N. Richet

Dual functions of the Hsm3 protein in chaperoning and scaffolding regulatory particle subunits during the proteasome assembly

The 26S proteasome, a molecular machine responsible for regulated protein degradation, consists of a proteolytic core particle (20S CP) associated with 19S regulatory particles (19S RPs) subdivided into base and lid subcomplexes. The assembly of 19S RP base subcomplex is mediated by multiple dedicated chaperones. Among these, Hsm3 is important for normal growth and directly targets the carboxyl-terminal (C-terminal) domain of Rpt1 of the Rpt1–Rpt2–Rpn1 assembly intermediate. Here, we report crystal structures of the yeast Hsm3 chaperone free and bound to the C-terminal domain of Rpt1. Unexpectedly, the structure of the complex suggests that within the Hsm3–Rpt1–Rpt2 module, Hsm3 also contacts Rpt2. We show that in both yeast and mammals, Hsm3 actually directly binds the AAA domain of Rpt2. The Hsm3 C-terminal region involved in this interaction is required in vivo for base assembly, although it is dispensable for binding Rpt1. Although Rpt1 and Rpt2 exhibit weak affinity for each other, Hsm3 unexpectedly acts as an essential matchmaker for the Rpt1-Rpt2-Rpn1 assembly by bridging both Rpt1 and Rpt2. In addition, we provide structural and biochemical evidence on how Hsm3/S5b may regulate the 19S RP association to the 20S CP proteasome. Our data point out the diverse functions of assembly chaperones.

The Atomic Structure of the Phage Tuc2009 Baseplate Tripod Suggests that Host Recognition Involves Two Different Carbohydrate Binding Modules

ABSTRACT The Gram-positive bacterium Lactococcus lactis, used for the production of cheeses and other fermented dairy products, falls victim frequently to fortuitous infection by tailed phages. The accompanying risk of dairy fermentation failures in industrial facilities has prompted in-depth investigations of these phages. Lactococcal phage Tuc2009 possesses extensive genomic homology to phage TP901-1. However, striking differences in the baseplate-encoding genes stimulated our interest in solving the structure of this host’s adhesion device. We report here the X-ray structures of phage Tuc2009 receptor binding protein (RBP) and of a “tripod” assembly of three baseplate components, BppU, BppA, and BppL (the RBP). These structures made it possible to generate a realistic atomic model of the complete Tuc2009 baseplate that consists of an 84-protein complex: 18 BppU, 12 BppA, and 54 BppL proteins. The RBP head domain possesses a different fold than those of phages p2, TP901-1, and 1358, while the so-called “stem” and “neck” domains share structural features with their equivalents in phage TP901-1. The BppA module interacts strongly with the BppU N-terminal domain. Unlike other characterized lactococcal phages, Tuc2009 baseplate harbors two different carbohydrate recognition sites: one in the bona fide RBP head domain and the other in BppA. These findings represent a major step forward in deciphering the molecular mechanism by which Tuc2009 recognizes its saccharidic receptor(s) on its host. IMPORTANCE Understanding how siphophages infect Lactococcus lactis is of commercial importance as they cause milk fermentation failures in the dairy industry. In addition, such knowledge is crucial in a general sense in order to understand how viruses recognize their host through protein-glycan interactions. We report here the lactococcal phage Tuc2009 receptor binding protein (RBP) structure as well as that of its baseplate. The RBP head domain has a different fold than those of phages p2, TP901-1, and 1358, while the so-called “stem” and “neck” share the fold characteristics also found in the equivalent baseplate proteins of phage TP901-1. The baseplate structure contains, in contrast to other characterized lactococcal phages, two different carbohydrate binding modules that may bind different motifs of the host’s surface polysaccharide. Understanding how siphophages infect Lactococcus lactis is of commercial importance as they cause milk fermentation failures in the dairy industry. In addition, such knowledge is crucial in a general sense in order to understand how viruses recognize their host through protein-glycan interactions. We report here the lactococcal phage Tuc2009 receptor binding protein (RBP) structure as well as that of its baseplate. The RBP head domain has a different fold than those of phages p2, TP901-1, and 1358, while the so-called “stem” and “neck” share the fold characteristics also found in the equivalent baseplate proteins of phage TP901-1. The baseplate structure contains, in contrast to other characterized lactococcal phages, two different carbohydrate binding modules that may bind different motifs of the host’s surface polysaccharide.

Hug1 is an intrinsically disordered protein that inhibits ribonucleotide reductase activity by directly binding Rnr2 subunit

Rad53 is a conserved protein kinase with a central role in DNA damage response and nucleotide metabolism. We observed that the expression of a dominant-lethal form of RAD53 leads to significant expression changes for at least 16 genes, including the RNR3 and the HUG1 genes, both of which are involved in the control of nucleotide metabolism. We established by multiple biophysical and biochemical approaches that Hug1 is an intrinsically disordered protein that directly binds to the small RNR subunit Rnr2. We characterized the surface of interaction involved in Hug1 binding to Rnr2, and we thus defined a new binding region to Rnr2. Moreover, we show that Hug1 is deleterious to cell growth in the context of reduced RNR activity. This inhibitory effect of Hug1 on RNR activity depends on the binding of Hug1 to Rnr2. We propose a model in which Hug1 modulates Rnr2–Rnr1 association by binding Rnr2. We show that Hug1 accumulates under various physiological conditions of high RNR induction. Hence, both the regulation and the mode of action of Hug1 are different from those of the small protein inhibitors Dif1 and Sml1, and Hug1 can be considered as a regulator for fine-tuning of RNR activity.