Structure and nucleic acid binding activity of the 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 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” 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 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 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). The structure of the ∼90 kDa Nup15770–893 was served residues in 1/ 5/ 6 and 9/ 11 that create a network of solved by single-wavelength anomalous dispersion (SAD) using a hydrogen bonds, which are further reinforced by a coating of seleno-methionine derivatized protein. The final model was re- neighboring van der Waals interactions (Fig. 2). The core of the fined at 2.4 Å resolution (Rwork/Rfree = 18.9%/23.1%). The data hydrogen bond network involves the absolutely conserved Arg718 collection and refinement statistics are summarized in Table S1. in α11 and Asp105 in α1 (Fig. 2C and Fig. S1). In addition, Arg718 forms a hydrogen bond with Glu639 of α9, which in turn forms α Architectural Overview. Nup15770–893 folds into a compact C-shaped a hydrogen bond with Lys549 of 5. These hydrophilic inter- structure with overall dimensions of ∼85 Å × 65 Å × 60 Å. actions are further enforced by neighboring hydrophobic pairs: – – – – Nup15770–893 contains two distinct domains: an N-terminal Phe97 Phe709, Leu101 Ala713, Phe590 Tyr635, and Tyr594 β-propeller followed by a C-terminal α-helical domain (Fig. 1B Tyr646 (Fig. 2D). In total, ∼40 residues contribute to forming the and Movie S1). The β-propeller domain contains seven blades interface between the β-propeller and α-helical domains, burying with several helical insertions in the interstrand or interblade loops. a total surface area of ∼3,100 Å2 (Fig. 2 and Fig. S1). The downstream α-helical domain forms an irregular α-helical stack that comprises 12 α-helices (Fig. 1B). Three α-helical inser- tions in the β-propeller domain create an interface between the two domains of Nup15770–893.Theα-helical domain extends from one end of the β-propeller domain and progresses toward the opposite end in a zigzag pattern. This arrangement of the two domains results in a cavity that measures ∼35Åinwidthand40Åin height when viewed from the front of the structure (Fig. 1B).

Domain Organization. The β-propeller domain conforms to the ca- nonical β-propeller fold in which seven blades of the four β-strands are radially arranged (Fig. 1C). Formed by the innermost strands from each blade, a small funnel-like channel runs through the central axis of the propeller domain. This water-rich channel is wider (∼20 Å) at the bottom and narrower (∼10 Å) toward the top face of the propeller domain. Notably, the Nup157 β-propeller domain contains an additional β-strand in blade 6 (6E). From the N terminus, the outermost strands (6E and 7D) interact with the inner strands (6D and 7C, respectively) to complete the seven- blade propeller structure (Fig. 1C). This structural arrangement, known as a double Velcro-type closure, is thought to contribute to the stability of the β-propeller fold (7). Radially arranged propeller proteins often accommodate inser- tions in their connecting loops (7). The β-propeller in Nup157 contains an α-helical extension (α1) and five α-helical inser- tions (α2toα6): α1 is positioned nearly perpendicular to the plane of the β-propeller domain, α2toα4 are found in the interblade loops (7D1A, 4D5A, and 5D6A, respectively), whereas α5andα6 lie within the interstrand loops (6CD and 7AB, re- spectively; Fig. 1C). Collectively, α1, α5, and α6 provide a packing platform for the α-helical domain (Fig. 1B). The Nup157 α-helical domain is composed of 12 α helices (α7–α18) that form a compact, unique fold (Fig. 1B). Exiting from the 7C strand of the β-propeller, the α-helical domain begins with α7/α8andα9/α10 that form consecutive pairs of antiparallel helices of varying lengths, with each pair orientated roughly perpendicular to the other. The α9/α10 pair is rotated ∼ α α α α by 90° with respect to the 7/ 8 pair. The long 11 to 13 he- Fig. 2. Interface between the Nup15770–893 domains. (A)Space-filling model lices in the middle of the domain further extend a zigzag pattern of Nup15770–893 (Center) and dissection of the buried interface between the that propagates through α16 to α18. Loosely packed α14 and β-propeller (orange; Right)andα-helical domain (yellow; Left). The α1exten- α15 deviate from this zigzag progression, and are likely to be sion and α2toα6 insertions are color-coded as in Fig. 1. Residues involved in flexible as evidenced by a higher B-factor (∼67 Å2)compared forming the interface between the two domains are in magenta and con- with the rest of the domain (∼38 Å2). toured using a black line. (B) Surface representation of Nup15770–893 is shown To investigate whether the Nup157 α-helical domain (∼280 res- in the same view as in A. Conserved residues from multiple sequence align- ment (Fig. S1) are mapped onto the surface and shaded in a color gradient idues) is related to any known protein folds, we performed se- from light yellow to dark red (40–100% sequence conservation). A magnified quence- and structure-based analyses. An iterative PSI-BLAST view in ribbon representation of the interdomain interface highlighting search using the α-helical domain returned only Nup157 ortho- hydrophilic (C) and hydrophobic (D) interactions formed by α1/α5/α6(blue) logues, indicating that its fold is not found in any other protein in the β-propeller and α9/α11 (yellow) in the α-helical domain.

16452 | www.pnas.org/cgi/doi/10.1073/pnas.1316607110 Seo et al. Downloaded by guest on October 2, 2021 Fig. 3. Surface properties of Nup15770–893.(A) Conserved residues from multiple sequence alignment (Fig. S1) are mapped onto the surface and shaded in a color gradient from light yellow to red (40–100% sequence conservation). (B) Electrostatic potential surface of Nup15770–893 is depicted in a gradient from red (−5 kT/e) to blue (+5 kT/e) as indicated (Right). The orientation of surface representations is identical in each column. The three most sequence conserved surface patches (areas 1–3) are encircled by a black line.

Surface Properties. The surface of Nup15770–893 features three Nup15770–893 with apparently comparable affinities, we further evolutionarily conserved patches, with two regions located in the inquired about the specificity of the Nup15770–893 interaction β-propeller domain and one region in the α-helical domain (Fig. with DNA. As expected, increasing concentrations of protein 3A). Specifically, area 1 is formed by α3 and blades 2 to 4 at the yielded an increase in formation of the DNA–protein complexes; front face of the β-propeller and spans the width of the domain. however, reversing the sequence of one of the zip code elements β Area 2, at the bottom of the -propeller domain, is comprised of (GRS-IRev) yielded no observable difference in binding affinity 7D1A and 1BC loops. Area 3, in the α-helical domain, consists of compared with the GRS-I sequence (Fig. 4C). Furthermore, the the last two helices of the α-helical domain (α17 and α18) and RNA equivalent of the GRS-I element also interacted with a connecting loop. Although this patch is surface exposed in Nup15770–893 (Fig. 4D). The binding of Nup15770–893 to dsDNA and Nup15770–893, it is likely to be buried by the remaining C-terminal RNA is indistinguishable in our assay. Taken together, we con- region of Nup157. clude that Nup15770–893 directly binds to nucleic acids in vitro in a We further analyzed the surface properties of Nup15770–893 sequence-independent manner. and identified a striking asymmetry in its charge distribution (Fig. 3B). The front side of the molecule is positively charged, whereas Discussion the backside is mostly negatively charged. Notably, a vast posi- NPCs and “nuclear envelope junctions” have previously been tively charged area encompasses the front and bottom surface of proposed to serve as orientation landmarks for the 3D organi- β the -propeller and largely overlaps with an evolutionarily con- zation of chromatids (9, 10). These reference points of the nu- served patch denoted as area 1 (Fig. 3 and Fig. S1). clear envelope might mediate direct or indirect binding to distinct portions of chromatid DNA. In yeast, Nup157 and Nup170 have Nup157 – Binds to Nucleic Acids. In addition to their role in the 70 893 been implicated in chromatin organization and gene expression symmetric core of the NPC, Nup157 and Nup170 have also been (“gene gating”) (5, 6, 11–13). Here, we show that the crystal implicated in distinct chromatid binding functions (5, 6). Specifi- NUP157 structure of recombinant ScNup157 (residues 70–893) consists cally, deletion of the gene leads to failure in the re- β α location of the inducible inositol-1-phosphate synthase (INO1) of a -propeller and a downstream -helical domain joined to- gene from the nuclear interior to the NPC. Distinct DNA gether in a compact C-shaped overall architecture. Notably, the elements of about 20 bp in length have been identified to serve surface of this protomer displays a highly asymmetric distribution BIOPHYSICS AND as “zip codes” to relocate the INO1 portion of the chromatid of charged residues. Consistent with a large patch of positively COMPUTATIONAL BIOLOGY (6). Given these observations and our finding of a large, charged surface residues, we observed that Nup157 directly binds to dsDNA or RNA without sequence specificity. A similar pos- positively charged surface patch on Nup15770–893,wetested whether Nup157 is directly involved in DNA binding. First, we itively charged surface has been previously observed in the β incubated a DNA library containing random sequences of 30 bp -propeller of Rae1, which is an RNA-binding nup involved in mRNA export (14). Moreover, the ability to interact with both in length with Nup15770–893 or Nup157894–1391 (the C-terminal portion of Nup157; Table S2). Subsequent analyses by EMSA types of nucleic acids was previously observed for a number of showed that DNA binding was detected only for Nup15770–893, proteins, including p53 tumor suppressor protein (reviewed in indicating that the crystallized portion of Nup157 is indispens- ref. 15). Considering the essential role of Nup157 in INO1 gene able for DNA binding (Fig. 4A). recruitment to the NPC and its sequence-independent binding to Next, we tested for binding of single- vs. double-stranded nucleic acids, we envision that Nup157 could serve as an anchor DNA (dsDNA) by using the recently identified zip code elements point for genes that are targeted to the nuclear periphery. Al- of the INO1 gene termed gene recruitment sequences (GRSs)— though the biological significance of the RNA-binding activity of GRS-I and GRS-II—and a memory recruitment sequence (MRS). Nup157 remains unknown, our finding is supported by the iden- fi We found Nup15770–893 binds to all three dsDNAs, but not to any ti cation of Nup155, a human orthologue of Nup157, as a member of the respective ssDNAs (Fig. 4B). As these zip code elements bind of the mRNA interactome (13).

Seo et al. PNAS | October 8, 2013 | vol. 110 | no. 41 | 16453 Downloaded by guest on October 2, 2021 Fig. 4. Nup157 directly interacts with nucleic acids in vitro. (A) EMSA was used to probe the interaction of Nup15770–893 and Nup157894–1391 with DNA. Protein samples were mixed with a DNA library (refer to Table S2) at a 4:1 molar ratio and analyzed by native PAGE. Free and bound DNAs were visualized by SYBR Gold staining as indicated by black arrows. (B) Three INO1 zip codes (GRS-I and –II and MRS) were tested for Nup157 binding. Eightfold molar excess

of Nup15770–893 over ssDNA (sense or antisense 21-mer DNA) or dsDNA was used for EMSA. Background staining of Nup15770–893 is marked by an asterisk. (C) Titration of Nup15770–893 to GRS-I and the reversed GRS-I (GRS-IRev). Both GRS elements were preincubated with increasing amounts of Nup15770–893 at 0-, 0.3-, 1-, 2-, 5-, 10-, and 20-fold molar excess and analyzed by EMSA. (D) Titration of Nup15770–893 to the RNA equivalent of the GRS-I element as in C.

Our discovery that Nup15770–893 binds to nucleic acids raises complexes remain to be established, such complexes might as- the question of how Nup157, located within the symmetric core semble near the nuclear side of NPCs (5, 6) or at nuclear en- of the NPC, could access DNA. We propose that Nup157 and velope junctions (10, 21). Alternatively, a pool of Nup157 could Nup170 have dual localizations and functions, as is already associate with chromatin and regulate gene expression away known for several other nups. For example, the β-propeller nups from the nuclear periphery, as was previously observed for Sec13 Sec13 and Seh1, other than being members of the Y-shaped and Nup98 (22, 23). complex of the symmetric core of the NPC, also interact with Modeling the C-terminal remainder of Nup157 (residues 894– a rather different set of proteins to assemble coat complexes for 1391) by using a previously published crystal structure of the vesicular traffic(16–20). Although alternative localizations of corresponding C-terminal protomer of the Nup170 paralogue Nup157 or Nup170 and their partitioning into distinct protein (24) yielded a highly asymmetric molecule of approximately

Fig. 5. A structural model of Nup157. (A) A schematic representation of the Nup157 domain architecture is shown as in Fig 1A. The dotted line above the α-helical region corresponds to the modeled C-terminal remainder of Nup157 (residues 894–1391). (B) Structural comparison between Nup170 (residues 979–1502; Protein Data Bank ID code 3I5P) in cyan and Nup157 (homology model) in gray. The structures align with 1.2 Å Cα rmsd. (C) A composite model of Nup157 (residues 70– 1391) is colored as in A. The N-terminal, 69-residue fragment of Nup157 is predicted to be disordered and was therefore omitted from the 3D structure prediction. Four hinge residues (hinge 1-T108, hinge 2-R615, hinge 3-R1053, hinge 4-L1241) predicted by elastic network models are indicated by red arrows.

16454 | www.pnas.org/cgi/doi/10.1073/pnas.1316607110 Seo et al. Downloaded by guest on October 2, 2021 200 Å in length. Taken together, Nup157 consists of an un- The crystal structure of Nup15770–893 presented here com- structured, N-terminal portion of ∼70 residues, followed by pletes a list of protomer structures determined for representative the compact C-shaped segment (the structure of which is reported adaptor nups (24, 26, 27, 31, 32). Future work will have to ad- here), and a long downstream superhelical stack extending from dress the challenge of determining atomic structures of binary the C-shaped compact region of the molecule (Fig. 5 and Fig. S2). and higher order interactomes between protomers of adaptor The predicted hinge residues in Nup157 indicate areas of in- nups and their neighboring nups. Exemplary analyses of higher- trinsic flexibility in this highly asymmetric molecule (Fig. 5C). order interactomes of nups located in various regions of the NPC According to elastic network models (25), rigid segments of Nup157 can bend around four hinge regions to adopt alternative have already provided remarkable insights into the structure and conformations (Movies S2 and S3). function of parts of the NPC, such as the proposed ring cycle As demonstrated here for Nup157, crystal structures of pro- among the channel Nup54 and Nup58 protomers for dilating and tomers derived from two other large adaptor nups, Nup188 and constricting the central channel (4), a model for a dynamic struc- its paralogue Nup192, and modeling the remaining parts of the ture of the coat cylinder nups (30, 33), and a structure of a node proteins, yielded similar highly asymmetric molecules consisting that joins three distinct asymmetric nups on the cytoplasmic side of a compact structure near the N terminus followed by a long of the NPC (34). superhelical stalk (26–28). A common trait of all four large adaptor nups (Nup157, Nup170, Nup188, and Nup192) appears Materials and Methods to be their plasticity, consistent with their conceived role as The details of molecular cloning, expression, purification, crystallization, adaptors for the large shape and diameter changes of the central X-ray diffraction data collection, structure determination, in vitro protein– transport channel. The dynamic nature of nups has also been DNA interaction analysis, and homology modeling are described in SI observed in the outer “coat” cylinder of the NPC. A 3D EM Materials and Methods.Briefly, recombinant proteins were expressed in reconstruction of the seven-membered Y complex at ∼35 Å E. coli with a cleavable N-terminal GST tag. The proteins were purified fi fi resolution identi ed two speci c hinge regions that permit the by affinity and size exclusion chromatography. The Nup15770–893 struc- heptamer to adapt alternative conformations (29). In addition, ture was solved by SAD. the unstructured region of the Y complex mediates the interaction between the adjacent heptamers in a head-to-tail arrangement ACKNOWLEDGMENTS. We thank members of the G.B. laboratory, especially (30). We envision that this flexible tether could also allow for Richard Wing, Junseock Koh, and Elias Coutavas, for discussions and com- ments on the manuscript; David King (University of California, Berkeley) for changes in the diameter of the Y complex oligomer to accommo- MS analysis; Wuxian Shi (National Synchrotron Light Source) for support date large structural rearrangements during the ring cycle of the during X-ray data collection; and Yingli Ma (GlaxoSmithKline) for her central channel. contributions at the beginning of this project.

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