Valine 114 replacements in archaeal elongation factor 1 alpha enhanced its ability to interact with aminoacyl-tRNA and kirromycin.
Valine 114 in the D(109)AAILVVA sequence of elongation factor 1alpha from the archaeon Sulfolobus solfataricus (SsEF-1alpha) was substituted with an acidic (V114E), basic (V114K), or cavity-forming (V114A) residue, and the effects on the biochemical properties of the factor were investigated. This sequence is well-conserved among most of eukaryal and eubacterial counterparts, and in the three-dimensional structure of SsEF-1alpha, V114 is located in a hydrophobic pocket near the first GDP-binding consensus sequence G(13)XXXXGK[T,S] [Vitagliano, L., Masullo, M., Sica, F., Zagari, A., and Bocchini, V. (2001) EMBO J. 20, 5305-5311]. These mutants displayed functions absent in the wild-type factor. In fact, although they exhibited a rate in poly(Phe) incorporation almost identical to that of SsEF-1alpha, V114K and V114A exhibited an affinity for GDP and GTP higher and a capability to bind heterologous aa-tRNA stronger than that elicited by SsEF-1alpha but similar to that of eubacterial EF-Tu. V114E instead displayed not only a weaker binding capability for aa-tRNA but also a lower affinity for GDP. The intrinsic GTPase activity of V114E was drastically reduced compared to those of SsEF-1alpha, V114K, and V114A. Interestingly, the decreased intrinsic GTPase activity of V114E was partially restored by kirromycin, an effect already observed for the G13A mutant of SsEF-1alpha [Masullo, M., Cantiello, P., de Paola, B., Catanzano, F., Arcari, P., and Bocchini, V. (2002) Biochemistry 41, 628-633]. Finally, the V114A substitution showed only a marginal effect on both the thermostability and thermophilicity of SsEF-1alpha, whereas V114K and V114E replacements strongly destabilized the molecule.
Masullo
Mariorosario
M
Dipartimento di Scienze Farmacobiologiche, Università di Catanzaro Magna Graecia, Roccelletta di Borgia, I-88021 Catanzaro, Italy.
Cantiello
Piergiuseppe
P
De Paola
Barbara
B
Fiengo
Antonio
A
Vitagliano
Luigi
L
Zagari
Adriana
A
Arcari
Paolo
P
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics*; D019843:Archaeal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000737:chemistry; D018698:Glutamic Acid / Q000235:genetics; D006358:Hot Temperature; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D020648:Peptide Elongation Factor 1 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D011728:Pyridones / Q000378:metabolism* / Q000494:pharmacology; D012345:RNA, Transfer, Amino Acid-Specific / Q000378:metabolism*; D013329:Structure-Activity Relationship; D016944:Sulfolobus; D014633:Valine / Q000737:chemistry* / Q000235:genetics
D019843:Archaeal Proteins; D020648:Peptide Elongation Factor 1; D011728:Pyridones; D012345:RNA, Transfer, Amino Acid-Specific; D018698:Glutamic Acid; D020558:GTP Phosphohydrolases; D014633:Valine; D008239:Lysine; D000409:Alanine; C008536:mocimycin