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

Structural insight into the functional mechanism of Nep1/Emg1 N1-specific pseudouridine methyltransferase in ribosome biogenesis

Nucleolar Essential Protein 1 (Nep1) is required for small subunit (SSU) ribosomal RNA (rRNA) maturation and is mutated in Bowen–Conradi Syndrome. Although yeast (Saccharomyces cerevisiae) Nep1 interacts with a consensus sequence found in three regions of SSU rRNA, the molecular details of the interaction are unknown. Nep1 is a SPOUT RNA methyltransferase, and can catalyze methylation at the N1 of pseudouridine. Nep1 is also involved in assembly of Rps19, an SSU ribosomal protein. Mutations in Nep1 that result in decreased methyl donor binding do not result in lethality, suggesting that enzymatic activity may not be required for function, and RNA binding may play a more important role. To study these interactions, the crystal structures of the scNep1 dimer and its complexes with RNA were determined. The results demonstrate that Nep1 recognizes its RNA site via base-specific interactions and stabilizes a stem-loop in the bound RNA. Furthermore, the RNA structure observed contradicts the predicted structures of the Nep1-binding sites within mature rRNA, suggesting that the Nep1 changes rRNA structure upon binding. Finally, a uridine base is bound in the active site of Nep1, positioned for a methyltransfer at the C5 position, supporting its role as an N1-specific pseudouridine methyltransferase

Structure of HoxA9 and Pbx1 bound to DNA: Hox hexapeptide and DNA recognition anterior to posterior

The HOX/HOM superfamily of homeodomain proteins controls cell fate and segmental embryonic patterning by a mechanism that is conserved in all metazoans. The linear arrangement of the Hox genes on the chromosome correlates with the spatial distribution of HOX protein expression along the anterior–posterior axis of the embryo. Most HOX proteins bind DNA cooperatively with members of the PBC family of TALE-type homeodomain proteins, which includes human Pbx1. Cooperative DNA binding between HOX and PBC proteins requires a residue N-terminal to the HOX homeodomain termed the hexapeptide, which differs significantly in sequence between anterior- and posterior-regulating HOX proteins. We report here the 1.9-Å-resolution structure of a posterior HOX protein, HoxA9, complexed with Pbx1 and DNA, which reveals that the posterior Hox hexapeptide adopts an altered conformation as compared with that seen in previously determined anterior HOX/PBC structures. The additional nonspecific interactions and altered DNA conformation in this structure account for the stronger DNA-binding affinity and altered specificity observed for posterior HOX proteins when compared with anterior HOX proteins. DNA-binding studies of wild-type and mutant HoxA9 and HoxB1 show residues in the N-terminal arm of the homeodomains are critical for proper DNA sequence recognition despite lack of direct contact by these residues to the DNA bases. These results help shed light on the mechanism of transcriptional regulation by HOX proteins and show how DNA-binding proteins may use indirect contacts to determine sequence specificity