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The Tripartite Motif Coiled-Coil Is an Elongated Antiparallel Hairpin Dimer

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The Tripartite Motif Coiled-Coil Is an Elongated Antiparallel Hairpin Dimer The tripartite motif coiled-coil is an elongated antiparallel hairpin dimer Jacint G. Sancheza,1, Katarzyna Okreglickaa,1, Viswanathan Chandrasekaranb, Jordan M. Welkera, Wesley I. Sundquistb, and Owen Pornillosa,2 aDepartment of Molecular Physiology and Biological Physics and The Myles H. Thaler Center for AIDS and Human Retrovirus Research, University of Virginia, Charlottesville, VA 22908; and bDepartment of Biochemistry, University of Utah, Salt Lake City, UT 84112 Edited by Alfonso Mondragon, Northwestern University, Evanston, IL, and accepted by the Editorial Board January 8, 2014 (received for review October 8, 2013) Tripartite motif (TRIM) proteins make up a large family of coiled- to be defined. Here, we report biochemical and crystallographic coil-containing RING E3 ligases that function in many cellular characterization of a coiled-coil-containing fragment of TRIM25. processes, particularly innate antiviral response pathways. Both The crystallized construct forms a stable dimer in solution, and the dimerization and higher-order assembly are important elements of structure reveals an elongated dimer composed of interdigitating TRIM protein function, but the atomic details of TRIM tertiary and hairpin-shaped subunits. We also present evidence that this dimer quaternary structure have not been fully understood. Here, we architecture is conserved across other TRIM family members, present crystallographic and biochemical analyses of the TRIM including TRIM5α. Finally, our studies allow us to assign the do- coiled-coil and show that TRIM proteins dimerize by forming main organization in the low-resolution EM reconstruction of the interdigitating antiparallel helical hairpins that position the N- TRIM5α lattice (18) and thereby gain new insights into the terminal catalytic RING domains at opposite ends of the dimer and mechanism of retroviral capsid pattern recognition. the C-terminal substrate-binding domains at the center. The dimer core comprises an antiparallel coiled-coil with a distinctive, sym- Results metric pattern of flanking heptad and central hendecad repeats TRIM25189–379 Forms an Elongated, Antiparallel Dimer. We chose that appear to be conserved across the entire TRIM family. Our human TRIM25 for analysis because it lacks the propensity of studies reveal how the coiled-coil organizes TRIM25 to polyubi- TRIM5α to form high-order assemblies and is therefore more quitylate the RIG-I/viral RNA recognition complex and how dimers tractable biochemically. In initial experiments, we found that of the TRIM5α protein are arranged within hexagonal arrays that a series of coiled-coil-containing TRIM25 constructs, including recognize the HIV-1 capsid lattice and restrict retroviral replication. the full tripartite motif and the full-length protein, behaved as single species during purification. All of these constructs eluted antiparallel dimer | disulfide crosslinking | X-ray crystallography rapidly on gel filtration chromatographs, indicating they had elongated shapes and/or were oligomers. The shortest well- ripartite motif (TRIM) proteins make up the largest super- behaved construct spanned residues 189–379, which includes the Tfamily of RING E3 ubiquitin ligases, with more than 100 entire coiled-coil region as well as the N-terminal half of the L2 members in the human proteome (1, 2). TRIM proteins function linker region that connects the coiled-coil to the B30.2/SPRY in a variety of cellular pathways, and many regulate innate im- domain (Fig. 1A). Analytical ultracentrifugation experiments re- munity and/or mediate antiviral responses. Antiviral TRIMs in- vealed that this construct is a stable dimer (Fig. 1C; Fig. S1 A–C), clude TRIM25, which regulates the IFN response to RNA viruses implying that the full-length protein is also probably dimeric. (3, 4), and TRIM5α, which senses and inhibits early stages of re- troviral replication (5, 6). Significance TRIM proteins share a common N-terminal domain organiza- tion, termed the tripartite or RBCC (RING, B-box, coiled-coil) Tripartite motif or TRIM proteins make up the largest super- motif, followed by variable C-terminal protein recognition domains family of RING-domain E3 ubiquitin ligases. These enzymes (Fig. 1 A and B). “Linker” segments of unknown structure typically function in a wide variety of important cellular processes, separate both the RING and B-box domains (L1) and the coiled- particularly innate antiviral response mechanisms. Dimerization coil and terminal effector domains (L2). The coiled-coil region is critical for the function of many TRIM proteins. Here we show mediates oligomerization, and both homooligomeric and hetero- how TRIM25 dimerizes and demonstrate that this dimerization oligomeric TRIMs have been described (7–13). Furthermore, many mode is apparently conserved across the entire TRIM protein TRIM proteins form higher-order assemblies in vitro and form family. Our results reveal how the dimerization domain positions punctate or fibrous structures in cells (14–16). For example, TRIM5α the other TRIM effector domains to recognize and ubiquitylate assembly allows the protein to function as a cytosolic pattern- substrates and how the TRIM5 family can form higher-order recognition receptor that can intercept the incoming capsids of hexagonal assemblies that increase the avidity of substrate diverse retroviruses, including HIV-1 (6). This results in recognition. species-specific “restriction” of viral replication (5), capsid dis- sociation (5, 6), and induction of innate immune responses (17). Author contributions: W.I.S. and O.P. designed research; J.G.S., K.O., V.C., J.M.W., and O.P. performed research; J.G.S., K.O., V.C., J.M.W., W.I.S., and O.P. analyzed data; and J.G.S., Retroviral capsids are recognized through a remarkable mecha- K.O., V.C., W.I.S., and O.P. wrote the paper. α nism of multivalent pattern recognition. TRIM5 forms a homo- The authors declare no conflict of interest. dimer (10, 11, 18), which can further assemble into a 2D lattice of This article is a PNAS Direct Submission. A.M. is a guest editor invited by the linked hexagons (18). The hexagonal TRIM5α net matches the Editorial Board. symmetry and spacing of the retroviral capsid surface lattice, Data deposition: The atomic coordinates and structure factors have been deposited in the thereby positioning multiple C-terminal B30.2/SPRY domains to Protein Data Bank, www.pdb.org (PDB ID code 4LTB). interact with their repeating binding epitopes on the capsid. 1J.G.S. and K.O. contributed equally to this work. Structures of isolated RING, B-box, and C-terminal domains of 2To whom correspondence should be addressed. E-mail: [email protected]. several TRIM proteins have been described, but the molecular This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. details of TRIM oligomerization and high-order assembly have yet 1073/pnas.1318962111/-/DCSupplemental. 2494–2499 | PNAS | February 18, 2014 | vol. 111 | no. 7 www.pnas.org/cgi/doi/10.1073/pnas.1318962111 Downloaded by guest on September 29, 2021 length (Fig. 2). Each subunit in the symmetrical dimer folds back into a hairpin configuration with long and short arms. The ele- ments annotated as the coiled-coil and L2 linker are structurally distinct, with the coiled-coil residues forming the long arm of each subunit (helix H1, colored green in Fig. 2A) and the L2 residues forming the short arm that folds back and packs against H1 (helices H2, H3, and an irregular but well-ordered inter- vening segment, colored gray). The two subunits dimerize inti- mately in an antiparallel orientation, similar to two interdigitated bobby pins (Fig. 2B). Almost all hydrophobic side-chains are involved in packing interactions, which occur along the entire length of each hairpin and bury a total surface area of 5,102 Å2. Polar and charged side-chains also form numerous hydrogen bonding and salt bridge interactions. TRIM proteins have been predicted to contain two distinct coiled-coil segments separated by a helical, but noncoil, segment (19, 20). The TRIM25 structure reveals that these segments actually make up a single contiguous coil, helix H1, which forms the long arm of each subunit. The dimeric H1–H1′ interaction is mediated by classic “knobs-into-holes” packing of both heptad repeats (wherein amino acid residue positions in each repeat are denoted by the letters abcdefg) and hendecad repeats (abcdefghijk) (Fig. 3; Fig. S2 C and D). Residues in the h positions of the hen- decads also form “knobs-to-knobs” interactions (21). The repeats are arranged in a symmetric 7-7-7-7-11-11-11-11-7-7-7-7 pattern, which produces a supercoil that is canonically left-handed at the ends but is underwound and slightly right-handed in the middle. Fig. 1. Domain organization and dimerization of TRIM proteins. (A) Sche- This unusual configuration likely explains why sequence analysis matic of the domain structure of TRIM25. The principal domains and linker regions are RING (red), L1 (gray), B-box 1 (yellow), B-box 2 (orange), coiled- programs failed to predict H1 as a single, contiguous coil. The hendecads mediate interactions at the center of the coiled-coil, and coil (green), L2 (gray), and B30.2/SPRY (blue). The TRIM25189–379 construct used in this study is shown beneath (black), with the secondary structure superhelical underwinding allows H1 and H1′ to sit side by side and derived from the crystal structure (rectangles represent helices). (B) Analo- form an amphipathic platform. Here, the terminal H3 helices from gous schematic of the domain structure of TRIM5α. TRIM5α does not contain the short arm of each hairpin pack against one side of the platform α a B-box 1 domain and has a shorter L2. The TRIM5 133–300 construct used in to form a 4-helix bundle. Thus, the structure indicates that both this study is shown beneath (black). The asterisk denotes a predicted helix ensuing C-terminal B30.2 domains will be centrally located on the (H3) that crosses from L2 into the B30.2 domain.
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