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Structure and Nucleic Acid Binding Activity of the Nucleoporin Nup157

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Structure and Nucleic Acid Binding Activity of the Nucleoporin Nup157 Structure and nucleic acid binding activity of the nucleoporin Nup157 Hyuk-Soo Seo1,2, Bartlomiej J. Blus1, Nina Z. Jankovic, and Günter Blobel2 Laboratory of Cell Biology and Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065 Contributed by Günter Blobel, September 2, 2013 (sent for review August 6, 2013) At the center of the nuclear pore complex (NPC) is a uniquely versatile three constricted rings was proposed, resulting in ∼30 nm changes central transport channel. Structural analyses of distinct segments in the diameter of the central transport channel. (“protomers”) of the three “channel” nucleoporins yielded a model Other than structurally and functionally distinguishing between for how this channel is constructed. Its principal feature is a midplane dilated and constricted forms of the nuclear pore, this model has ring that can undergo regulated diameter changes of as much as an implications for the function of a large network of nups adjacent to estimated 30 nm. To better understand how a family of “adaptor” the central channel that constitute the symmetric core of the NPC. nucleoporins—concentrically surrounding this channel—might cush- Only the channel nups “line” the channel wall, whereas the ion these huge structural changes, we determined the crystal struc- concentrically surrounding nups, linked to each other in a Rube ture of one adaptor nucleoporin, Nup157. Here, we show that a Goldberg-like network, are envisioned to accommodate and or- recombinant Saccharomyces cerevisiae Nup157 protomer, repre- chestrate the vast shape and diameter changes of the central channel. – senting two-thirds of Nup157 (residues 70 893), folds into a This involves not only conformational plasticity of individual nups, β α seven-bladed -propeller followed by an -helical domain, which but likely requires reversible disruptions of interacting sites between together form a C-shaped architecture. Notably, the structure con- nups. Complementarity between interacting sites of distinct nups may tains a large patch of positively charged residues, most of which are establish alternative interactomes (reviewed in ref. 1). evolutionarily conserved. Consistent with this surface feature, we The Saccharomyces cerevisiae (Sc) Nup157 and its paralogue found that Nup15770–893 binds to nucleic acids, although in a se- Nup170 are among the family of “adaptor” nups, which concen- quence-independent manner. Nevertheless, this interaction sup- trically surround the central transport channel (reviewed in ref. 1). ports a previously reported role of Nup157, and its paralogue Here, we describe the crystal structure of a protomer of Nup157 Nup170, in chromatin organization. Based on its nucleic acid bind- – ing capacity, we propose a dual location and function of Nup157. (residues 70 893) that represents approximately two-thirds of the Finally, modeling the remaining C-terminal portion of Nup157 molecule and will be referred to as Nup15770–893 in the remainder of the text. We show that Nup15770–893 consists of a seven-bladed shows that it projects as a superhelical stack from the compact β C-shaped portion of the molecule. The predicted four hinge re- -propeller and a downstream helical domain with a unique fold. gions indicate an intrinsic flexibility of Nup157, which could con- Multiple van der Waals and electrostatic interactions between tribute to structural plasticity within the NPC. these two domains form a compact C-shaped architecture that features large patches of predominantly positively or negatively gene gating | X-ray crystallography | DNA-binding protein | charged residues. Consistent with a large positively charged surface RNA-binding protein patch and reports that Nup157/Nup170 are involved in chromatid fi ultiple copies of only ∼30 distinct proteins, collectively Signi cance Mtermed nucleoporins (nups), form a large nuclear pore complex (NPC) that, in vertebrates, amounts to an estimated The nuclear pore complex (NPC) is a multiprotein gating com- mass of more than 100 MDa. Purification of sufficient quantities plex that allows for bidirectional transport across the nuclear of intact and monodisperse NPCs that would be suitable for membrane. A key feature of the NPC is a central transport crystallographic analyses is presently not feasible. In an alter- channel that can undergo regulated diameter changes, thus native approach, a recombinant full-length nup, or a nup frag- enabling the trafficking of cargo of various sizes. Surrounding ment (“protomer”), or complexes thereof, are crystallized and this channel is a group of proteins, named “adaptor” nucleo- their atomic structures are modeled into higher-order assemblies porins, which are envisioned to accommodate and orchestrate that represent distinct regions of the NPC (reviewed in ref. 1). these structural changes. Here we show the crystal structure of Arguably, the most notable insights stemming from the afore- a fragment of an adaptor nucleoporin, Nup157, which forms mentioned strategy were obtained from the crystal structures of a compact C-shaped architecture. Notably, Nup157 contains a protomers representing several structured regions of the three positively charged surface consistent with its nucleic acid “channel” nups, Nup58, Nup54, and Nup62 (2-4). The outcome binding capacity. Furthermore, the predicted hinge regions in of these crystallographic analyses was a model of the atomic structure Nup157 suggest its flexibility in agreement with the plastic of the central transport channel, the heart of the NPC. The crucial nature of the NPC. features of this model are midplane rings that undergo dramatic Author contributions: H.-S.S., B.J.B., and G.B. designed research; H.-S.S., B.J.B., and N.Z.J. structural rearrangements from the dilated to constricted state of performed research; H.-S.S., B.J.B., and G.B. analyzed data; and H.-S.S., B.J.B., N.Z.J., and the nuclear pore. In the dilated state, helical segments of four G.B. wrote the paper. Nup58 and eight Nup54 protomers form a dodecameric module. The authors declare no conflict of interest. The arrangement of eight such dodecamers results in a single, Freely available online through the PNAS open access option. heterooligomeric midplane ring with a flexible diameter of Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, 40 to 50 nm. Such a ring can then be rearranged into three www.pdb.org (PDB ID code 4MHC). smaller homooligomeric rings that collectively represent the 1H.-S.S. and B.J.B. contributed equally to this work. constricted form of the nuclear pore with a diameter of 10 to 20 2To whom correspondence may be addressed. E-mail: [email protected] or blobel@ nm. One ring consists of eight modules of Nup58 tetramers and rockefeller.edu. the other two are each comprised of eight modules of Nup54 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tetramers (4). A “ring cycle” between a single dilated ring and 1073/pnas.1316607110/-/DCSupplemental. 16450–16455 | PNAS | October 8, 2013 | vol. 110 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1316607110 Downloaded by guest on October 2, 2021 organization (5, 6), we found that Nup15770–893 preferentially suggest an intrinsic flexibility of Nup157, supporting a perceived interacts with double-stranded nucleic acids without sequence role for adaptor nups in cushioning the shape and diameter changes specificity. We also modeled the C-terminal remainder of Nup157 of the central channel. andcombineditwiththeNup15770–893 structure. As a result, we obtained a highly asymmetric model of Nup157 with a long su- Results perhelical stack extending from the C-shaped compact portion of Structure Determination. Saccharomyces cerevisiae Nup157 is pre- the molecule. In this model, there are four hinge residues that dicted to contain two distinct domains: an N-terminal, ∼610-residue BIOPHYSICS AND COMPUTATIONAL BIOLOGY Fig. 1. Structure of the S. cerevisiae Nup15770–893.(A) Schematic representation of the Nup157 domain architecture. The boundaries for each domain are color-coded and marked with residue numbers. Starting from the N terminus: the unstructured fragment (gray) denoted by “U”, β-propeller domain (orange), α-helical domain (yellow), and a predicted α-helical region (gray) are indicated. Positions of the N-terminal α-helical extension (α1) and five α-helical insertions (α2–α6) in the propeller domain are indicated: α1/α5/α6, in blue, create an interface between the two domains in Nup157, whereas α2toα4, in green, are found on the side of the propeller. The bar above the domain architecture corresponds to the crystallized fragment (residues 70–893). (B) Structure of Nup15770–893 in ribbon representation, colored as in A.(Right) A 90°-rotated view. (C) Ribbon representation of the Nup157 β-propeller domain. Seven blades of the β-propeller core (orange), the α-helical insertions (blue and green) are indicated. (Right) Schematic representation of the Nup15770–893 β-propeller domain and locations of its α-helical elements. Dotted gray lines indicate disordered regions. The double Velcro-type closure is highlighted by asterisks. Seo et al. PNAS | October 8, 2013 | vol. 110 | no. 41 | 16451 Downloaded by guest on October 2, 2021 β-strand–rich region followed by a C-terminal, ∼780-residue families. Moreover, a search of the PDBeFold database (www. α-helical fragment (Fig. 1A). Based on this prediction, we de- ebi.ac.uk/msd-srv/ssm/) (8) did not yield any matches with mean- signed a series of Nup157 constructs for recombinant expression ingful structural similarity to the α-helical domain, further sup- in Escherichia coli, and identified a stable fragment encompassing porting that the Nup157 α-helical domain structure represents a residues 70 to 893. Nup15770–893 crystals grew in the primitive unique fold. monoclinic space group P21, with one molecule in the asymmetric unit. To improve the signal from anomalous scattering, two non- Interface Between the Two Domains of Nup157. The interface be- conserved serine residues were mutated to methionines (S217M tween the β-propeller and the α-helical domain consists of con- α α α α α and S638M).
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