Structural Model of the Human BTG2-PABPC1 Complex by Combining Mutagenesis, NMR Chemical Shift Perturbation Data and Molecular Docking

Aalam Ameerul; Hibah Almasmoum; Lorenzo Pavanello; Cyril Dominguez; Gerlof Sebastiaan Winkler

doi:10.1016/j.jmb.2022.167662

Structural Model of the Human BTG2-PABPC1 Complex by Combining Mutagenesis, NMR Chemical Shift Perturbation Data and Molecular Docking

J Mol Biol. 2022 Jul 30;434(14):167662. doi: 10.1016/j.jmb.2022.167662. Epub 2022 May 28.

Authors

Aalam Ameerul¹, Hibah Almasmoum², Lorenzo Pavanello¹, Cyril Dominguez³, Gerlof Sebastiaan Winkler⁴

Affiliations

¹ School of Pharmacy, University of Nottingham, East Drive, University Park, Nottingham NG7 2RD, UK.
² School of Pharmacy, University of Nottingham, East Drive, University Park, Nottingham NG7 2RD, UK; Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Umm Al-Qura University, Makkah, Saudi Arabia.
³ Leicester Institute of Structural & Chemical Biology and Department of Molecular & Cell Biology, University of Leicester, Leicester LE1 7RH, UK.
⁴ School of Pharmacy, University of Nottingham, East Drive, University Park, Nottingham NG7 2RD, UK. Electronic address: sebastiaan.winkler@nottingham.ac.uk.

PMID: 35640718
DOI: 10.1016/j.jmb.2022.167662

Abstract

Degradation of cytoplasmic mRNA in eukaryotes involves the shortening and removal of the mRNA poly(A) tail by poly(A)-selective ribonuclease (deadenylase) enzymes. In human cells, BTG2 can stimulate deadenylation of poly(A) bound by cytoplasmic poly(A)-binding protein PABPC1. This involves the concurrent binding by BTG2 of PABPC1 and the Caf1/CNOT7 nuclease subunit of the Ccr4-Not deadenylase complex. To understand in molecular detail how PABPC1 and BTG2 interact, we set out to identify amino acid residues of PABPC1 and BTG2 contributing to the interaction. To this end, we first used algorithms to predict PABPC1 interaction surfaces. Comparison of the predicted interaction surface with known residues involved in the binding to poly(A) resulted in the identification of a putative interaction surface for BTG2. Subsequently, we used pulldown assays to confirm the requirement of PABPC1 residues for the interaction with BTG2. Analysis of RNA-binding by PABPC1 variants indicated that PABPC1 residues required for interaction with BTG2 do not interfere with poly(A) binding. After further defining residues of BTG2 that are required for the interaction with PABPC1, we used information from published NMR chemical shift perturbation experiments to guide docking and generate a structural model of the BTG2-PABPC1 complex. A quaternary poly(A)-PABPC1-BTG2-Caf1/CNOT7 model showed that the 3' end of poly(A) RNA is directed towards the catalytic centre of Caf1/CNOT7, thereby providing a rationale for enhanced deadenylation by Caf1/CNOT7 in the presence of BTG2 and PABPC1.

Keywords: deadenylation; mRNA; poly(A); protein complex; ribonuclease.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Humans
Immediate-Early Proteins* / chemistry
Immediate-Early Proteins* / genetics
Models, Structural
Molecular Docking Simulation
Mutagenesis
Poly A / chemistry
Poly A / metabolism
Poly(A)-Binding Proteins* / chemistry
Poly(A)-Binding Proteins* / genetics
Protein Conformation
RNA, Messenger / chemistry
RNA, Messenger / metabolism
Tumor Suppressor Proteins* / chemistry
Tumor Suppressor Proteins* / genetics

Substances

Immediate-Early Proteins
PABPC1L protein, human
Poly(A)-Binding Proteins
RNA, Messenger
Tumor Suppressor Proteins
BTG2 protein, human
Poly A