Exploration of the TRIM fold of MuRF1 using EPR reveals a canonical antiparallel structure and extended COS-box

MuRF1 (TRIM63) is a RING-type E3 ubiquitin ligase with a predicted tripartite TRIM fold. TRIM proteins rely upon the correct placement of an N-terminal RING domain, with respect to C-terminal, specific substrate-binding domains. The TRIM domain organization is orchestrated by a central helical domain that forms an antiparallel coiled-coil motif and mediates the dimerization of the fold. MuRF1 has a reduced TRIM composition characterized by a lack of specific substrate binding domains, but contains in its helical domain a conserved sequence motif termed COS-box that has been speculated to fold independently into an α-hairpin. These characteristics had led to question whether MuRF1 adopts a canonical TRIM fold. Using a combination of electron paramagnetic resonance, on spin-labeled protein, and disulfide crosslinking, we show that TRIM63 follows the structural conservation of the TRIM dimerization domain, observed in other proteins. We also show that the COS-box motif folds back onto the dimerization coiled-coil motif, predictably forming a four-helical bundle at the center of the protein and emulating the architecture of canonical TRIMs.publishe

TRIM5α self-assembly and compartmentalization of the HIV-1 viral capsid.

The tripartite-motif protein, TRIM5α, is an innate immune sensor that potently restricts retrovirus infection by binding to human immunodeficiency virus capsids. Higher-ordered oligomerization of this protein forms hexagonally patterned structures that wrap around the viral capsid, despite an anomalously low affinity for the capsid protein (CA). Several studies suggest TRIM5α oligomerizes into a lattice with a symmetry and spacing that matches the underlying capsid, to compensate for the weak affinity, yet little is known about how these lattices form. Using a combination of computational simulations and electron cryo-tomography imaging, we reveal the dynamical mechanisms by which these lattices self-assemble. Constrained diffusion allows the lattice to reorganize, whereas defects form on highly curved capsid surfaces to alleviate strain and lattice symmetry mismatches. Statistical analysis localizes the TRIM5α binding interface at or near the CypA binding loop of CA. These simulations elucidate the molecular-scale mechanisms of viral capsid cellular compartmentalization by TRIM5α

TRIM5α SPRY/coiled-coil interactions optimize avid retroviral capsid recognition

<div><p>Restriction factors are important components of intrinsic cellular defense mechanisms against viral pathogens. TRIM5α is a restriction factor that intercepts the incoming capsid cores of retroviruses such as HIV and provides an effective species-specific barrier to retroviral infection. The TRIM5α SPRY domain directly binds the capsid with only very weak, millimolar-level affinity, and productive capsid recognition therefore requires both TRIM5α dimerization and assembly of the dimers into a multivalent hexagonal lattice to promote avid binding. Here, we explore the important unresolved question of whether the SPRY domains are flexibly linked to the TRIM lattice or more precisely positioned to maximize avidity. Biochemical and biophysical experiments indicate that the linker segment connecting the SPRY domain to the coiled-coil domain adopts an α-helical fold, and that this helical portion mediates interactions between the two domains. Targeted mutations were generated to disrupt the putative packing interface without affecting dimerization or higher-order assembly, and we identified mutant proteins that were nevertheless deficient in capsid binding <i>in vitro</i> and restriction activity in cells. Our studies therefore support a model wherein substantial avidity gains during assembly-mediated capsid recognition by TRIM5α come in part from tailored spacing of tethered recognition domains.</p></div

Dimerization of CC-L2 mutants.

<p>Purified mutant proteins were analyzed by using SEC-MALS (size exclusion chromatography coupled with multi-angle light scattering). The solid curves represent the normalized UV absorbance trace (arbitrary units) of eluting components. The dotted curves show the population averaged molecular mass calculated from the measured protein concentration and light scattering data. Dashed gray lines indicate the expected masses of the monomer and dimer species. (A) Wildtype control. (B) D186A. (C) I193A. (D) E197A. The major peak had a substantial trailing edge indicating dissociation into monomers. (E) E201A.</p

Differential scanning fluorimetry thermal stability profiles of purified TRIM5α proteins.

<p>(A) Comparison of CC-L2, CC-L2-SPRY, and SPRY constructs. (B) Effect of model-based mutations in context CC-L2. (C) Effect of model-based mutations in context of the restriction-competent TRIM5-21R protein.</p

<i>In vitro</i> assembly activities of class II TRIM5-21R mutants.

<p>Purified TRIM5-21R proteins were incubated in assembly buffer overnight and the resulting precipitates were examined by negative stain electron microscopy. (A) I193A. (B) E201A. Insets: Fourier transforms of the associated images. Scale bars = 200 nm.</p

Capsid binding activities of TRIM5-21R proteins.

<p>Representative results of pull-down assays. Purified TRIM5-21R (5 μM) was incubated with disulfide-stabilized HIV-1 CA tubes, fractionated by centrifugation, and visualized by SDS-PAGE with Coommassie staining. L, load; S, soluble fraction; P, pellet fraction. Band intensities were quantified by densitometry. Experiments were repeated at least 2 times for each mutant using independent protein preparations, with similar results.</p

Cytoplasmic body assembly activities of YFP-TRIM5α proteins.

<p>(A) The number of cytoplasmic bodies was counted in each cell and normalized to the intracellular YFP concentration as described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006686#ppat.1006686.ref024" target="_blank">24</a>]. (B) Representative image of HeLa cells stably expressing the wildtype control. (C) D186A. (D) I193A. (E) E201A. Cytoplasmic bodies appear as green puncta. DAPI was used to stain nuclei blue. Scale bars = 10 μ.</p

Functional phenotypes of TRIM5α mutants.

<p>Functional phenotypes of TRIM5α mutants.</p

Restriction activities of TRIMCyp proteins.

<p>(A) HeLa cells that stably expressed the indicated HA-tagged owl monkey TRIM5α proteins were infected with GFP-labeled HIV and the extent of viral replication was quantified. (B) Expression levels were quantified by immunoblotting. Experiments were repeated 2 times independently with similar results.</p