Crystal structures of 7-methylguanosine 5′-triphosphate (m7GTP)- and P1-7-methylguanosine-P3-adenosine-5′,5′-triphosphate (m7GpppA)-bound human full-length eukaryotic initiation factor 4E: biological importance of the C-terminal flexible region

Abstract

The crystal structures of the full-length human eukaryotic initiation factor (eIF) 4E complexed with two mRNA cap analogues [7-methylguanosine 5'-triphosphate (m(7)GTP) and P(1)-7-methylguanosine-P(3)-adenosine-5',5'-triphosphate (m(7)GpppA)] were determined at 2.0 A resolution (where 1 A=0.1 nm). The flexibility of the C-terminal loop region of eIF4E complexed with m(7)GTP was significantly reduced when complexed with m(7)GpppA, suggesting the importance of the second nucleotide in the mRNA cap structure for the biological function of eIF4E, especially the fixation and orientation of the C-terminal loop region, including the eIF4E phosphorylation residue. The present results provide the structural basis for the biological function of both N- and C-terminal mobile regions of eIF4E in translation initiation, especially the regulatory function through the switch-on/off of eIF4E-binding protein-eIF4E phosphorylation.

Phosphorylation of eIF4E attenuates its interaction with mRNA 5′ cap analogs by electrostatic repulsion: Intein-mediated protein ligation strategy to obtain phosphorylated protein

“…According to the fluorescence results (Table 1) m 7 GpppG binds to eIF4E two times more tightly than m 7 GpppA. No interaction between the guanosine and the truncated eIF4E is observed in the crystal structure (Niedzwiecka et al 2002), contrary to the crystallographic data for the full-length human eIF4E bound with m 7 GpppA (Tomoo et al 2002). In the latter complex, a conformational change of the C-terminal protein loop stabilizes the adenosine by hydrogen bonding with Thr 205 and Thr 211.…”

Novel “anti-reverse” cap analogs with superior translational properties

“…If one takes into account some favorable effect of a smaller stacked population (Table 1) and the unfavorable entropic effect of the phosphate chain extension, which partially cancel each other out, the more negative ⌬G°for the pentaphosphate might be caused by an additional stabilizing contact between the negatively charged chain and the positively charged protein surface, that is, Lys 159. It is clear that groups in capped mRNA "downstream" of the m 7 Guo interact with eIF4E, because (1) capped oligonucleotides bind more strongly than cap dinucleotides (Carberry et al 1992), and (2) the Ado moiety in m 7 Gp 3 A is stabilized by two H-bonds to Thr 205 and Thr 211 and van der Walls contacts at the surface of full-length human eIF4E (Tomoo et al 2002) and at the surface of yeast eIF4E (Matsuo et al 2000). On the other hand, no such interactions were observed in the latter study for m 7 Gp 3 ACC.…”

Starting the protein synthesis machine: eukaryotic translation initiation

“…The opposite convex face of the protein is the contact region with eIF4G. (28)(29)(30) Its primary function in translation is to enhance the binding of the eIF4F complex to the 5 0 end of the mRNA. eIF4A is a DEAD box helicase that can unwind RNA secondary structure in the cap-proximal region of the mRNA.…”