Mass spectrometric identification of leucine zipper-like homodimer complexes of the autoantigen L7.
The eucaryotic protein L7 has been shown to associate in the cytoplasm with the large subunit of ribosomes and to interact specifically with as yet unknown cognate sites of mRNA, thereby inhibiting cell-free translation (Neumann, F., Hemmerich, P., von Mikecz, A., Peter, H. H., and Krawinkel, U.(1995) Nucleic Acids Res. 23, 195-202). The N-terminal region of protein L7 contains a sequence motif similar to the leucine zipper domain of eucaryotic transcription factors, which promotes dimerization through alpha-helical coiled coil formation. Using electrospray-ionization mass spectrometry as a method of molecular specificity, we have directly identified the dimeric complexes comprising the leucine zipper-like region of protein L7 and have determined the dissociation constant of L7 homodimers in an affinity binding assay. We also demonstrate the high content of alpha-helicity of the dimer by circular dichroism spectra and computer-based structure simulation and show that the leucine zipper region of protein L7 is fully sufficient to mediate the inhibition of cell-free mRNA translation. A structural basis for the function of L7 to regulate translation is discussed. From the present results we conclude that L7 interacts with double stranded mRNA in a similar fashion as leucine zipper proteins with specific cognate sites on double stranded DNA.
Witte
S
S
Fakultät für Biologie and the Fakultät für Chemie, Universität Konstanz, Postfach 5560, 78434 Konstanz, Germany.
Neumann
F
F
Krawinkel
U
U
Przybylski
M
M
D000595:Amino Acid Sequence; D001324:Autoantigens / Q000737:chemistry* / Q000235:genetics; D016350:Leucine Zippers / Q000235:genetics; D008156:Luciferases / Q000096:biosynthesis; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics
D001324:Autoantigens; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D008156:Luciferases