The proteasome holoenzyme is the major non-lysosomal protease; its proteolytic activity is essential for cellular homeostasis. Thus, it is an attractive target for the development of chemotherapeutics. While the structural basis of core particle (CP) inhibitors is largely understood, their structural impact on the proteasome holoenzyme remains entirely elusive. Here, we determined the structure of the 26S proteasome with and without the inhibitor Oprozomib. Drug binding modifies the energy landscape of conformational motion in the proteasome regulatory particle (RP). Structurally, the energy barrier created by Oprozomib triggers a long-range allosteric regulation, resulting in the stabilization of a non-productive state. Thereby, the chemical drug-binding signal is converted, propagated and amplified into structural changes over a distance of more than 150 Å from the proteolytic site to the ubiquitin receptor Rpn10. The direct visualization of changes in conformational dynamics upon drug binding allows new ways to screen and develop future allosteric proteasome inhibitors.

Wax esters (WE) are neutral lipids that are formed by the transesterification of an activated fatty acyl moiety to a fatty alcohol. Due to their diverse physicochemical properties, WE are used as industrial lubricants, in cosmetics, or for coating. There is substantial interest in producing WE in bacteria and plants by genetic engineering to improve their sustainability and to reduce production costs. However, we lack a detailed understanding of the catalytic mechanism and structural determinants that influence substrate specificities of WE-synthesizing enzymes, which is essential for tailored WE production. One class of well-studied WE-producing enzymes are the bifunctional bacterial wax synthases/acyl-CoA:diacylglycerol O-acyltransferases (WSD). Here, we report the 1.95 Å crystal structure of Acinetobacter baylyi WSD1 (AbWSD1) with a fatty acid molecule bound in the active site. The location of a cocrystallized myristic acid confirms a previously proposed acyl-CoA binding site. A comparison of this AbWSD1 structure to a published Marinobacter aquaeolei WSD1 (MaWSD1) structure of the apoenzyme revealed a major structural difference in the C-terminal part of AbWSD1. This leads us to propose a conformational change in AbWSD1 induced by substrate binding. This conformational change forms then a potential coenzyme A (CoA) binding site. Furthermore, we have identified an additional cavity in AbWSD1 and could show through mutational studies that two amino acids lining the cavity are crucial for the acyl-CoA:diacylglycerol O-acyltransferase (DGAT) activity of the enzyme. Our findings provide a foundation for designing WSD variants that lack DGAT activity.

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