Abstract
Rio1 kinase is an essential ribosome-processing factor required for proper maturation of 40 S ribosomal subunit. Although its structure is known, several questions regarding its functional remain to be addressed. We report that both Archaeoglobus fulgidus and human Rio1 bind more tightly to an adenosine analog, toyocamycin, than to ATP. Toyocamycin has antibiotic, antiviral and cytotoxic properties, and is known to inhibit ribosome biogenesis, specifically the maturation of 40 S. We determined the X-ray crystal structure of toyocamycin bound to Rio1 at 2.0 Å and demonstrated that toyocamycin binds in the ATP binding pocket of the protein. Despite this, measured steady state kinetics were inconsistent with strict competitive inhibition by toyocamycin. In analyzing this interaction, we discovered that Rio1 is capable of accessing multiple distinct oligomeric states and that toyocamycin may inhibit Rio1 by stabilizing a less catalytically active oligomer. We also present evidence of substrate inhibition by high concentrations of ATP for both archaeal and human Rio1. Oligomeric state studies show both proteins access a higher order oligomeric state in the presence of ATP. The study revealed that autophosphorylation by Rio1 reduces oligomer formation and promotes monomerization, resulting in the most active species. Taken together, these results suggest the activity of Rio1 may be modulated by regulating its oligomerization properties in a conserved mechanism, identifies the first ribosome processing target of toyocamycin and presents the first small molecule inhibitor of Rio1 kinase activity.
Highlights
Protein kinases are involved in a large variety of cellular processes including regulation of gene expression, DNA transcription, signal transduction, cell cycle progression and ribosome biogenesis [1,2,3]
Using thermal shift assays that monitor the shift in melting temperature (Tm) of bound Archaeoglobus fulgidus Rio1 (afRio1) in comparison to unbound afRio1, we identified two adenosine analogs, sangivamycin and toyocamycin, that resulted in a significant increase in the Tm of afRio1 compared to the unbound protein (Fig. 1B, Fig. S1A)
Previous afRio1 crystal structures that have been solved in the presence of adenosine, ADP and ATP show that N6 position of the adenine moiety requires a hydrogen donor to interact with the peptidyl carbonyl oxygen of Glu 148 [26]
Summary
Protein kinases are involved in a large variety of cellular processes including regulation of gene expression, DNA transcription, signal transduction, cell cycle progression and ribosome biogenesis [1,2,3]. 40 are classified as atypical protein kinases (aPKs), while the rest are considered classical eukaryotic protein kinases (ePKs) [4]. Both classes of protein kinases catalyze the phosphorylation of serine, threonine or tyrosine residues. The ePK catalytic domain is comprised of 250 to 300 amino acids consisting of twelve subdomains that are composed of conserved structural elements [7]. The twelve subdomains include a nucleotide-binding loop, a hinge region between the N- and C-terminal domains that participates in binding the adenine moiety of ATP, a catalytic loop consisting of catalytic Asp and Asn residues, a metal binding loop (DFG loop), substrate binding subdomains and the activation loop (APE loop) that is usually involved in regulation of kinase activity [7]. Rio autophosphorylates in trans, such that one molecule of Rio will autophosphorylate another [9]
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