Nuclear import of proteins containing a classical nuclear localization signal (NLS) is an energy-dependent process that requires the heterodimer importin ␣/␤.Three to six basic contiguous arginine/lysine residues characterize a classical NLS and are thought to form a basic patch on the surface of the import cargo. In this study, we have characterized the NLS of phospholipid scramblase 1 (PLSCR1), a lipid-binding protein that enters the nucleus via the nonclassical NLS 257 GKISKH-WTGI 266 . This import sequence lacks a contiguous stretch of positively charged residues, and it is enriched in hydrophobic residues. We have determined the 2.2 Å crystal structure of a complex between the PLSCR1 NLS and the armadillo repeat core of vertebrate importin ␣. Our crystallographic analysis reveals that PLSCR1 NLS binds to armadillo repeats 1-4 of importin ␣, but its interaction partially overlaps the classical NLS binding site. Two PLSCR1 lysines occupy the canonical positions indicated as P2 and P5. Moreover, we present in vivo evidence that the critical lysine at position P2, which is essential in other known NLS sequences, is dispensable in PLSCR1 NLS. Taken together, these data provide insight into a novel nuclear localization signal that presents a distinct motif for binding to importin ␣.Nuclear transport is an active signal-mediated process that requires, in most cases, soluble transport factors and specific import signals. Two families of transport receptors have been identified in the importin ␤ superfamily, which is involved in both nuclear import and export, and the TAP superfamily that mediates nuclear export. Transport receptors recognize specific nuclear localization signals (NLSs) 1 and nuclear export signals exposed on the molecular surface of cargoes. In the classical nuclear import pathway proteins bearing a classical SV40-like NLS (PKKKRKV) are recognized by the importin ␣/␤ heterodimer (also known as karyopherin ␣/␤) (1-3). Importin ␣ (4) acts as an adaptor that recognizes NLS sequences after association with the receptor importin ␤ (5). The importin ␣/␤-NLS cargo complex is then translocated through the nuclear pore complex in a process that requires multiple rounds of interaction of the receptor importin ␤ with nucleoporins, likely via their exposed hydrophobic FG-rich motifs (6, 7

The nuclear import of uridine-rich ribonucleoproteins is mediated by the transport adaptor snurportin 1 (SNP1). Similar to importin ␣, SNP1 uses an N-terminal importin ␤ binding (sIBB) domain to recruit the receptor importin ␤ and gain access to the nucleus. In this study, we demonstrate that the sIBB domain has a bipartite nature, which contains two distinct binding determinants for importin ␤. The first determinant spans residues 25-65 and includes the previously identified importin ␣ IBB (␣IBB) region of homology. The second binding determinant encompasses residues 1-24 and resembles region 1011-1035 of the nucleoporin 153 (Nup153). The two binding determinants synergize within the sIBB domain to confer a low nanomolar binding affinity for importin ␤ (K d ϳ 2 nM) in an interaction that, in vitro, is displaced by RanGTP. We propose that in vivo the synergy of Nup153 and nuclear RanGTP promotes translocation of uridine-rich ribonucleoproteins into the nucleus.Nuclear import of proteins and nucleic acids is an active signal-mediated process that requires, in most cases, soluble transport factors and GTP hydrolysis by the GTPase Ran (1-4). Nuclear transport factors are sorted into two categories: importins and exportins (also known as karyopherins), which include 14 members in budding yeast and at least 20 in humans (5, 6). All karyopherins fold into superhelical solenoids, which present an external convex surface involved in nucleoporin binding and a concave internal face that interacts with transport cargos and RanGTP. The two best characterized importins, human importin ␤1 and yeast karyopherin ␤2, have been visualized using crystallographic methods bound to specific import cargos (7-10) and RanGTP (11,12). These structures have provided invaluable information on the structural flexibility of karyopherins and their ability to undergo conformational changes during the import process (13-16).The best understood nuclear import pathway involves cargos bearing a classical SV40-like nuclear localization sequence (PKKKRKV) (17). These cargos are imported into the nucleus by the adaptor importin ␣ and the receptor importin ␤ with the aid of RanGTP. GTP hydrolysis by Ran energizes the import reaction by promoting both release of the import complex from nucleoporins as well as unloading of import cargos into the nucleus (1-3). In addition to classical nuclear localization sequence cargos, uridine-rich ribonucleoproteins (U snRNPs) 2 represent an important class of import cargos. Mature U snRNP particles are assembled in the cytoplasm and imported into the nucleus in at least two distinct pathways, both dependent on importin ␤ (18). In the first pathway, the import signal is in the proteinaceous core of the U snRNP formed by the Sm proteins (18 -21). In the second pathway, the trimethylated guanosine cap of the mature U snRNP is recognized by the adaptor snurportin 1 (SNP1) (18 -21), which, in turn, recruits importin ␤. SNP1 consists of an N-terminal IBB domain (sIBB) similar to that found in importin ␣ (␣IBB) and a larg...

Importin ␤ mediates active passage of cellular substrates through the nuclear pore complex (NPC). Adaptors such as importin ␣ and snurportin associate with importin ␤ via an importin ␤ binding (IBB) domain. The intrinsic structural flexibility of importin ␤ allows its concerted interactions with IBB domains, phenylalanine-glycine nucleoporins, and the GTPase Ran during transport. In this paper, we provide evidence that the nature of the IBB domain modulates the affinity of the import complex for the NPC. In permeabilized cells, importin ␤ imports a cargo fused to the snurportin IBB (sIBB) with ϳ70% reduced energy requirement as compared with the classical importin ␣ IBB. At the molecular level, this is explained by ϳ200-fold reduced affinity of importin ␤ for Nup62, when bound to the sIBB. Consistently, in vivo, the importin ␤⅐sIBB complex has greatly reduced persistence inside the central channel of the NPC. We propose that by controlling the degree of strain in the tertiary structure of importin ␤, the IBB domain modulates the affinity of the import complex for nucleoporins, thus dictating its persistence inside the NPC.

Circular permutation of a protein covalently links its original termini and creates new ends at another location. To maintain the stability of the permuted structure, the termini are typically bridged by a peptide long enough to span the original distance between them. Here, we take the opposite approach and employ a very short linker to introduce conformational strain into a protein by forcing its termini together. We join the N- and C-termini of the small ribonuclease barnase (normally 27.2 Å distant) with a single Cys residue and introduce new termini at a surface loop, to create pBn. Compared to a similar variant permuted with an 18-residue linker, permutation with a single amino acid dramatically destabilizes barnase. Surprisingly, pBn is folded at 10 °C and possesses near wild-type ribonuclease activity. The 2.25 Å X-ray crystal structure of pBn reveals how the barnase fold is able to adapt to permutation, partially defuse conformational strain, and preserve enzymatic function. We demonstrate that strain in pBn can be relieved by cleaving the linker with a chemical reagent. Catalytic activity of both uncleaved (strained) pBn and cleaved (relaxed) pBn is proportional to their thermodynamic stabilities, i.e., the fraction of folded molecules. The stability and activity of cleaved pBn are dependent on protein concentration. At concentrations above ∼2 μM, cleaving pBn is predicted to increase the fraction of folded molecules and thus enhance ribonuclease activity at 37 °C. This study suggests that introducing conformational strain by permutation, and releasing strain by cleavage, is a potential mechanism for engineering an artificial zymogen.

No abstract

No abstract

No abstract

No abstract