Cotranslational chaperones assist in de novo folding of nascent polypeptides in all organisms. In yeast, the heterodimeric ribosome-associated complex (RAC) forms a unique chaperone triad with the Hsp70 homologue Ssb. We report the X-ray structure of full length Ssb in the ATP-bound open conformation at 2.6 Å resolution and identify a positively charged region in the α-helical lid domain (SBDα), which is present in all members of the Ssb-subfamily of Hsp70s. Mutational analysis demonstrates that this region is strictly required for ribosome binding. Crosslinking shows that Ssb binds close to the tunnel exit via contacts with both, ribosomal proteins and rRNA, and that specific contacts can be correlated with switching between the open (ATP-bound) and closed (ADP-bound) conformation. Taken together, our data reveal how Ssb dynamics on the ribosome allows for the efficient interaction with nascent chains upon RAC-mediated activation of ATP hydrolysis.

In eukaryotes, N-terminal acetylation is one of the most common protein modifications involved in a wide range of biological processes. Most N-acetyltransferase complexes (NATs) act co-translationally, with the heterodimeric NatA complex modifying the majority of substrate proteins. Here we show that the Huntingtin yeast two-hybrid protein K (HypK) binds tightly to the NatA complex comprising the auxiliary subunit Naa15 and the catalytic subunit Naa10. The crystal structures of NatA bound to HypK or to a N-terminal deletion variant of HypK were determined without or with a bi-substrate analogue, respectively. The HypK C-terminal region is responsible for high-affinity interaction with the C-terminal part of Naa15. In combination with acetylation assays, the HypK N-terminal region is identified as a negative regulator of the NatA acetylation activity. Our study provides mechanistic insights into the regulation of this pivotal protein modification.

Cotranslational chaperones assist de novo folding of nascent polypeptides, prevent them from aggregating and modulate translation. The ribosome-associated complex (RAC) is unique in that the Hsp40 protein Zuo1 and the atypical Hsp70 chaperone Ssz1 form a stable heterodimer, which acts as a cochaperone for the Hsp70 chaperone Ssb. Here we present the structure of the Chaetomium thermophilum RAC core comprising Ssz1 and the Zuo1 N terminus. We show how the conserved allostery of Hsp70 proteins is abolished and this Hsp70-Hsp40 pair is molded into a functional unit. Zuo1 stabilizes Ssz1 in trans through interactions that in canonical Hsp70s occur in cis. Ssz1 is catalytically inert and cannot adopt the closed conformation, but the substrate binding domain β is completed by Zuo1. Our study offers insights into the coupling of a special Hsp70-Hsp40 pair, which evolved to link protein folding and translation.

In humans and plants, N-terminal acetylation plays a central role in protein homeostasis, affects 80% of proteins in the cytoplasm and is catalyzed by five ribosome-associated Nacetyltransferases (NatA-E). Humans also possess a Golgi-associated NatF (HsNAA60) that is essential for Golgi integrity. Remarkably, NAA60 is absent in fungi and has not been identified in plants. Here we identify and characterize the first plasma membrane-anchored post-translationally acting N-acetyltransferase AtNAA60 in the reference plant Arabidopsis thaliana by the combined application of reverse genetics, global proteomics, live-cell imaging, microscale thermophoresis, circular dichroism spectroscopy, nano-differential scanning fluorometry, intrinsic tryptophan fluorescence and X-ray crystallography. We demonstrate that AtNAA60, like HsNAA60, is membrane-localized in vivo by an a-helical membrane anchor at its C-terminus, but in contrast to HsNAA60, AtNAA60 localizes to the plasma membrane. The AtNAA60 crystal structure provides insights into substrate-binding, the broad substrate specificity and the catalytic mechanism probed by structure-based mutagenesis. Characterization of the NAA60 loss-of-function mutants (naa60-1 and naa60-2) uncovers a plasma membrane-localized substrate of AtNAA60 and the importance of NAA60 during high salt stress. Our findings provide evidence for the plant-specific evolution of a plasma membrane-anchored N-acetyltransferase that is vital for adaptation to stress.

Endotoxins are cell wall components of Gram-negative bacteria. A release of endotoxins into the human blood stream results in an inflammation reaction that can lead to life-threatening conditions like sepsis. Therefore, control for endotoxin contamination of intravenously administered drugs is crucial. Drugs are usually tested for putative endotoxin contamination with Limulus-based tests. However, validity of the compendial test procedures is questioned in the case of low endotoxin recovery (LER). To assure validity, regulatory authorities request hold-time studies of endotoxin in addition to pharmacopoeial requirements. Within these studies, endotoxin is added (spiked) to an undiluted product. The spiked product is held for a certain period of time and subsequently diluted for endotoxin determination. Due to the known heterogeneity of endotoxin the question has been raised as to which source represents the most adequate endotoxin spike. In the present study, endotoxin hold-time studies were analyzed by using different sources of endotoxin. Highly purified endotoxin, crude endotoxin extracts (Naturally Occurring Endotoxin) from different bacterial species and varied growth conditions as well as endogenous endotoxin contaminations were investigated. The results clearly demonstrate that endotoxin masking—an effect of LER—is dependent on the endotoxin source used. Various parameters such as bacterial strain and growth conditions lead to different masking susceptibilities. Due to these effects it is impossible to predict the susceptibility of bacterial endotoxin contamination to LER. In order to determine whether a sample is prone to LER, an endotoxin spike that is susceptible to LER is required.