Editorial overview: Macromolecular assemblies: clues from structural insights

Nowadays, college biology textbooks teach us that most biomolecules, acting on no matter which biochemical process, generally function not as single entities, but rather, as constituents of macromolecular assemblies of variable complexity, whereby biochemical activities are promoted, regulated and fine-tuned in order to yield precise biological outcomes. In this respect, the past decades have witnessed a broad development of novel experimental approaches that, accompanied by an unprecedented surge of computational capabilities, have enabled extensive and multifaceted analyses on a large plethora of biomolecular interactions. These have substantially fostered our understanding of essential mechanistic and functional aspects underlying the activity of biomolecules that participate in key cellular processes. And in some cases, have provided the clue for deciphering the molecular essence of pathologies arising when such biomolecules go awry.

Throughout 2020, structural biology assumed a prominent role by defining the iconic representation of COVID-19, making SARS-CoV-2 never an unknown threat to humanity but rather a call for scientific action. In this same tumultuous year, the 50-year challenge of computationally predicting the 3-dimensional structure of a protein from its amino acid sequence was achieved to greater than 85% accuracy by using artificial intelligence. This achievement was made possible by the large number of structures in deposited into the Protein Data Bank by experimentalists, enabled by the past century of developed technology aimed to resolve the 3-dimensional structures of biomolecules by x-ray diffraction, nuclear magnetic resonance spectrometry, and cryoelectron microscopy. As solving structures of individual protein domains has become more achievable by computational approaches, experimental structural biology has advanced to solving the structures of biomolecular machines in their native states, with significant progress towards observing them at atomic level resolution in their native or cellular environments. The output of such studies is providing key mechanistic and functional insights into biological activities as well as yielding modern medicines. This issue of Current Opinion in Structural Biology (COSB) features a series of articles that demonstrate the power of structural and computational biology in resolving at atomic level resolution macromolecular complexes, providing new insights into how these biomolecules drive function and in some cases pathology. The knowledge garnered by these and similar studies has motivated powerful approaches for therapeutic intervention, ushering in a new era of precision medicine.

Thematic in this issue is the machinery that defines the cellular lifespan of protein and RNA molecules. These degradation processes involve multi-component assemblies and coordinated activities. The RNA exosome acts on diverse RNA substrates, catalyzing their maturation and/or decay by 3′–5′ RNA processing. Weick and Lima describe the structural underpinnings by which Ski2-like helicases recruit substrates to the RNA exosome. They highlight mutually exclusive interactions that they propose may be used to orchestrate hierarchical activity and thus substrate specificity. Regulated protein degradation in eukaryotes is performed by the 26S proteasome, which is composed of a proteolytic core particle that is capped by a regulatory particle. Davis et al. highlight the structural features required for substrate recognition by the proteasome regulatory particle, which includes a ubiquitin tag added post-translationally to the substrate and a disordered initiation sequence within the substrate that can engage the proteasome ATPase ring.

Approximately 1000 enzymes regulate the ubiquitination status of cellular proteins, and ~20% of all protein degradation by the proteasome involves the action of cullin-RING ligase (CRL) enzymes. These enzymes are themselves modified and in turn activated by the ubiquitin-like protein NEDD8. Baek et al. describe the molecular architecture and impressive gymnastics of multiprotein CRL complexes that drives their neddylation and NEDD8-mediated catalysis of substrate ubiquitination. An exciting therapeutical application of the ubiquitin-proteasome pathway has been realized through proteolysis targeting chimeras (PROTACs) degraders and small-molecule glues including immunomodulatory drugs (IMiDs). Ramachandran and Ciulli describe mechanisms by which E3 ligase substrate receptors bind substrates and how molecular glues and PROTACs direct ubiquitination machinery to non-native substates.

Mechanisms of proteolysis in pathogenic bacteria are described by Kahne and Darwin and include the bacterial proteasome and ClpP proteases. The authors highlight the role of activators for these two degrader mechanisms in defining substrate specificity with interesting comparison to each other and also to features highlighted by Davis et al. for the eukaryotic 26S proteasome. Structure-based design of modulators for bacterial degradation systems is proposed by Kahne and Darwin as a potential antimicrobial approach. Another molecular machine unique to bacteria are the Ton molecular motor complexes. Ratliff et al. highlight advancements and resolution of longstanding questions on how Ton motors actively transport nutrients into Gram-negative bacteria.

The hierarchical interaction network of the degradation machines is mirrored in the motor protein myosin VI, which functions in myriad cellular processes including clathrin-mediated endocytosis, autophagy, and cell migration. Magistrati and Polo describe the impressive structural and functional plasticity of myosin VI enabled in part by alternative splicing. As the structural underpinnings of molecular machinery has come to be increasing defined, so too has an appreciation for the functional importance of intrinsically disordered regions of proteins in driving liquid-liquid phase separation. Borcherds et al. describe the dramatic advancements made in defining the sequence properties within intrinsically disordered regions that can drive phase separation. This foundational knowledge has implications for biological function as well as dysfunction, such as in neurological diseases.

The disturbance of macromolecular protein assemblies underlies many diseases, including cancer. Rukhlenko et al. using the RAS-ERK pathway as a model discuss how systems biology, integrating mathematical modelling, computational simulations and structural studies, has contributed to our understanding of cancer-related signaling networks. Though often regarded as static molecular machines, signaling complexes exhibit intricate dynamics that are critical for their function, and both spatial and temporal dynamics govern the molecular interactions necessary for correct signal flux and for the processing and fine-tuning of signal timing, duration and intensity. As exemplified, by the RAS-ERK pathway, oncogenic mutations distort molecular interactions, and subvert these dynamic processes, contributing to tumorigenesis and drug resistance acquisition.

Tumor heterogeneity, a major hurdle in our struggle against cancer, results as a consequence of mutations in host genomes induced by cytosine deaminase activity of mislocalized or unregulated APOBEC3 (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3) enzymes. These enzymes are otherwise key regulators of our innate immune system that play major roles in the response against HIV and other viral infections. Maiti et al. explain how recent crystal and NMR structures of APOBEC3 complexes have unveiled the structural features of the diverse epitopes, at distinct protein domains, involved in binding nucleic acids and how an elaborate interplay between these diverse interactions underlies the mechanisms whereby APOBEC3s recognize and process their substrates.

Cancer and developmental disorders can also arise as a consequence of the malfunction of the histone methyl-transferase activities of Polycomb Repressive Complex 2 (PRC2), an essential factor in the development and maintenance of eukaryotic cellular identity. As noted by Bracken et al., recent advances in mass spectrometry and structural biology techniques have led to an explosion of studies revealing novel insights into PRC2 organisation and engagement with chromatin and have shed new light on how core and accessory PRC2 subunits coordinate accurate H3K27 methylation throughout the genome.

Protein synthesis is central in all cellular processes. In it, ribosome biogenesis requires the orchestrated activity of hundreds of assembly factors. Many of these organize into macromolecular machines that participate in several stages of ribosome synthesis to ensure precise and productive assembly. Frazier et al. summarize how recent advances in structural biology, in particular cryo-EM, have provided detailed information about the structure and function of AAA-ATPase machines that function in every cellular compartment involved in ribosome biogenesis, and on how such techniques have made it possible to distinguish between multiple conformational states at high-resolution, providing valuable mechanistic and regulatory insights.

Somewhat related, RUVBL1 and RUVBL2 are two highly conserved AAA + ATPases that form a hetero-hexameric complex involved in a wide range of completely unrelated cellular processes. How the RUVBL1-RUVBL2 complex is capable of undertaking such a broad spectrum of activities, sometimes antagonistic, has been a major conundrum. Here, Dauden et al.. describe how recent cryo-electron microscopy (cryo-EM) studies have shed light onto how RUVBL1-RUVBL2 interaction forms a scaffold that facilitates complex protein-protein interactions and how the ATPase activity of RUVBL1-RUVBL2 can be distinctively modulated by structural changes dependent on the interacting partner.

Altogether, this set of essays accurately reflects how the development of sophisticated techniques has provided invaluable structural information on distinct macromolecular complexes, paving the road for a better understanding of structure-function relationships. The outcome has made possible novel mechanistic and functional insights into key biochemical and cellular processes.

Biographies

Kylie Walters graduated from Wesleyan University, where funded by the Howard Hughes Medical Institute she did biophysical research with Irina Russu. She obtained a Ph. D. degree with supervision by Gerhard Wagner from the Harvard University Biophysics Program in 1999. With American Cancer Society funding, Kylie was a postdoctoral fellow with Peter Howley at Harvard Medical School. In 2002, she joined the University of Minnesota as a faculty member and in 2013 moved to the National Cancer Institute as a Senior Investigator and Head of the Protein Processing Section. Her research interests are in the area of structural biology and targeted protein degradation.

Piero Crespo graduated from Madrid University (Spain) and obtained his Ph.D in Biochemistry and Molecular Biology from the University of Cantabria (Spain) in 1991. Always interested in signal transduction, in 1992 he moved to the United States as a Fulbright scholar, to join Silvio Gutkind’s laboratory at the National Institutes of Health. Upon his return to Spain in 1998, he obtained tenure at the Spanish Research Council (CSIC). In 2008 he was promoted to Full Professor at the Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), where he is currently working as a group leader and as director of the institute. Prof. Crespo’s research has focused on the regulation of RAS-ERK pathway, in particular on how subcellular localization and signal compartmentalization affect its biochemical and biological outputs, both in physiological and pathological contexts, and on how space-regulated effects can be exploited as antitumoral strategies.