Interaction of Rio1 Kinase with Toyocamycin Reveals a Conformational Switch That Controls Oligomeric State and Catalytic Activity

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

Steady State Analysis for afRio1.

<p>Steady State Analysis for afRio1.</p

Analysis of Sedimentation Equilibrium Data for Various afRio1 Complexes.

<p>Analysis of Sedimentation Equilibrium Data for Various afRio1 Complexes.</p

Interactions between afRio1 and toyocamycin.

<p>A. Active site of afRio1 overlaid with difference density (F_o-F_c) map calculated using model in which toyocamycin (toyo) is omitted (contoured at 2.5σ). Some of the residues lining the hydrophobic pocket of the active site are labeled. B. Hydrogen bonding interactions (dashed lines) observed between toyocamycin and afRio1.</p

Dimer of afRio1/toyocamycin (toyo) complex observed in the asymmetric unit.

<p>Dimer of afRio1/toyocamycin (toyo) complex observed in the asymmetric unit.</p

Adenosine analogs screened for Rio1 binding activity.

<p>A. Chemical structures of molecules used in this study. Thermal shift data for B. afRio1 and C. hRio1. Difference between melting temperatures (T_m) for unbound afRio1 and complexes with each compound plotted. Actual thermal shift plots provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037371#pone.0037371.s001" target="_blank">Fig. S1</a>.</p

Steady State Analysis of Phosphorylated afRio1.

<p>Steady State Analysis of Phosphorylated afRio1.</p

Steady state analyses of afRio1.

<p>A. V_o vs [ATP] curves for afRio1 in the presence and absence of 20, 40 and 60 nM toyocamycin. B. Autoradiogram showing the transfer of ³²P-labelled phosphate to afRio1 (by autophosphorylation) and MBP as a function of increasing ATP concentration [ATP]. C. V_o vs [ATP] curves for afRio1 showing substrate inhibition at high concentrations of ATP. D. Autoradiogram showing autophosphorylation of human Rio1 as a function of increasing [ATP]. E.V_o vs [ATP] curves for phosphorylated afRio1 in the presence and absence of 40 and 60 toyocamycin.</p