Dysregulation of polyamine homeostasis strongly associates with human diseases. ATP13A2, which is mutated in juvenile-onset Parkinson’s disease and autosomal recessive spastic paraplegia 78, is a transporter with a critical role in balancing the polyamine concentration between the lysosome and the cytosol. Here, to better understand human ATP13A2-mediated polyamine transport, we use single-particle cryo-electron microscopy to solve high-resolution structures of human ATP13A2 in six intermediate states, including the putative E2 structure for the P5 subfamily of the P-type ATPases. These structures comprise a nearly complete conformational cycle spanning the polyamine transport process and capture multiple substrate binding sites distributed along the transmembrane regions, suggesting a potential polyamine transport pathway. Integration of high-resolution structures, biochemical assays, and molecular dynamics simulations allows us to obtain a better understanding of the structural basis of how hATP13A2 transports polyamines, providing a mechanistic framework for ATP13A2-related diseases.

Cross-linking mass spectrometry (XL-MS) is widely used in the analysis of protein structure and protein−protein interactions (PPIs). Throughout the entire workflow, the utilization of cross-linkers and the interpretation of cross-linking data are the core steps. In recent years, the development of crosslinkers and analytical software mostly follow up on the classical models of non-cleavable cross-linkers such as BS3/DSS and MScleavable cross-linkers such as DSSO. Although such a paradigm promotes the maturity and robustness of the XL-MS field, it confines the innovation and flexibility of new cross-linkers and analytical software. This critical insight will discuss the classification, advantages, and disadvantages of existing data analysis search engines. Take the new platinum-based metal cross-linker as an example, potential pitfalls in characterization of cross-linked peptides using existing software are discussed. Finally, ideas on developing more flexible, comprehensive, and userfriendly cross-linkers and software tools are proposed.