To maintain metabolic homeostasis, enzymes must adapt to fluctuating nutrient levels through mechanisms beyond gene expression. Here, we demonstrate that human glutamine synthetase (GS) can reversibly polymerize into filaments aided by a composite binding site formed at the filament interface by the product, glutamine. Time-resolved cryo-electron microscopy (cryo-EM) confirms that glutamine binding stabilizes these filaments, which in turn exhibit reduced catalytic specificity for ammonia at physiological concentrations. This inhibition appears induced by a conformational change that remodulates the active site loop ensemble gating substrate entry. Metadynamics ensemble refinement revealed >10 Å conformational range for the active site loop and that the loop is stabilized by transient contacts. This disorder is significant, as we show that the transient contacts which stabilize this loop in a closed conformation are essential for catalysis both in vitro and in cells. We propose that GS filament formation constitutes a negative-feedback mechanism, directly linking product concentration to the structural and functional remodeling of the enzyme.

Glutamine synthetase (GS) is a ubiquitous enzyme central to nitrogen metabolism, catalyzing the ATP-dependent formation of glutamine from glutamate and ammonia. Positioned at the intersection of nitrogen metabolism with carbon metabolism, the activity of GS is subject to sophisticated regulation. While the intricate regulatory pathways that govern Escherichia coli GS were established long ago, recent work has demonstrated that homologues are controlled by multiple distinct regulatory patterns, such as the metabolite induced oligomeric state formation in archaeal GS by 2-oxoglutarate. Such work was enabled in large part by advances in cryo-electron microscopy (cryoEM) that allowed greater structural access to this large enzyme complex, such as assessment of the large heterogeneous oligomeric states of GS and protein-interactor-GS complexes. This perspective highlights recent advances in understanding GS regulation, focusing on the dynamic interplay between its oligomeric state, metabolite binding, and protein interactors. These interactions modulate GS activity, influencing cellular processes such as nitrogen assimilation, carbon metabolism, and stress responses. Furthermore, we explore the emerging concept of GS “moonlighting” functions, revealing its roles in palmitoylation, cell cycle regulation, and ion channel modulation. These diverse functions highlight a newfound versatility of GS beyond its primary catalytic role and suggest complex roles in health and disease that warrant further study.

Deep-learning language models have shown promise in various biotechnological applications, including protein design and engineering. Here we describe ProGen, a language model that can generate protein sequences with a predictable function across large protein families, akin to generating grammatically and semantically correct natural language sentences on diverse topics. The model was trained on 280 million protein sequences from >19,000 families and is augmented with control tags specifying protein properties. ProGen can be further fine-tuned to curated sequences and tags to improve controllable generation performance of proteins from families with sufficient homologous samples. Artificial proteins fine-tuned to five distinct lysozyme families showed similar catalytic efficiencies as natural lysozymes, with sequence identity to natural proteins as low as 31.4%. ProGen is readily adapted to diverse protein families, as we demonstrate with chorismate mutase and malate dehydrogenase.

Changes in the cellular environment modulate protein energy landscapes to drive important biology, with consequences for signaling, allostery and other vital processes. The effects of ubiquitination are particularly important because of their potential influence on degradation by the 26S proteasome. Moreover, proteasomal engagement requires unstructured initiation regions that many known proteasome substrates lack. To assess the energetic effects of ubiquitination and how these manifest at the proteasome, we developed a generalizable strategy to produce isopeptide-linked ubiquitin within structured regions of a protein. The effects on the energy landscape vary from negligible to dramatic, depending on the protein and site of ubiquitination. Ubiquitination at sensitive sites destabilizes the native structure and increases the rate of proteasomal degradation. In well-folded proteins, ubiquitination can even induce the requisite unstructured regions needed for proteasomal engagement. Our results indicate a biophysical role of site-specific ubiquitination as a potential regulatory mechanism for energy-dependent substrate degradation.

The 26S proteasome is essential for proteostasis and the regulation of vital processes through ATP-dependent degradation of ubiquitinated substrates. To accomplish the multi-step degradation process, the proteasome’s regulatory particle, consisting of lid and base subcomplexes, undergoes major conformational changes whose origin is unknown. Investigating the Saccharomyces cerevisiae proteasome, we found that peripheral interactions between the lid subunit Rpn5 and the base AAA+ ATPase ring are important for stabilizing the substrate-engagement-competent state and coordinating the conformational switch to processing states upon substrate engagement. Disrupting these interactions perturbs the conformational equilibrium and interferes with degradation initiation, while later processing steps remain unaffected. Similar defects in early degradation steps are observed when eliminating hydrolysis in the ATPase subunit Rpt6, whose nucleotide state seems to control proteasome conformational transitions. These results provide important insight into interaction networks that coordinate conformational changes with various stages of degradation, and how modulators of conformational equilibria may influence substrate turnover.

The 26S proteasome is the essential compartmental protease in eukaryotic cells required for the ubiquitin-dependent clearance of damaged polypeptides and obsolete regulatory proteins. Recently, a combination of high-resolution structural, biochemical, and biophysical studies has provided crucial new insights into the mechanisms of this fascinating molecular machine. A multitude of new cryo-electron microscopy structures provided snapshots of the proteasome during ATP-hydrolysis-driven substrate translocation, and detailed biochemical studies revealed the timing of individual degradation steps, elucidating the mechanisms for substrate selection and the commitment to degradation through conformational transitions. It was uncovered how ubiquitin removal from substrates is mechanically coupled to degradation, and cryo-electron tomography studies gave a glimpse of active proteasomes inside the cell, their subcellular localization, and interactions with protein aggregates. Here, we summarize these advances in our mechanistic understanding of the proteasome, with a particular focus on how its structural features and conformational transitions enable the multi-step degradation process.

Cellular environments modulate protein energy landscapes to drive important biology, where small perturbations are consequential for biological signaling, allostery, and other vital processes. The energetic effects of ubiquitination are interesting due to its potential influence on degradation by the 26S proteasome, which requires intrinsically flexible or unstructured initiation regions that many known proteasome substrates lack. We generated proteins with natively attached, isopeptide-linked ubiquitin in structured domains to assess the energetic changes contributed by ubiquitin and how such changes manifest at the proteasome. Ubiquitination at sensitive sites destabilizes the native structure, and thereby increases the rate of degradation for substrates containing unstructured initiation regions. Importantly, this ubiquitination can even induce those requisite regions in well-folded proteins for proteasomal engagement. Our results indicate a biophysical role of site-specific ubiquitination as a potential regulatory mechanism for energy-dependent substrate degradation.

As the endpoint for the ubiquitin-proteasome system, the 26S proteasome is the principal proteolytic machine responsible for regulated protein degradation in eukaryotic cells. The proteasome’s cellular functions range from general protein homeostasis and stress response to the control of vital processes such as cell division and signal transduction. To reliably process all the proteins presented to it in the complex cellular environment, the proteasome must combine high promiscuity with exceptional substrate selectivity. Recent structural and biochemical studies have shed new light on the many steps involved in proteasomal substrate processing, including recognition, deubiquitination, and ATP-driven translocation and unfolding. In addition, these studies revealed a complex conformational landscape that ensures proper substrate selection before the proteasome commits to processive degradation. These advances in our understanding of the proteasome’s intricate machinery set the stage for future studies on how the proteasome functions as a major regulator of the eukaryotic proteome.