Substrate recognition and processing by the AAA-ATPase Drg1

The widespread and evolutionary conserved protein family of AAA-ATPases transforms chemical energy into mechanical work to unfold or remodel protein substrates. Although functionally well characterized, the exact mode of substrate-recognition and processing, including the substrate-induced stimulation of ATPase activity is not well understood for most AAA-ATPases. In this proposal we aim at a detailed understanding of the substrate processing mechanism of the yeast AAA-ATPase Drg1 which releases Rlp24 from pre-ribosomal particles and hence plays an essential role in eukaryotic ribosome biogenesis. Our proposed combination of biochemical and structural biology methods will allow us to identify binding and activity-modulating domains of the Rlp24 substrate and to visualize conformational changes in the course of its processing. Together this will give us a conclusive picture of what happens when the substrate protein is processed by the AAA-ATPase Drg1. Deciphering the complex interplay between the AAA-ATPase and its substrate will help us to close an existing knowledge gap in our understanding of the general mode of action of this widespread protein family Moreover, we will address the function of the human orthologue of Drg1, the SPATA5 protein. Although there are hints that SPATA5 might also be involved in ribosome biogenesis, little is known about its actual function in the cell. For this purpose, we will include functional studies to resolve the role of SPATA5 in ribosome biogenesis. Elucidating the cellular function will be an important step towards understanding the pathogenesis of the severe neurological development disorders caused by homo- and heterozygous mutations in the SPATA5 gene. Interestingly, most described mutations are found in regions of the protein that are highly conserved between SPATA5 and Drg1. In the proposed project, the impact of the individual mutations on the enzymatic properties of SPATA5 will be addressed by combining a detailed biochemical analysis with a genetic yeast model to test if and how heterozygous mutations interact to compromise the function of the AAA-ATPase. These analyses will help us to understand the pathogenesis mechanisms behind the mutations in the SPATA5 gene and their deleterious effects on the protein.

Ribosomes synthesize proteins in the cell and are therefore essential. They consist of a small and a large subunit, each composed of ribosomal RNA and ribosomal proteins. The formation of ribosomes is the most energy-intensive process in the cell and is therefore closely coordinated with cell growth and division. The formation of mature ribosomes occurs in many sequential steps, conducted by more than 300 maturation factors in baker's yeast. These maturation factors bind the ribosomal precursors in a strict order and must be released after the reactions they catalyze to allow maturation to proceed. Most of these maturation factors act in the nucleolus or in the nucleoplasm, but some, known as "shuttling factors," accompany the ribosomal precursors into the cytoplasm, where they must be released and recycled. In previous work, we demonstrated that the AAA-ATPase Drg1 binds to the precursor of the large ribosomal subunit immediately after nuclear export and is essential for the release of such "shuttling factors." In this project, we determined the release mechanism in detail. Using cryo-electron microscopy, we resolved the structure of the AAA-ATPase in complex with the ribosomal precursor. Drg1 consists of an N-terminal domain and two consecutive ATPase domains, forming a ring-shaped hexamer. This associates through its N-terminal domains near the "polypeptide exit tunnel" on the ribosomal precursor and feeds the C-terminal residues of the maturation factor Rlp24 into the central hole of the hexamer. The aromatic amino acid residues present in this region of the AAA-ATPase successively engage the peptide chain of Rlp24 in an ATP-dependent manner, pulling it away from the ribosomal precursor. Through structural elucidation of several intermediate states, we could clarify the mechanism of substrate processing. The release of Rlp24 is a prerequisite for release and recycling of all other "shuttling factors," as well as for downstream cytoplasmic maturation steps. Inhibition of this process by the drug diazaborine is therefore fatal for the cell. In this project, we also elucidated the structure of Drg1 with the inhibitor and showed that diazaborine specifically binds to the second ATPase domain of Drg1 and forms a covalent adduct with the bound nucleotide. This adduct prevents the release of the nucleotide, leading to a marked stiffening and inactivation of the second ATPase domain. This example illustrates the importance of small molecule inhibitors for elucidating the processes involved in ribosome maturation and the significant potential that inhibitors of ribosome biogenesis hold for medical research. In fact, we recently showed in further studies that usnic acid, a compound isolated from lichens, inhibits early steps of ribosome biogenesis. Since the formation of new ribosomes is particularly important for rapidly dividing cells, this finding could explain the known anti-tumor activity of usnic acid.