Rio1 kinase is an atypical protein kinase that is essential for the proper maturation of the 40S subsunit during ribosome biogenesis. Ribosome biogenesis is linked to cell cycle progression and studies have shown that the process is upregulated in both normal and abnormal highly proliferative cells in order to meet cell demands. In an effort to regulate cell proliferation especially in abnormal dividing cells, the work described here takes the approach of modulating ribosome biogenesis by targeting Rio1 kinase. Several small molecules were tested for binding to afRio1 and toyocamycin, an adenosine analog, had the highest binding affinity. A co-crystal structure of afRio1 with toyocamycin bound was determined at 2.0Å revealing occupancy of toyocamycin in the ATP binding pocket. Steady state kinetic studies revealed that inhibition occurred via both competitive and non-competitive mechanisms. This analysis also revealed that Rio1 accesses multiple oligomeric states and autophosphorylation reduces formation of oligomers. Autophosphorylation may therefore modulate Rio1 enzymatic activity through regulating oligomerization. Additional inhibition studies revealed that afRio1 was susceptible to substrate inhibition at high concentrations of ATP.

The crystal structure of human Rio1 kinase (RioK1) was also determined, providing the first structure of a eukaryotic Rio1 kinase. This work was complemented with biochemical studies that revealed RioK1 shared similar trends as afRio1 including affinity for toyocamycin, formation of oligomers in the presence of different ligands and substrate inhibition at high concentrations of ATP suggesting that both afRio1 and RioK1 may share the same substrate inhibition mechanism.

In addition to work done on Rio1 kinase, structural determination of EspB, a Mycobacterium tuberculosis virulence protein involved in ESX-1 secretion system, is reported.