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Acyldepsipeptide (ADEP) is an exploratory antibiotic with a novel mechanism of action. ClpP, the proteolytic core of the caseinolytic protease, is deregulated towards unrestrained proteolysis. Here, we report on the mechanism of ADEP resistance in Firmicutes. This bacterial phylum contains important pathogens that are relevant for potential ADEP therapy. For Staphylococcus aureus, Bacillus subtilis, enterococci and streptococci, spontaneous ADEP‐resistant mutants were selected in vitro at a rate of 10−6. All isolates carried mutations in clpP. All mutated S. aureus ClpP proteins characterised in this study were functionally impaired; this increased our understanding of the mode of operation of ClpP. For molecular insights, crystal structures of S. aureus ClpP bound to ADEP4 were determined. Well‐resolved N‐terminal domains in the apo structure allow the pore‐gating mechanism to be followed. The compilation of mutations presented here indicates residues relevant for ClpP function and suggests that ADEP resistance will occur at a lower rate during the infection process., ClpP mutant analysis: ADEP antibiotics select ClpP out‐of‐function mutations in Firmicutes in vitro. Studying these ClpP defects provides a map of amino acids relevant for the function of ClpP. Several mutated ClpP proteins were purified, and their diverse impairments were characterised. A new S. aureus ClpP:ADEP co‐crystal structure shows an electrostatic network stabilising the N‐terminal gate.

In systemic light chain amyloidosis, an overexpressed antibody light chain (LC) forms fibrils which deposit in organs and cause their failure. While it is well-established that mutations in the LC’s VL domain are important prerequisites, the mechanisms which render a patient LC amyloidogenic are ill-defined. In this study, we performed an in-depth analysis of the factors and mutations responsible for the pathogenic transformation of a patient-derived λ LC, by recombinantly expressing variants in E. coli. We show that proteolytic cleavage of the patient LC resulting in an isolated VL domain is essential for fibril formation. Out of 11 mutations in the patient VL, only one, a leucine to valine mutation, is responsible for fibril formation. It disrupts a hydrophobic network rendering the C-terminal segment of VL more dynamic and decreasing domain stability. Thus, the combination of proteolytic cleavage and the destabilizing mutation trigger conformational changes that turn the LC pathogenic., eLife digest Amyloid light chain amyloidosis, shortened to AL amyloidosis, is a rare and often fatal disease. It is caused by a disorder of the bone marrow. Usually, cells in the bone marrow produce Y-shaped proteins called antibodies to fight infections. In AL amyloidosis, these cells release too much of the short arm of the antibody, known as its light chain, and the light chains also carry mutations. The antibodies are no longer able to assemble properly, and instead misfold and form structures, known as amyloid fibrils. The fibrils build up outside the cells, gradually causing damage to tissues and organs that can lead to life-threatening organ failure. Due to the rareness of the disease, diagnosis is often overlooked and delayed. People experience widely varying symptoms, depending on the organs affected. Also, given the diversity of antibodies people make, every person with AL amyloidosis has a variety of mutations implicated in their disease. It is thought that mutations in the antibody light chain make it unstable and prone to misfolding, but it remains unclear which specific mutations trigger a cascade of amyloid fibril formation. Now, Kazman et al. have pinpointed the exact mechanism in one case of the disease. First, tissue biopsies from a woman with advanced AL amyloidosis were analyzed, and the defunct antibody light chain was isolated. Eleven mutations were identified in the antibody light chain, only one of which was found to be responsible for the formation of the harmful fibrils. The next step was to determine how this one small change was so damaging. The experiments showed that after the antibody light chain was cut in two, a process that happens naturally in the body, this single mutation transforms it into a protein capable of causing disease. In this ‘bedside to lab bench’ study, Kazman et al. have succeeded in determining the molecular origin of one case of AL amyloidosis. The results have also shown that the instability of antibodies due to mutation does not alone explain the formation of amyloid fibrils in this disease and that the cutting of this protein in two is also important. It is hoped that, in the long run, this work will lead to new diagnostics and treatment options for people with AL amyloidosis.

Journal of molecular biology 430(5), 611 - 627 (2018). doi:10.1016/j.jmb.2017.11.016, Ancestral β-subunit (Anbu) is homologous to HslV and 20S proteasomes. Based on its phylogenetic distribution and sequence clustering, Anbu has been proposed as the “ancestral” form of proteasomes. Here, we report biochemical data, small-angle X-ray scattering results, negative-stain electron microscopy micrographs and a crystal structure of the Anbu particle from Yersinia bercovieri (YbAnbu). All data are consistent with YbAnbu forming defined 12–14 subunit multimers that differ in shape from both HslV and 20S proteasomes. The crystal structure reveals that YbAnbu subunits form tight dimers, held together in part by the Anbu specific C-terminal helices. These dimers (“protomers”) further assemble into a low-rise left-handed staircase. The lock-washer shape of YbAnbu is consistent with the presence of defined multimers, X-ray diffraction data in solution and negative-stain electron microscopy images. The presented structure suggests a possible evolutionary pathway from helical filaments to highly symmetric or pseudosymmetric multimer structures. YbAnbu subunits have the Ntn-hydrolase fold, a putative S1 pocket and conserved candidate catalytic residues Thr1, Asp17 and Lys32(33). Nevertheless, we did not detect any YbAnbu peptidase or amidase activity. However, we could document orthophosphate production from ATP catalyzed by the ATP-grasp protein encoded in the Y. bercovieri Anbu operon., Published by Elsevier, Amsterdam [u.a.]