Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is now a critical tool in molecular biology and structural proteomics. It is routinely used to probe protein and conformational dynamics through a well-established experiment where amide hydrogens exchange with deuterium atoms in a buffer containing DO. Although there have been numerous advances in the field, data analysis still poses challenges mainly due to the need for manual curation of the data and the lack of standardized statistics and accessible software. In response, we developed Kingfisher, an open-source, user-friendly, web-based solution that facilitates downstream analysis using well-established statistics and provides advanced high-resolution representations of the HDX results. Kingfisher is able to read data directly as exported from common software packages and usually takes less than a minute to run the analysis, without the need to download the raw code or install any software. We foresee Kingfisher as a valuable tool for both newcomers and experts in the field of Hydrogen Exchange Mass Spectrometry. Kingfisher is available to all users as an interactive web application at https://kingfisher.wustl.edu/.

The partner and localizer of BRCA2 (PALB2) is a scaffold protein linking BRCA1 with BRCA2 and RAD51 during homologous recombination (HR). PALB2 interaction with DNA strongly enhances HR in cells, while the PALB2 DNA-binding domain (PALB2-DBD) supports DNA strand exchange . We determined that PALB2-DBD is intrinsically disordered beyond a single N-terminal α-helix. Coiled-coil mediated dimerization is stabilized by interaction between intrinsically disordered regions (IDRs) leading to a 2-fold structural compaction. Single-stranded (ss)DNA binding promotes additional structural compaction and protein tetramerization. Using confocal single-molecule FRET, we observed bimodal and oligomerization-dependent compaction of ssDNA bound to PALB2-DBD, suggesting a novel strand exchange mechanism. Bioinformatics analysis and preliminary observations indicate that PALB2 forms protein-nucleic acids condensates. Intrinsically disordered DBDs are prevalent in the human proteome. PALB2-DBD and similar IDRs may use a chaperone-like mechanism to aid formation and resolution of DNA and RNA multichain intermediates during DNA replication, repair and recombination.

Single-stranded DNA (ssDNA) intermediates which emerge during DNA metabolic processes are shielded by replication protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper to direct the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent functional specificity requires knowledge of the structural conformation of ssDNA when RPA-bound. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPA-ssDNA complexes using single-molecule FRET and double electron-electron resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.

The Partner and Localizer of BRCA2 (PALB2) tumor suppressor is a scaffold protein that links BRCA1 with BRCA2 to initiate homologous recombination (HR). PALB2 interaction with DNA strongly enhances HR efficiency. The PALB2 DNA-binding domain (PALB2-DBD) supports DNA strand exchange, a complex multistep reaction supported by only a few protein families such as RecA-like recombinases or Rad52. The mechanisms of PALB2 DNA binding and strand exchange are unknown. We performed circular dichroism, electron paramagnetic spectroscopy, and small-angle X-ray scattering analyses and determined that PALB2-DBD is intrinsically disordered, even when bound to DNA. The intrinsically disordered nature of this domain was further supported by bioinformatics analysis. Intrinsically disordered proteins (IDPs) are prevalent in the human proteome and have many important biological functions. The complexity of the strand exchange reaction significantly expands the functional repertoire of IDPs. The results of confocal single-molecule FRET indicated that PALB2-DBD binding leads to oligomerization-dependent DNA compaction. We hypothesize that PALB2-DBD uses a chaperone-like mechanism to aid formation and resolution of complex DNA and RNA multichain intermediates during DNA replication and repair. Since PALB2-DBD alone or within the full-length PALB2 is predicted to have strong liquid-liquid phase separation (LLPS) potential, protein-nucleic acids condensates are likely to play a role in complex functionality of PALB2-DBD. Similar DNA-binding intrinsically disordered regions may represent a novel class of functional domains that evolved to function in eukaryotic nucleic acid metabolism complexes.

Spinster (Spns) lipid transporters are critical for transporting sphingosine-1-phosphate (S1P) across cellular membranes. In humans, Spns2 functions as the main S1P transporter in endothelial cells, making it a potential drug target for modulating S1P signaling. Here, we employed an integrated approach in lipid membranes to identify unknown conformational states of a bacterial Spns from Hyphomonas neptunium (HnSpns) and to define its proton- and substrate-coupled conformational dynamics. Our systematic study reveals conserved residues critical for protonation steps and their regulation, and how sequential protonation of these proton switches coordinates the conformational transitions in the context of a noncanonical ligand-dependent alternating access. A conserved periplasmic salt bridge (Asp60:Arg289) keeps the transporter in a closed conformation, while proton-dependent conformational dynamics are significantly enhanced on the periplasmic side, providing a pathway for ligand exchange.