Steffen Lindert

Ion mobility coupled to mass spectrometry informs on the shape and size of protein structures in the form of a collision cross section (CCSIM). Although there are several computational methods for predicting CCSIM based on protein structures, including our previously developed projection approximation using rough circular shapes (PARCS), the process usually requires prior experience with the command-line interface. To overcome this challenge, here we present a web application on the Rosetta Online Server that Includes Everyone (ROSIE) webserver to predict CCSIM from protein structure using projection approximation with PARCS. In this web interface, the user is only required to provide one or more PDB files as input. Results from our case studies suggest that CCSIM predictions (with ROSIE-PARCS) are highly accurate with an average error of 6.12%. Furthermore, the absolute difference between CCSIM and CCSPARCS can help in distinguishing accurate from inaccurate AlphaFold2 protein structure predictions. ROSIE-PARCS is designed with a user-friendly interface, is available publicly and is free to use. The ROSIE-PARCS web interface is supported by all major web browsers and can be accessed via this link (https://rosie.graylab.jhu.edu).

Amadori rearrangement products are stable sugar‐amino acid conjugates that are formed nonenzymatically during preparation, dehydration, and storage of foods. Because Amadori compounds such as fructose‐lysine (F‐Lys), an abundant constituent in processed foods, shape the animal gut microbiome, it is important to understand bacterial utilization of these fructosamines. In bacteria, F‐Lys is first phosphorylated, either during or after uptake to the cytoplasm, to form 6‐phosphofructose‐lysine (6‐P‐F‐Lys). FrlB, a deglycase, then converts 6‐P‐F‐Lys to L‐lysine and glucose‐6‐phosphate. Here, to elucidate the catalytic mechanism of this deglycase, we first obtained a 1.8‐Å crystal structure of Salmonella FrlB (without substrate) and then used computational approaches to dock 6‐P‐F‐Lys on this structure. We also took advantage of the structural similarity between FrlB and the sugar isomerase domain of Escherichia coli glucosamine‐6‐phosphate synthase (GlmS), a related enzyme for which a structure with substrate has been determined. An overlay of FrlB—6‐P‐F‐Lys on GlmS—fructose‐6‐phosphate structures revealed parallels in their active‐site arrangement and guided our selection of seven putative active‐site residues in FrlB for site‐directed mutagenesis. Activity assays with eight recombinant single‐substitution mutants identified residues postulated to serve as the general acid and general base in the FrlB active site and indicated unexpectedly significant contributions from their proximal residues. By exploiting native mass spectrometry (MS) coupled to surface‐induced dissociation, we distinguished mutations that impaired substrate binding versus cleavage. As demonstrated with FrlB, an integrated approach involving x‐ray crystallography, in silico approaches, biochemical assays, and native MS can synergistically aid structure–function and mechanistic studies of enzymes.