Structural characterization of TRIM family proteins

TRIM proteins are E3 ubiquitin ligases that participate in many cellular processes and signaling pathways, such as immune system response. Several TRIM proteins were shown to form antiparallel coiled-coil dimers which position catalytic RING domains on the opposite sides of an elongated rod. As RING dimerization is necessary for its enzymatic activity, higher-order oligomerization is an attractive model for RING activation. In this dissertation, I present structural studies of two members of the TRIM superfamily.
TRIM5α is a restriction factor that leads to premature disassembly of the HIV-1 capsid at a post-entry but pre-integration step. The ubiquitination activity was linked to aborted reverse transcription of retroviral RNA. Despite multiple studies, understanding the mechanism of capsid recognition has remained incomplete. By cryo-electron tomographic reconstructions and subtomogram averaging I show how TRIM5α interacts with HIV-1 CA tubes that mimic the HIV-1 capsid. This work defines the spatial organization of the TRIM5α domains within a full-length protein, consistent with the models predicted from crystal structures of individual domains. Results further show that TRIM5α forms a continuous cage around the CA tubes. In contrast to models proposed before, the capsid-like tubes are not destabilized upon TRIM5α binding as no apparent discontinuities in the CA lattice were observed. I suggest that additional events are necessary to trigger capsid dissociation. I also show that the SPRY domains interact with the capsid lattice in a degenerate manner, probably binding different interfaces. These studies provide new insights into retroviral capsid recognition that is the first step in species-specific restriction by TRIM5α.
TRIM63 is a protein that is upregulated in response to atrophic stimuli and is responsible for muscle catabolism mediated by the proteasome system. I analyze the self-assembly properties of TRIM63 by cross-linking probing and DEER-EPR. Results show that contrary to previously published work, TRIM63 dimerizes in an antiparallel orientation, consistent with other characterized members of the TRIM family. Similar to TRIM5α, TRIM63 may also require higher-order self-assembly architectures for its catalytic activity. Such a mechanism of E3 ligase activation may be conserved across the entire TRIM family.