Importin-␣ is the nuclear import receptor that recognizes cargo proteins carrying conventional basic monopartite and bipartite nuclear localization sequences (NLSs) and facilitates their transport into the nucleus. Bipartite NLSs contain two clusters of basic residues, connected by linkers of variable lengths. To determine the structural basis of the recognition of diverse bipartite NLSs by mammalian importin-␣, we co-crystallized a non-autoinhibited mouse receptor protein with peptides corresponding to the NLSs from human retinoblastoma protein and Xenopus laevis phosphoprotein N1N2, containing diverse sequences and lengths of the linker. We show that the basic clusters interact analogously in both NLSs, but the linker sequences adopt different conformations, whereas both make specific contacts with the receptor. The available data allow us to draw general conclusions about the specificity of NLS binding by importin-␣ and facilitate an improved definition of the consensus sequence of a conventional basic/bipartite NLS (KRX 10 -12 KRRK) that can be used to identify novel nuclear proteins.Nucleocytoplasmic transport occurs through nuclear pore complexes, large proteinaceous structures that penetrate the double lipid layer of the nuclear envelope. Most macromolecules require an active, signal-mediated transport process that enables the passage of particles up to 25 nm in diameter (ϳ25 MDa). The best characterized nuclear targeting signals are the conventional nuclear localization sequences (NLSs) 1 that contain one or more clusters of basic amino acids (1). The NLSs fall into two distinct classes termed monopartite NLSs, containing a single cluster of basic amino acids, and bipartite NLSs, comprising two basic clusters separated by a spacer. Despite the variability, the conventional basic NLSs are recognized by the same receptor protein termed importin or karyopherin, a heterodimer of ␣ and ␤ subunits (for recent reviews, see Refs. 2-4). Importin-␣ (Imp␣) contains the NLSbinding site, and importin-␤ (Imp␤) is responsible for the translocation of the importin-substrate complex through the nuclear pore complex. Once inside the nucleus, Ran-GTP binds to Imp␤ and causes the dissociation of the import complex. Imp␣ becomes autoinhibited, and both importin subunits return to the cytoplasm separately without the import cargo. The directionality of nuclear import is conferred by an asymmetric distribution of the GTP-and GDP-bound forms of Ran between the cytoplasm and the nucleus. This distribution is in turn controlled by various Ran-binding regulatory proteins.Imp␣ consists of two structural and functional domains, a short basic N-terminal Imp␤-binding domain (5-7) and a large NLS-binding domain built of armadillo (Arm) repeats (8). The structural basis of monopartite and bipartite NLS recognition by Imp␣ has been studied crystallographically in yeast and mouse Imp␣ proteins (9 -11). The two basic clusters of the bipartite NLSs bind to two separate binding sites on Imp␣, involving Arm repeats 1-4 and 4 -8, respectivel...

Classification of rare missense variants as neutral or disease causing is a challenge and has important implications for genetic counseling. A multifactorial likelihood model for classification of unclassified variants in BRCA1 and BRCA2 has previously been developed, which uses data on cooccurrence of the unclassified variant with pathogenic mutations in the same gene, cosegregation of the unclassified variant with affected status, and Grantham analysis of the fit between the missense substitution and the evolutionary range of variation observed at its position in the protein. We have further developed this model to take into account relevant features of BRCA1-and BRCA2-associated tumors, such as the characteristic histopathology and immunochemical profiles associated with pathogenic mutations in BRCA1, and the fact that f80% of tumors from BRCA1 and BRCA2 carriers undergo inactivation of the wild-type allele by loss of heterozygosity. We examined 10 BRCA1 and 15 BRCA2 unclassified variants identified in Australian, multiple-case breast cancer families. By a combination of genetic, in silico, and histopathologic analyses, we were able to classify one BRCA1 variant as pathogenic and six BRCA1 and seven BRCA2 variants as neutral. Five of these neutral variants were also found in at least 1 of 180 healthy controls, suggesting that screening a large number of appropriate controls might be a useful adjunct to other methods for evaluation of unclassified

The large number of protein kinases makes it impractical to determine their specificities and substrates experimentally. Using the available crystal structures, molecular modeling, and sequence analyses of kinases and substrates, we developed a set of rules governing the binding of a heptapeptide substrate motif (surrounding the phosphorylation site) to the kinase and implemented these rules in a web-interfaced program for automated prediction of optimal substrate peptides, taking only the amino acid sequence of a protein kinase as input. We show the utility of the method by analyzing yeast cell cycle control and DNA damage checkpoint pathways. Our method is the only available predictive method generally applicable for identifying possible substrate proteins for protein serine͞threonine kinases and helps in silico construction of signaling pathways. The accuracy of prediction is comparable to the accuracy of data from systematic large-scale experimental approaches.

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