Highlights d Smc4 and Smc2 ATPase head structures reorganize upon ATP binding and dimerization d A Q-loop-mediated switch releases the Ycs4 HEAT-repeat subunit from the Smc4 head d The Smc2 head engages with the ATP-bound Smc4 head into an asymmetric heterodimer d Head dimerization releases the Brn1 kleisin from Smc2 via coiled-coil rotation

Glycosylation is a universal strategy to posttranslationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline-specific bacterial translation elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from Pseudomonas putida. The enzyme is composed of two opposing domains with Rossmann folds, thus constituting a B pattern-type glycosyltransferase (GT-B). While dTDP-β-l-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the KOW-like N-domain of EF-P. Based on our data, we propose a structural model for arginine glycosylation by EarP. As EarP is essential for pathogenicity in P. aeruginosa, our study provides the basis for targeted inhibitor design.

Here we describe a potent tool to investigate arginine rhamnosylation and develop novel antibiotics.

Tripeptides with two consecutive prolines are the shortest and most frequent sequences causing ribosome stalling. The bacterial translation elongation factor P (EF-P) relieves this arrest, allowing protein biosynthesis to continue. A seven amino acids long loop between beta-strands β3/β4 is crucial for EF-P function and modified at its tip by lysylation of lysine or rhamnosylation of arginine. Phylogenetic analyses unveiled an invariant proline in the -2 position of the modification site in EF-Ps that utilize lysine modifications such as Escherichia coli . Bacteria with the arginine modification like Pseudomonas putida on the contrary have selected against it. Focusing on the EF-Ps from these two model organisms we demonstrate the importance of the β3/β4 loop composition for functionalization by chemically distinct modifications. Ultimately, we show that only two amino acid changes in E. coli EF-P are needed for switching the modification strategy from lysylation to rhamnosylation.

Molecular chaperones are the key instruments of bacterial protein homeostasis. Chaperones not only facilitate folding of client proteins, but also transport them, prevent their aggregation, dissolve aggregates and resolve misfolded states. Despite this seemingly large variety, single chaperones can perform several of these functions even on multiple different clients, thus suggesting a single biophysical mechanism underlying. Numerous recently elucidated structures of bacterial chaperone–client complexes show that dynamic interactions between chaperones and their client proteins stabilize conformationally flexible non-native client states, which results in client protein denaturation. Based on these findings, we propose chaotropicity as a suitable biophysical concept to rationalize the generic activity of chaperones. We discuss the consequences of applying this concept in the context of ATP-dependent and -independent chaperones and their functional regulation.