Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins.
mRNA 5'-cap recognition by the eukaryotic translation initiation factor eIF4E has been exhaustively characterized with the aid of a novel fluorometric, time-synchronized titration method, and X-ray crystallography. The association constant values of recombinant eIF4E for 20 different cap analogues cover six orders of magnitude; with the highest affinity observed for m(7)GTP (approximately 1.1 x 10(8) M(-1)). The affinity of the cap analogues for eIF4E correlates with their ability to inhibit in vitro translation. The association constants yield contributions of non-covalent interactions involving single structural elements of the cap to the free energy of binding, giving a reliable starting point to rational drug design. The free energy of 7-methylguanine stacking and hydrogen bonding (-4.9 kcal/mol) is separate from the energies of phosphate chain interactions (-3.0, -1.9, -0.9 kcal/mol for alpha, beta, gamma phosphates, respectively), supporting two-step mechanism of the binding. The negatively charged phosphate groups of the cap act as a molecular anchor, enabling further formation of the intermolecular contacts within the cap-binding slot. Stabilization of the stacked Trp102/m(7)G/Trp56 configuration is a precondition to form three hydrogen bonds with Glu103 and Trp102. Electrostatically steered eIF4E-cap association is accompanied by additional hydration of the complex by approximately 65 water molecules, and by ionic equilibria shift. Temperature dependence reveals the enthalpy-driven and entropy-opposed character of the m(7)GTP-eIF4E binding, which results from dominant charge-related interactions (DeltaH degrees =-17.8 kcal/mol, DeltaS degrees= -23.6 cal/mol K). For recruitment of synthetic eIF4GI, eIF4GII, and 4E-BP1 peptides to eIF4E, all the association constants were approximately 10(7) M(-1), in decreasing order: eIF4GI>4E-BP1>eIF4GII approximately 4E-BP1(P-Ser65) approximately 4E-BP1(P-Ser65/Thr70). Phosphorylation of 4E-BP1 at Ser65 and Thr70 is insufficient to prevent binding to eIF4E. Enhancement of the eIF4E affinity for cap occurs after binding to eIF4G peptides.
Niedzwiecka
Anna
A
Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki & Wigury Street, 02-089 Warsaw, Poland.
Marcotrigiano
Joseph
J
Stepinski
Janusz
J
Jankowska-Anyszka
Marzena
M
Wyslouch-Cieszynska
Aleksandra
A
Dadlez
Michal
M
Gingras
Anne-Claude
AC
Mak
Pawel
P
Darzynkiewicz
Edward
E
Sonenberg
Nahum
N
Burley
Stephen K
SK
Stolarski
Ryszard
R
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D018797:Cell Cycle Proteins; D018360:Crystallography, X-Ray; D004573:Electrolytes / Q000378:metabolism; D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D039642:Eukaryotic Initiation Factors; D006860:Hydrogen Bonding; D008745:Methylation; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012314:RNA Cap Analogs / Q000737:chemistry / Q000378:metabolism; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps / Q000737:chemistry* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C499527:Eif4ebp1 protein, mouse; D004573:Electrolytes; D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D039642:Eukaryotic Initiation Factors; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D010710:Phosphates; D010750:Phosphoproteins; D012314:RNA Cap Analogs; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps; D016601:RNA-Binding Proteins; C017516:7-methylguanosine triphosphate