Intragenic Suppressor Mutations Restore GTPase and Translation Functions of a Eukaryotic Initiation Factor 5B Switch II Mutant

ABSTRACT

Structural studies of GTP-binding proteins identified the Switch I and Switch II elements as contacting the γ-phosphate of GTP and undergoing marked conformational changes upon GTP versus GDP binding. Movement of a universally conserved Gly at the N terminus of Switch II is thought to trigger the structural rearrangement of this element. Consistently, we found that mutation of this Gly in the Switch II element of the eukaryotic translation initiation factor 5B (eIF5B) from Saccharomyces cerevisiae impaired cell growth and the guanine nucleotide-binding, GTPase, and ribosomal subunit joining activities of eIF5B. In a screen for mutations that bypassed the critical requirement for this Switch II Gly in eIF5B, intragenic suppressors were identified in the Switch I element and at a residue in domain II of eIF5B that interacts with Switch II. The intragenic suppressors restored yeast cell growth and eIF5B nucleotide-binding, GTP hydrolysis, and subunit joining activities. We propose that the Switch II mutation distorts the geometry of the GTP-binding active site, impairing nucleotide binding and the eIF5B domain movements associated with GTP binding. Accordingly, the Switch I and domain II suppressor mutations induce Switch II to adopt a conformation favorable for nucleotide binding and hydrolysis and thereby reestablish coupling between GTP binding and eIF5B domain movements.