Journal of Biochemistry Advance Access originally published online on May 26, 2009
Journal of Biochemistry 2009 146(3):359-368; doi:10.1093/jb/mvp078
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Domain-dependent Interaction of Eukaryotic Initiation Factor eIF4A for Binding to Middle and C-terminal Domains of eIF4G
1Department of Physical Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan; and 2Riken Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa, 230-0045, Japan
To whom correspondence should be addressed. Tel: +81-72-690-1069, Fax: +81-72-690-1068, E-mail: tomoo{at}gly.oups.ac.jp
Received April 28, 2009; Accepted May 11, 2009
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Abstract |
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The interactions of recombinant human eIF4A (4A) and its N- and C-terminal side domains (AN and AC, respectively) with the middle- and C-terminal-domain-linked fragment (GMC) of eIF4G and its middle and C-terminal domains (GM and GC, respectively) were investigated by surface plasmon resonance (SPR) analysis and isothermal titration calorimetry (ITC). It is remarkable that the kinetic parameter-dependent SPR profile observed for the 4A–GMC pair was quite different from the steady affinity profiles of the 4A–GM/GC pairs, suggesting the simultaneous contribution of the middle and C-terminal domains of eIF4G for the binding with eIF4A. On the other hand, ITC yielded the enthalpy energies of –1.5 x 104 to –2.5 x 104 J/mol for the domain–domain interactions of 4A with GMC. Although the ITC profile of the 4A–GM pair reflects well the structural feature shown previously by NMR and X-ray analyses, it was essentially different from that of the 4A-GMC pair. The present results suggest that the intimate interaction between the eIF4A N- and C-terminal domains and the eIF4G middle and C-terminal domains is necessary to reveal the biologically active function of the eIF4A–eIF4G complex.
Key Words: eIF4A, eIF4G, association, domain–domain interaction, surface plasmon resonance, isothermal titration calorimetry
Abbreviations: 4A, eukaryotic initiation factor 4A; AN, eIF4A N-terminal domain; AC, eIF4A C-terminal domain; GM, eIF4G middle domain; GC, eIF4G C-terminal domain; GMC, middle- and C-terminal-domain-linked fragment of eIF4G; GST, glutathione S-transferase; CD, circular dichroism; CM5, carboxymethylated dextran; eIF4A, eukaryotic initiation factor 4A; eIF4E, eukaryotic initiation factor 4E; eIF4G, eukaryotic initiation factor 4G; HEPES, 2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid; IPTG, isopropyl-β-D-thio-galacto- pyranoside; ITC, isothermal titration calorimetry; mRNA, messenger RNA; NTA, nitrilotriacetic acid; PABP, poly(A)-binding protein; PCR, polymerase chain reaction; SPR, surface plasmon resonance
*Present address: Screening Science, Lead Discovery Research Labs. Drug Discovery Research, Astellas Pharma Inc. 21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan.