A helical LC3-interacting region mediates the interaction between the retroviral restriction factor Trim5α and mammalian autophagy-related ATG8 proteins

Jeremy R Keown,Moyra M Black,Aaron Ferron,Melvyn Yap,Michael J Barnett,F Grant Pearce,Jonathan P Stoye,David C Goldstone

doi:10.1074/jbc.ra118.004202

Abstract

The retroviral restriction factor tripartite motif–containing 5α (Trim5α) acts during the early postentry stages of the retroviral life cycle to block infection by a broad range of retroviruses, disrupting reverse transcription and integration. The mechanism of this restriction is poorly understood, but it has recently been suggested to involve recruitment of components of the autophagy machinery, including members of the mammalian autophagy-related 8 (ATG8) family involved in targeting proteins to the autophagosome. To better understand the molecular details of this interaction, here we utilized analytical ultracentrifugation to characterize the binding of six ATG8 isoforms and determined the crystal structure of the Trim5α Bbox coiled-coil region in complex with one member of the mammalian ATG8 proteins, autophagy-related protein LC3 B (LC3B). We found that Trim5α binds all mammalian ATG8s and that, unlike the typical LC3-interacting region (LIR) that binds to mammalian ATG8s through a β-strand motif comprising approximately six residues, LC3B binds to Trim5α via the α-helical coiled-coil region. The orientation of the structure demonstrated that LC3B could be accommodated within a Trim5α assembly that can bind the retroviral capsid. However, mutation of the binding interface does not affect retroviral restriction. Comparison of the typical linear β-strand LIR with our atypical helical LIR reveals a conservation of the presentation of residues that are required for the interaction with LC3B. This observation expands the range of LC3B-binding proteins to include helical binding motifs and demonstrates a link between Trim5α and components of the autophagosome.

Highlights

The retroviral restriction factor tripartite motif– containing 5␣ (Trim5␣) acts during the early postentry stages of the retroviral life cycle to block infection by a broad range of retroviruses, disrupting reverse transcription and integration
We found that Trim5␣ binds all mammalian autophagy-related 8 (ATG8) and that, unlike the typical LC3-interacting region (LIR) that binds to mammalian ATG8s through a ␤-strand motif comprising approximately six residues, LC3 B (LC3B) binds to Trim5␣ via the ␣-helical coiled-coil region
We utilized a construct of Trim5␣ from Rhesus macaque encompassing the Bbox coiled-coil regions with an E120K/R121D mutation (RhT5 88 –296 EK/RD) that blocks the higher-order assembly of the protein [16, 29], and LC3B expressed in Escherichia coli

Summary

Results

The interaction between mATG8 family members and Trim5␣ was identified by co-immunoprecipitation studies [9]. To determine whether the interaction with Trim5␣ is common to all mATG8s, we undertook further sedimentation velocity experiments employing RhT5 88 –296 EK/RD and LC3A, LC3C, GABARAP, GABARAPL1, and GABARAPL2 and analyzed the effect on the position of the fast-moving peak. To measure the affinity of the interaction, two representative mATG8 proteins, LC3B and GABARAPL1, were chosen and titrated (0 –320 ␮M) against a 20 ␮M RhT5 88 –296 EK/RD and analyzed binding by SV–AUC In both the LC3B and GABARAPL1 titrations, a concentration-dependent shift was observed for the fast-moving species with the S value of the peak increasing as the concentration of either protein was increased. Two copies of RhT5 88 –296 EK/RD and two copies of LC3B are present in the asymmetric unit

Data collection statistics Diffraction source

Discussion

Crystallization and structure determination

Protein purification

Analytical ultracentrifugation

Restriction assays