Evolutionarily Conserved Binding of Translationally Controlled Tumor Protein to Eukaryotic Elongation Factor 1B

Huiwen Wu,Weibin Gong,Xingzhe Yao,Jinfeng Wang,Sarah Perrett,Yingang Feng

doi:10.1074/jbc.m114.628594

Abstract

Translationally controlled tumor protein (TCTP) is an abundant protein that is highly conserved in eukaryotes. However, its primary function is still not clear. Human TCTP interacts with the metazoan-specific eukaryotic elongation factor 1Bδ (eEF1Bδ) and inhibits its guanine nucleotide exchange factor (GEF) activity, but the structural mechanism remains unknown. The interaction between TCTP and eEF1Bδ was investigated by NMR titration, structure determination, paramagnetic relaxation enhancement, site-directed mutagenesis, isothermal titration calorimetry, and HADDOCK docking. We first demonstrated that the catalytic GEF domain of eEF1Bδ is not responsible for binding to TCTP but rather a previously unnoticed central acidic region (CAR) domain in eEF1Bδ. The mutagenesis data and the structural model of the TCTP-eEF1Bδ CAR domain complex revealed the key binding residues. These residues are highly conserved in eukaryotic TCTPs and in eEF1B GEFs, including the eukaryotically conserved eEF1Bα, implying the interaction may be conserved in all eukaryotes. Interactions were confirmed between TCTP and the eEF1Bα CAR domain for human, fission yeast, and unicellular photosynthetic microalgal proteins, suggesting that involvement in protein translation through the conserved interaction with eEF1B represents a primary function of TCTP.

Highlights

The primary function of the abundant and highly conserved protein Translationally controlled tumor protein (TCTP) is not clear
central acidic region (CAR) Domain of eEF1B␦ Is the Region Responsible for TCTP Binding—The C-terminal region containing the CAR and guanine nucleotide exchange factor (GEF) domains of eEF1B␦ was previously identified as the TCTP binding region [10, 11]
We found that the CAR domain and the GEF domain are independent structural domains, as the peaks in the 1H-15N HSQC spectrum of the isolated CAR domain overlay well with the corresponding peaks in the 1H-15N HSQC spectrum of the CAR-GEF region (Fig. 2)

Summary

Background

The primary function of the abundant and highly conserved protein TCTP is not clear. Results: TCTP binds to a conserved central acidic region of eukaryotic elongation factor 1B␣/␤/␦. The mutagenesis data and the structural model of the TCTP-eEF1B␦ CAR domain complex revealed the key binding residues These residues are highly conserved in eukaryotic TCTPs and in eEF1B GEFs, including the eukaryotically conserved eEF1B␣, implying the interaction may be conserved in all eukaryotes. Interactions were confirmed between TCTP and the eEF1B␣ CAR domain for human, fission yeast, and unicellular photosynthetic microalgal proteins, suggesting that involvement in protein translation through the conserved interaction with eEF1B represents a primary function of TCTP. By employing various structural techniques, including NMR titration, chemical shift mapping, paramagnetic relaxation enhancement, and HADDOCK docking, as well as isothermal titration calorimetry (ITC) and site-directed mutagenesis, we identified the binding interfaces and key residues in the interaction between TCTP and eEF1B␦.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION