization that was observed on mutating residues 365 KKK 367 . Acidic and polar residues in the X 10 linker region close to the basic clusters play an important role in binding to importin . These results suggest that the basic residues in the N-terminal basic cluster of bipartite NLSs may play roles that are more critical than those of the many basic residues in the C-terminal basic cluster.

Xeroderma pigmentosum type G (XPG) proteins are involved in DNA lesion recognition and promotion of nucleotide excision repair. Specific mutations in these proteins may lead to Cockayne syndrome, in which the patients may display severe developmental retardation and neurological abnormalities. No structural information is available for their spacer region or the C-terminal domain, which are important, respectively, for specific nucleotide excision repair activity and substrate specificity, as well as nuclear translocation. Immunofluorescence studies suggested two specific regions of the XPG C-terminus as potential bipartite nuclear localization sequences, which would be responsible for its translocation to the nucleus by the classical nuclear import pathway mediated by the importin-α (Impα). Thus, in order to test these hypotheses and gain insight into the structural basis for the nuclear import process for the XPG protein, we solved the crystal structures of complexes formed by the Impα and peptides corresponding to both putative nuclear localization signal (NLS) sequences (XPG1 and XPG2) and performed isothermal titration calorimetry assays to determine their binding affinities. Structural experiments confirm the binding of both NLS peptides to Impα but, unexpectedly, they bind to the receptor as monopartite NLSs. The isothermal titration calorimetry assays demonstrated that XPG1 and XPG2 peptides bind to two separate binding sites, but with high affinity to the major NLS-binding site of the Impα, resembling classical monopartite SV40 TAg NLS. The results lead to insights about what distinguishes monopartite and bipartite NLSs, as well as the differential roles of XPG1 and XPG2 NLSs in the nuclear localization of XPG.

Environmental pH induces a stress response triggering a signaling pathway whose components have been identified and characterized in several fungi. Neurospora crassa shares all six components of the Aspergillus nidulans pH signaling pathway, and we investigate here their regulation during an alkaline pH stress response. We show that the N. crassa pal mutant strains, with the exception of Δpal-9, which is the A. nidulans palI homolog, exhibit low conidiation and are unable to grow at alkaline pH. Moreover, they accumulate the pigment melanin, most likely via regulation of the tyrosinase gene by the pH signaling components. The PAC-3 transcription factor binds to the tyrosinase promoter and negatively regulates its gene expression. PAC-3 also binds to all pal gene promoters, regulating their expression at normal growth pH and/or alkaline pH, which indicates a feedback regulation of PAC-3 in the pal gene expression. In addition, PAC-3 binds to the pac-3 promoter only at alkaline pH, most likely influencing the pac-3 expression at this pH suggesting that the activation of PAC-3 in N. crassa results from proteolytic processing and gene expression regulation by the pH signaling components. In N. crassa, PAC-3 is proteolytically processed in a single cleavage step predominately at alkaline pH; however, low levels of the processed protein can be observed at normal growth pH. We also demonstrate that PAC-3 preferentially localizes in the nucleus at alkaline pH stress and that the translocation may require the N. crassa importin-α since the PAC-3 nuclear localization signal (NLS) has a strong in vitro affinity with importin-α. The data presented here show that the pH signaling pathway in N. crassa shares all the components with the A. nidulans and S. cerevisiae pathways; however, it exhibits some properties not previously described in either organism.