Structure of substrate-bound SMG1-8-9 kinase complex reveals molecular basis for phosphorylation specificity.

Lukas M Langer,Fabien Bonneau,Yair Gat,Elena Conti

doi:10.7554/elife.57127

Abstract

PI3K-related kinases (PIKKs) are large Serine/Threonine (Ser/Thr)-protein kinases central to the regulation of many fundamental cellular processes. PIKK family member SMG1 orchestrates progression of an RNA quality control pathway, termed nonsense-mediated mRNA decay (NMD), by phosphorylating the NMD factor UPF1. Phosphorylation of UPF1 occurs in its unstructured N- and C-terminal regions at Serine/Threonine-Glutamine (SQ) motifs. How SMG1 and other PIKKs specifically recognize SQ motifs has remained unclear. Here, we present a cryo-electron microscopy (cryo-EM) reconstruction of a human SMG1-8-9 kinase complex bound to a UPF1 phosphorylation site at an overall resolution of 2.9 Å. This structure provides the first snapshot of a human PIKK with a substrate-bound active site. Together with biochemical assays, it rationalizes how SMG1 and perhaps other PIKKs specifically phosphorylate Ser/Thr-containing motifs with a glutamine residue at position +1 and a hydrophobic residue at position -1, thus elucidating the molecular basis for phosphorylation site recognition.

Highlights

Members of phosphatidylinositol 3-kinase-related kinases (PIKKs) activate distinct signaling pathways that promote cellular survival in different environmental and endogenous stress conditions (Bareticand Williams, 2014; Imseng et al, 2018; Lempiainen and Halazonetis, 2009)
We studied the interaction between recombinant human SMG1-SMG8-SMG9 with UPF1 peptides using cryoelectron microscopy (cryo-EM) and mass spectrometry to identify the molecular basis with which SMG1, and potentially other PIKKs, recognizes specific phosphorylation sites in its substrate
Cryo-EM structure of the human SMG1-8-9 kinase complex bound to a UPF1 peptide

Summary

Introduction

Members of phosphatidylinositol 3-kinase-related kinases (PIKKs) activate distinct signaling pathways that promote cellular survival in different environmental and endogenous stress conditions (Bareticand Williams, 2014; Imseng et al, 2018; Lempiainen and Halazonetis, 2009). Cells have a special detection system that spots and removes any messenger RNA molecules that contain errors, which would result in the protein produced being too short For this error-detecting system to work, a protein called UPF1 must be modified by an enzyme called SMG1. To further understand the role of the amino acids, Langer et al replaced them one-by-one with different amino acids to see how each affected the interaction between the two proteins This revealed that SMG1 preferred leucine at the beginning of the recognized pattern and glutamine at the end when binding to UPF1. We studied the interaction between recombinant human SMG1-SMG8-SMG9 with UPF1 peptides using cryo-EM and mass spectrometry to identify the molecular basis with which SMG1, and potentially other PIKKs, recognizes specific phosphorylation sites in its substrate

Results and discussion

Conclusions

Materials and methods