Structure of the tripartite motif of KAP1/TRIM28 identifies molecular interfaces required for transcriptional silencing of retrotransposons

Abstract

Transcription of transposable elements is tightly regulated to prevent genome damage. KRAB domain-containing zinc finger proteins (KRAB-ZFPs) and KRAB-associated protein 1 (KAP1/TRIM28) play a key role in regulating retrotransposons. KRAB-ZFPs recognize specific 15 retrotransposon sequences and recruit KAP1, inducing the assembly of an epigenetic silencing complex, with chromatin remodeling activities that repress transcription of the targeted retrotransposon and adjacent genes. Our biophysical and structural data show that the tripartite motif (TRIM) of KAP1 forms antiparallel dimers, which further assemble into tetramers and higherorder oligomers in a concentration-dependent manner. Structure-based mutations in the B-box 1 20 domain prevent higher-order oligomerization without significant loss of retrotransposon silencing activity, indicating that, in contrast to other TRIM-family proteins, self-assembly is not essential for KAP1 function. The crystal structure of the KAP1 TRIM dimer identifies the KRAB domain binding site, in the coiled-coil domain near the dyad. Mutations at this site abolished KRAB binding and transcriptional silencing activity of KAP1. This work identifies the interaction interfaces in the 25 KAP1 TRIM responsible for self-association and KRAB binding and establishes their role in retrotransposon silencing. Keywords Transcriptional repressor; epigenetic silencing; endogenous retrovirus; transposable element; ubiquitin E3 ligase 30 2 Running title Structure and function of KAP1/TRIM28 Significance Retroviruses can integrate their DNA into the host-cell genome. Inherited retroviral DNA and other transposable elements account for over half of the human genome. Transposable elements must be tightly regulated to restrict their proliferation and prevent toxic gene expression. KAP1/TRIM2835 is an essential regulator of transposable element transcription. We determined the crystal structure of the KAP1 TRIM. The structure identifies a protein-protein interaction site required for recruitment of KAP1 to transposable elements. An epigenetic gene silencing assay confirms the importance of this site for KAP1-dependent silencing. We also show that KAP1 self-assembles in solution, but this self-assembly is not required for silencing. Our work provides insights into KAP1-40 dependent silencing, and tools for expanding our mechanistic understanding of this process. /body

Structure of KAP1 tripartite motif identifies molecular interfaces required for retroelement silencing

“…The structure factors and atomic coordinates were deposited in the Protein Data Bank (PDB ID code 6QAJ) (42). The original experimental X-ray diffraction images were deposited in the SBGrid Data Bank (dataset 637) (43).…”

The RING domain of TRIM69 promotes higher-order assembly

“…The coiled-coil region forms a highly extended antiparallel dimer. High-resolution structures of the coiled-coil region are available for TRIM5, TRIM20, TRIM25, TRIM28 and TRIM69 (Goldstone et al, 2014;Weinert et al, 2015;Sanchez et al, 2014;Li et al, 2014;Stoll et al, 2018). TRIM proteins exhibit a highly conserved heptad and hendecad repeat, strongly suggesting that all members share this architecture.…”