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. 2015 Jun 4:5:10819.
doi: 10.1038/srep10819.

Crystal structure of TRIM20 C-terminal coiled-coil/B30.2 fragment: implications for the recognition of higher order oligomers

Christopher Weinert  1 , Damien Morger  1 , Aleksandra Djekic  1 , Markus G Grütter  1 , Peer R E Mittl  1
Affiliations

Crystal structure of TRIM20 C-terminal coiled-coil/B30.2 fragment: implications for the recognition of higher order oligomers

Christopher Weinert et al. Sci Rep. .

Abstract

Many tripartite motif-containing (TRIM) proteins, comprising RING-finger, B-Box, and coiled-coil domains, carry additional B30.2 domains on the C-terminus of the TRIM motif and are considered to be pattern recognition receptors involved in the detection of higher order oligomers (e.g. viral capsid proteins). To investigate the spatial architecture of domains in TRIM proteins we determined the crystal structure of the TRIM20Δ413 fragment at 2.4 Å resolution. This structure comprises the central helical scaffold (CHS) and C-terminal B30.2 domains and reveals an anti-parallel arrangement of CHS domains placing the B-box domains 170 Å apart from each other. Small-angle X-ray scattering confirmed that the linker between CHS and B30.2 domains is flexible in solution. The crystal structure suggests an interaction between the B30.2 domain and an extended stretch in the CHS domain, which involves residues that are mutated in the inherited disease Familial Mediterranean Fever. Dimerization of B30.2 domains by means of the CHS domain is crucial for TRIM20 to bind pro-IL-1β in vitro. To exemplify how TRIM proteins could be involved in binding higher order oligomers we discuss three possible models for the TRIM5α/HIV-1 capsid interaction assuming different conformations of B30.2 domains.

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Figures

Figure 1
Figure 1. Central position of the CHS domain in TRIM proteins and its impact on dimerization.
(A) Schematic representation of the domains of TRIM proteins comprising RING, B-Box, and CHS domains. In the literature the CHS domain is referred to as coiled-coil domain with a L2 linker that connects to the C-terminal B30.2 domain. TRIM20 is a RING-less TRIM member and has a long N-terminal extension composed of a PYD domain and a long linker region. For crystallization a construct starting at position 414 was designed. (B) Sedimentation velocity analytical ultracentrifugation data of the isolated B30.2 and CHS-B30.2 (TRIM20Δ413) fragments of TRIM20 show a monomer and a dimer, respectively. The molecular masses of monomers calculated from the B30.2 and CHS-B30.2 sequences are 23 305 Da and 42 737 Da, respectively.
Figure 2
Figure 2. Crystal structure of TRIM20Δ413.
The crystal structure of one out of three dimers from the asymmetric unit is shown. Chains A and B are colored in cyan and red, respectively. Helices of each CHS domain are numbered α1 to α4. The components of the CHS domain that participate in 2-helical (2-CC) or 3-helical coiled coils (3-CC) are indicated and depicted in more detail in B. (A) The dimer is shown from a side and a top view and its dimensions are indicated. (B) Residues involved in the 2-CC (top) and 3-CC (bottom) are shown as sticks and labeled in the corresponding color of the helix. (C) Topology of the CHS domain including its structural motifs are shown schematically.
Figure 3
Figure 3. Interface between the B30.2 domain and the extended proline-rich stretch.
(A) The crystal contact between two dimers is shown in a cartoon representation. Chains A and B are colored as in Fig. 2. The second dimer made of chain C and D is colored in orange and green, respectively. The black box indicates the display detail shown in B. The transition from the closed to the open state would require a swapping of the B30.2 domains shown in orange and red. (B) Detailed view of the B30.2/CHS domain interface. Residues involved in the interface are depicted in sticks and hydrogen bonds are indicated by black dashed lines. A water molecule between the B30.2 domains is shows as a red sphere.
Figure 4
Figure 4. Solution structure of TRIM20Δ413.
(A) Data extrapolated to infinite dilution is shown together with the theoretical curves calculated from the crystal structure, rigid body and ensemble optimized structures together with the χ2 values. (B) The crystal structure is shown in a cartoon representation next to the rigid body model (C), three representatives of the ensemble (D), and a schematic illustration of the distances between the B30.2 domains found in the crystal structure and the widest arrangement of the ensemble (E). The orientation found to be most similar to the crystal structure is shown in a blue surface representation. Other orientations of the B30.2 domain found in the rigid body and ensemble fit are colored violet and red.
Figure 5
Figure 5. Interaction between TRIM20 and pro-IL-1β.
(A) 10 μM of the TRIM20 constructs were incubated with 10 μg FLAG-tagged pro-IL-1β in a total volume on 100 μl. Complex was co-immunoprecipitated using ANTI-FLAG® M2 Affinity Gel (Sigma-Aldrich) and subjected to SDS-PAGE analysis. (B) TRIM20Δ413 and pro-IL-1β were incubated as indicated and their SEC profiles are shown together with the corresponding retention volumes of 1.27 ml, 1.64 ml, and 1.81 ml for the complex after 1 h incubation, TRIM20Δ413 and pro-IL-1β, respectively. Co-elution of both proteins was verified by SDS-PAGE of the indicated fractions. (C) Elution profiles of the individual proteins incubated for 4 h at 25° C show no signs of peak shifts.
Figure 6
Figure 6
(A) Superposition of CHS domains of TRIM20Δ413 (chains C and D in cyan and green, respectively), TRIM5α B-box/coiled-coil (CHS-domain and B-box domains in salmon and red, respectively), TRIM25 coiled-coil (orange), and TRIM69 (grey). Helices α1 (N- and C-termini), α3, α4 and the extended stretch are labeled. (B) Illustration of open- and closed states. Crystal structure of TRIM20Δ413 (chains C and D are shown in red and cyan, and the B30.2 domains of chains B and E in orange and blue, respectively). B30.2 domains in orange/blue and red/cyan indicate the open- and closed states, respectively. The side chain of M694 from the putative peptide binding site of TRIM20 is shown as blue spheres. The hinge region for swapping between open and closed states is indicated by grey spheres. Distances between M694 residues are shown as dashed lines. (C) Three docking models for the binding of TRIM5α to HIV-1 CA. 19 CA hexamers (equivalent to 114 CA subunits) are shown as spheres in salmon and grey. TRIM5α is shown as ribbons. The asymmetric unit of the lattice is indicated by a blue dashed line (a = b = 187 Å, γ = 120°). Some 6-, 3- and 2-fold axes are shown as blue hexagons, triangles and ellipses, respectively. A second unit cell is shown in lighter colors. (i.) Model of the symmetric dimer in the closed state. (ii.) Model of the symmetric tetramer. (iii.) Model of the asymmetric dimer. Ribbons in magenta are experimental structures representing the closed state. Ribbons in green represent the open state that was modeled assuming a domain swapping of the B30.2 domain as described in the text.
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References

    1. Han K., Lou D. I. & Sawyer S. L. Identification of a genomic reservoir for new TRIM genes in primate genomes. PLoS Genet. 7, e1002388 (2011). - PMC - PubMed
    1. Ozato K., Shin D. M., Chang T. H. & Morse H. C. 3rd. TRIM family proteins and their emerging roles in innate immunity. Nat. Rev. Immunol. 8, 849–860 (2008). - PMC - PubMed
    1. Kohl A. & Grütter M. G. Fire and death: the pyrin domain joins the death-domain superfamily. C. R. Biol. 327, 1077–1086 (2004). - PubMed
    1. Perfetto L. et al.. Exploring the diversity of SPRY/B30.2-mediated interactions. Trends Biochem. Sci. 38, 38–46 (2013). - PubMed
    1. Rajsbaum R. & Garcia-Sastre A. Viral evasion mechanisms of early antiviral responses involving regulation of ubiquitin pathways. Trends Microbiol. 21, 421–429 (2013). - PMC - PubMed

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