Intramolecular Cohesion of Coils Mediated by Phenylalanine–Glycine Motifs in the Natively Unfolded Domain of a Nucleoporin

et al. (2008) Intramolecular Cohesion of Coils Mediated by Phenylalanine-Glycine Motifs in the Natively Unfolded Domain of a Nucleoporin. PLoS Comput Biol 4(8): e1000145. doi:10.1371/journal.pcbi.1000145 Intramolecular Cohesion of Coils Mediated by Phenylalanine-Glycine Motifs in the Natively Unfolded Domain of a Nucleoporin V. V. Krishnan Edmond Y. Lau Justin Yamada Daniel P. Denning Samir S. Patel Michael E. Colvin Michael F. Rexach Philip E. Bourne, University of California San Diego, United States of America The nuclear pore complex (NPC) provides the sole aqueous conduit for macromolecular exchange between the nucleus and the cytoplasm of cells. Its diffusion conduit contains a size-selective gate formed by a family of NPC proteins that feature large, natively unfolded domains with phenylalanine-glycine repeats (FG domains). These domains of nucleoporins play key roles in establishing the NPC permeability barrier, but little is known about their dynamic structure. Here we used molecular modeling and biophysical techniques to characterize the dynamic ensemble of structures of a representative FG domain from the yeast nucleoporin Nup116. The results showed that its FG motifs function as intramolecular cohesion elements that impart order to the FG domain and compact its ensemble of structures into native premolten globular configurations. At the NPC, the FG motifs of nucleoporins may exert this cohesive effect intermolecularly as well as intramolecularly to form a malleable yet cohesive quaternary structure composed of highly flexible polypeptide chains. Dynamic shifts in the equilibrium or competition between intra- and intermolecular FG motif interactions could facilitate the rapid and reversible structural transitions at the NPC conduit needed to accommodate passing karyopherin-cargo complexes of various shapes and sizes while simultaneously maintaining a size-selective gate against protein diffusion. - Funding: This work was supported in part by NIH Grant #GM061900 and #GM077520 awarded to MR. This work was also performed in part under the auspices of the United States Department of Energy through the University of California Lawrence Livermore National Laboratory under contract number W-7405-ENG-48. This work was also supported in part by the U.S. Department of Energy, Office of Science, Offices of Advanced Scientific Computing Research, and Biological & Environmental Research through the University of California Merced Center for Computational Biology. These sponsors or funders had no role in the design and conduct of this study, or in the collection, analysis, and interpretation of the data, or in the prepartaion, review, or approval of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The nuclear pore complex is a supramolecular protein structure in the nuclear envelope that controls nucleo-cytoplasmic traffic and communication (Figure 1A) [1]. A key NPC architectural feature is a poorly understood semi-permeable diffusion barrier at its center, which allows passive diffusion of particles less than 3 4 nm in diameter (or 3040 kDa in mass for a folded protein) and opens to allow facilitated transport of larger particles up to 39 nm in diameter [2]. The NPC is composed of ,30 proteins or nucleoporins (nups) that are present in multiple copies [3,4]. Among these, a group that contains numerous phenylalanineglycine repeats (FG nups) (a subset is shown in Figure 1B) line the transport conduit of the NPC (Figure 1A). These FG nups function as stepping-stones for karyopherin movement across the NPC [5,6] and as structural elements of the NPC protein diffusion barrier [7,8]. The three dimensional structure of S. cerevisiae FG nups is unusual because their 150700 amino acid (AA) FG domains are natively unfolded [9] in their functional state [6]. Since there are ,150 FG nups in each NPC [4], it is currently hypothesized that its transport conduit is lined and/or flanked by 150 natively unfolded FG domains. Together these FG domains constitute ,12% of the total NPC mass or .6.5 MDa of its ,55 MDa structure in yeast [10]. The FG domains of nups were initially hypothesized to function as repulsive entropic bristles that create a virtual gate at the NPC periphery [11,12], and later as cohesive polypeptide chains that form a hydrogel at the NPC center [8,13,14]. More recently, an analysis of all nup FG domains in S. cerevisiae indicated that some FG domains (the GLFG-rich domains) bind to each other weakly via hydrophobic attractions between their FG motifs, whereas other FG domains (the FxFGrich domains) do not form such cohesions [7]. Despite the fact that different subtypes of FG domains are defined by their content of FxFG, GLFG or SAFGxPSFG motifs, their ability to interact with each other (i.e., their cohesiveness) seems to correlate best with the AA composition of the sequences between FG motifs, rather than with the specific FG motif [7]. Hence, the human FG nups may The nuclear pore complex is a molecular filter that gates macromolecular exchange between the cytoplasm and the nucleoplasm of cells. It contains a size-selective diffusion barrier at its center composed of proteins named FG nucleoporins. These nucleoporins feature large, structurally disordered domains that are highly decorated with phenylalanineglycine (FG) sequence motifs. The dynamic structure of these disordered FG domains excludes them from classical structural biology analyses such as X-ray crystallography; thus, new approaches are needed to characterize their shape. Here computational and biophysical approaches were used to elucidate the ensemble of structures adopted by the FG domain of a nucleoporin. The analyses showed that the FG motifs function as intramolecular cohesion elements that compact the shape of the FG domain, forcing it to adopt loosely knit globular configurations that are constantly reconfiguring. Within the nuclear pore complex, dozens of these nucleoporin FG domains may stack as loosely knit globules forming a porous sieve that gates molecular diffusion by size exclusion. also interact with each other, despite having only one GLFG-rich nup among its eleven members [3]. It is generally assumed that natively unfolded proteins have some preferred 3-D structures dictated by intra-molecular cohesion [15,16]. Current evidence that the FG domains of nups have some structure is based on CD and FTIR spectroscopic analysis, which indicates that FG domains have anywhere from 5% to 20% a-helical and b-sheet content at any given moment [9], yet the locations of such structures in the protein are probably ever-changing. The conformational flexibility inherent to natively unfolded proteins and protein domains such as those in the FG nups, places them beyond the reach of classical structural biology tools such as X-ray crystallography and homology-based computational methods [1720]. However, it is clear that these and other unfolded (...truncated)