Periodic Reporting for period 1 - hyP5 (Adopting orphan pumps: Structural and functional characterization of P5-ATPases)
Reporting period: 2021-09-01 to 2023-08-31
The aim of this project was the structural and functional analysis of yeast and human P5 ATPases. P5 ATPases belong to the P-type ATPase superfamily of essential membrane transport proteins and are conserved in all eukaryotes. This class of proteins utilizes ATP to transport specific substrates across membranes. The substrates that are being transported by P-type ATPases are very diverse, ranging from small Metal-ions to large substrates such as lipids. P5 ATPases can be further divided into P5A and P5B ATPases. It has been shown that the yeast P5A Spf1p extracts single-spanning transmembrane proteins out of the ER membrane, while members of P5B subclass transport polyamines. This project was focusing on investigating Spf1p, the yeast P5A ATPase and ATP13A2, the human P5B ATPase.
It has been found that mutations in human P5 ATPases cause severe neurodegenerative diseases, such as familial early-onset parkinsonism and autism/ language disorders. To better understand how mutations alter the protein and influence the substrate transport, it is important to obtain structural information on the protein. Protein structures help to better understand the function of the protein and to design potential drugs.
The specific objectives of this project were to 1) functionally characterize P5 ATPases to better understand their transport mechanism, 2) to obtain structures of different states during the catalytic cycle and co-structures with substrates/ inhibitors to help better targeting drug development, and 3) to analyze the interaction network as malfunction of this class of proteins have been shown to have a broad phenotype and therefore influence many downstream processes.
It has been found that mutations in human P5 ATPases cause severe neurodegenerative diseases, such as familial early-onset parkinsonism and autism/ language disorders. To better understand how mutations alter the protein and influence the substrate transport, it is important to obtain structural information on the protein. Protein structures help to better understand the function of the protein and to design potential drugs.
The specific objectives of this project were to 1) functionally characterize P5 ATPases to better understand their transport mechanism, 2) to obtain structures of different states during the catalytic cycle and co-structures with substrates/ inhibitors to help better targeting drug development, and 3) to analyze the interaction network as malfunction of this class of proteins have been shown to have a broad phenotype and therefore influence many downstream processes.
I was working extensively on optimizing expression and purification strategies of P5 ATPases. As expression system, purification methods and detergent can have a great impact on the overall structure and functionality of a membrane protein, it was important to identify the strategy, that would lead to the most ‘native-like’ protein. Only this way can we truly study and understand its function. Instead of focusing on ‘artificial’ detergent for the purification, I established many nanodisc reconstitution methods, that allow for the study of membrane proteins in their native membrane environment.
Furthermore, I managed to obtain cryo-EM structures of ATP13A2 in different states and was driving new activity assays.
Furthermore, I managed to obtain cryo-EM structures of ATP13A2 in different states and was driving new activity assays.
We are currently preparing manuscripts about our findings on structural and functional studies of ATP13A2 and new project ideas were developed during this funding period and are currently being worked on. Our results shed light onto the function and importance of P5 ATPases.