eIF2B Conformation and Assembly State Regulate the Integrated Stress Response

Abstract

The integrated stress response (ISR) is activated by phosphorylation of the translation initiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2’s nucleotide exchange factor eIF2B, a two-fold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2B’s α-subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing an allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-EM structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into ‘conjoined tetramers’ with greatly diminished activity. Thus, ISRIB’s effects in disease models could arise from eIF2B decamer stabilization, allosteric modulation, or both.

Viral Evasion of the Integrated Stress Response Through Antagonistic eIF2-P Mimicry

“…As previously reported, the NSs with a C-terminal FLAG tag (NSs::FLAG) should retain its PKR-evading properties while tagging at the N-terminus (FLAG::NSs) blocks this functionality [29]. These constructs were genomically integrated into our previously generated ISR reporter system, in which both changes in ATF4 translation and general translation can be monitored [5]. Both NSs::FLAG and FLAG::NSs were stably expressed in these cells without impacting the levels of key ISR components (eIF2B, eIF2, PKR, PERK) (Fig.…”

“…While earlier models suggested eIF2B assembly to be rate-limiting and a potential regulatory step, recent work by us and others show that eIF2B in cells primarily exists in its fully assembled decameric, enzymatically active state [5,6]. Cryo-EM studies of various eIF2B complexes elucidated the mechanisms of nucleotide exchange and ISR inhibition through eIF2-P binding [5,6,[12][13][14][15]. Under non-stress conditions, eIF2 engages eIF2B through multiple interfaces along a path spanning the heterodecamer.…”

“…Upon S51 phosphorylation, eIF2 is converted from a substrate to an inhibitor of its dedicated nucleotide exchange factor (GEF) eIF2B. GEF inhibition results from binding of eIF2-P in a new, inhibitory binding orientation on eIF2B, where it elicits allosteric changes to antagonize eIF2 binding and additionally compromise eIF2B's intrinsic enzymatic activity [5,6]. eIF2B is a two-fold symmetric heterodecamer composed of 2 copies each of α, β, δ, γ, and ε subunits [7][8][9][10].…”

“…eIF2B is a two-fold symmetric heterodecamer composed of 2 copies each of α, β, δ, γ, and ε subunits [7][8][9][10]. eIF2B can exist in a range of stable subcomplexes (eIF2Bβδγε tetramers and eIF2Bα2 dimers) if the concentrations of its constituent subunits are altered [5,8,9,11]. While earlier models suggested eIF2B assembly to be rate-limiting and a potential regulatory step, recent work by us and others show that eIF2B in cells primarily exists in its fully assembled decameric, enzymatically active state [5,6].…”

“…eIF2B can exist in a range of stable subcomplexes (eIF2Bβδγε tetramers and eIF2Bα2 dimers) if the concentrations of its constituent subunits are altered [5,8,9,11]. While earlier models suggested eIF2B assembly to be rate-limiting and a potential regulatory step, recent work by us and others show that eIF2B in cells primarily exists in its fully assembled decameric, enzymatically active state [5,6]. Cryo-EM studies of various eIF2B complexes elucidated the mechanisms of nucleotide exchange and ISR inhibition through eIF2-P binding [5,6,[12][13][14][15].…”