Transient Structural Properties of the Rho GDP-Dissociation Inhibitor.

Rho GTPases, master spatial regulators of a wide range of cellular processes, are orchestrated by complex formation with guanine nucleotide dissociation inhibitors (RhoGDIs). These have been thought to possess an unstructured N-terminus that inhibits nucleotide exchange of their client upon binding/folding. Via NMR analyses, molecular dynamics simulations, and biochemical assays, we reveal instead pertinent structural properties transiently maintained both, in the presence and absence of the client, imposed onto the terminus context-specifically by modulating interactions with the surface of the folded C-terminal domain. These observations revise the long-standing textbook picture of the GTPases' mechanism of membrane extraction. Rather than by a disorder-to-order transition upon binding of an inhibitory peptide, the intricate and highly selective extraction process of RhoGTPases is orchestrated via a dynamic ensemble bearing preformed transient structural properties, suitably modulated by the specific surrounding along the multi-step process.

Structure of the RZZ complex and molecular basis of Spindly-driven corona assembly at human kinetochores.

In metazoans, a ≈1 megadalton (MDa) multiprotein complex comprising the dynein-dynactin adaptor Spindly and the ROD-Zwilch-ZW10 (RZZ) complex is the building block of a fibrous biopolymer, the kinetochore fibrous corona. The corona assembles on mitotic kinetochores to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. We report here a high-resolution cryo-EM structure that captures the essential features of the RZZ complex, including a farnesyl-binding site required for Spindly binding. Using a highly predictive in vitro assay, we demonstrate that the SAC kinase MPS1 is necessary and sufficient for corona assembly at supercritical concentrations of the RZZ-Spindly (RZZS) complex, and describe the molecular mechanism of phosphorylation-dependent filament nucleation. We identify several structural requirements for RZZS polymerization in rings and sheets. Finally, we identify determinants of kinetochore localization and corona assembly of Spindly. Our results describe a framework for the long-sought-for molecular basis of corona assembly on metazoan kinetochores.

Exploring protein-protein interactions at the proteome level.

Proteins are central to all of the processes of life. For their activity, they almost invariably need to interact with other macromolecules, be they nucleic acids, membranes, glycans, or other proteins. The interaction between proteins is indeed the most common mode of macromolecular interaction underpinning living systems. To understand these systems at a molecular level, it is therefore essential to identify and characterize their constituent protein-protein interactions. Despite an unprecedented growth in our knowledge of complete proteomes across all domains of life, both at the sequence level and increasingly at the structure level, the inherently low accuracy and molecular resolution of many techniques have made the characterization of protein-protein interactions one of the grand challenges of molecular biology. In this review, we survey both computational and experimental techniques for the medium- to high-throughput characterization of protein-protein interactions and discuss the potential of integrative approaches, given recent advances in sequence analysis and structure prediction.

Mechanism of actin-dependent activation of nucleotidyl cyclase toxins from bacterial human pathogens.

Bacterial human pathogens secrete initially inactive nucleotidyl cyclases that become potent enzymes by binding to actin inside eukaryotic host cells. The underlying molecular mechanism of this activation is, however, unclear. Here, we report structures of ExoY from Pseudomonas aeruginosa and Vibrio vulnificus bound to their corresponding activators F-actin and profilin-G-actin. The structures reveal that in contrast to the apo-state, two flexible regions become ordered and interact strongly with actin. The specific stabilization of these regions results in an allosteric stabilization of the nucleotide binding pocket and thereby to an activation of the enzyme. Differences in the sequence and conformation of the actin-binding regions are responsible for the selective binding to either F- or G-actin. Other nucleotidyl cyclase toxins that bind to calmodulin rather than actin undergo a similar disordered-to-ordered transition during activation, suggesting that the allosteric activation-by-stabilization mechanism of ExoY is conserved in these enzymes, albeit the different activator.

A barbed end interference mechanism reveals how capping protein promotes nucleation in branched actin networks.

Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP potentiates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we combine structural biology with in vitro reconstitution to demonstrate that CP not only terminates filament elongation, but indirectly stimulates the activity of Arp2/3 activating nucleation promoting factors (NPFs) by preventing their association to filament barbed ends. Key to this function is one of CP's C-terminal "tentacle" extensions, which sterically masks the main interaction site of the terminal actin protomer. Deletion of the ß tentacle only modestly impairs capping. However, in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes-capping and nucleation-in branched actin network assembly.

An assessment of esophageal balloon use for the titration of airway pressure release ventilation and controlled mechanical ventilation in a patient with extrapulmonary acute respiratory distress syndrome: a case report.

BACKGROUND: Esophageal pressure measurement is a minimally invasive monitoring process that assesses respiratory mechanics in patients with acute respiratory distress syndrome. Airway pressure release ventilation is a relatively new positive pressure ventilation modality, characterized by a series of advantages in patients with acute respiratory distress syndrome. CASE PRESENTATION: We report a case of a 55-year-old chilean female, with preexisting hypertension and recurrent renal colic who entered the cardiosurgical intensive care unit with signs and symptoms of urinary sepsis secondary to a right-sided obstructive urolithiasis. At the time of admission, the patient showed signs of urinary sepsis, a poor overall condition, hemodynamic instability, tachycardia, hypotension, and needed vasoactive drugs. Initially the patient was treated with volume control ventilation. Then, ventilation was with conventional ventilation parameters described by the Acute Respiratory Distress Syndrome Network. However, hemodynamic complications led to reduced airway pressure. Later she presented intraabdominal hypertension that compromised the oxygen supply and her ventilation management. Considering these records, an esophageal manometry was used to measure distending lung pressure, that is, transpulmonary pressure, to protect lungs. Initial use of the esophageal balloon was in a volume-controlled modality (deep sedation), which allowed the medical team to perform inspiratory and expiratory pause maneuvers to monitor transpulmonary plateau pressure as a substitute for pulmonary distension and expiratory pause and determine transpulmonary positive end-expiratory pressure. On the third day of mechanical respiration, the modality was switched to airway pressure release ventilation. The use of airway pressure release ventilation was associated with reduced hemodynamic complications and kept transpulmonary pressure between 0 and 20 cmH2O despite a sustained high positive end-expiratory pressure of 20 cmH2O. CONCLUSION: The application of this technique is shown in airway pressure release ventilation with spontaneous ventilation, which is then compared with a controlled modality that requires a lesser number of sedative doses and vasoactive drugs, without altering the criteria for lung protection as guided by esophageal manometry.

The breakthrough in protein structure prediction.

Proteins are the essential agents of all living systems. Even though they are synthesized as linear chains of amino acids, they must assume specific three-dimensional structures in order to manifest their biological activity. These structures are fully specified in their amino acid sequences - and therefore in the nucleotide sequences of their genes. However, the relationship between sequence and structure, known as the protein folding problem, has remained elusive for half a century, despite sustained efforts. To measure progress on this problem, a series of doubly blind, biennial experiments called CASP (critical assessment of structure prediction) were established in 1994. We were part of the assessment team for the most recent CASP experiment, CASP14, where we witnessed an astonishing breakthrough by DeepMind, the leading artificial intelligence laboratory of Alphabet Inc. The models filed by DeepMind's structure prediction team using the program AlphaFold2 were often essentially indistinguishable from experimental structures, leading to a consensus in the community that the structure prediction problem for single protein chains has been solved. Here, we will review the path to CASP14, outline the method employed by AlphaFold2 to the extent revealed, and discuss the implications of this breakthrough for the life sciences.

Structure of the Lifeact-F-actin complex.

Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact-F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as the hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.

Structural basis of TRPC4 regulation by calmodulin and pharmacological agents.

Canonical transient receptor potential channels (TRPC) are involved in receptor-operated and/or store-operated Ca2+ signaling. Inhibition of TRPCs by small molecules was shown to be promising in treating renal diseases. In cells, the channels are regulated by calmodulin (CaM). Molecular details of both CaM and drug binding have remained elusive so far. Here, we report structures of TRPC4 in complex with three pyridazinone-based inhibitors and CaM. The structures reveal that all the inhibitors bind to the same cavity of the voltage-sensing-like domain and allow us to describe how structural changes from the ligand-binding site can be transmitted to the central ion-conducting pore of TRPC4. CaM binds to the rib helix of TRPC4, which results in the ordering of a previously disordered region, fixing the channel in its closed conformation. This represents a novel CaM-induced regulatory mechanism of canonical TRP channels.

Structural Effects and Functional Implications of Phalloidin and Jasplakinolide Binding to Actin Filaments.

Actin undergoes structural transitions during polymerization, ATP hydrolysis, and subsequent release of inorganic phosphate. Several actin-binding proteins sense specific states during this transition and can thus target different regions of the actin filament. Here, we show in atomic detail that phalloidin, a mushroom toxin that is routinely used to stabilize and label actin filaments, suspends the structural changes in actin, likely influencing its interaction with actin-binding proteins. Furthermore, high-resolution cryoelectron microscopy structures reveal structural rearrangements in F-actin upon inorganic phosphate release in phalloidin-stabilized filaments. We find that the effect of the sponge toxin jasplakinolide differs from the one of phalloidin, despite their overlapping binding site and similar interactions with the actin filament. Analysis of structural conformations of F-actin suggests that stabilizing agents trap states within the natural conformational space of actin.

Towards a structural understanding of the remodeling of the actin cytoskeleton.

Actin filaments (F-actin) are a key component of eukaryotic cells. Whether serving as a scaffold for myosin or using their polymerization to push onto cellular components, their function is always related to force generation. To control and fine-tune force production, cells have a large array of actin-binding proteins (ABPs) dedicated to control every aspect of actin polymerization, filament localization, and their overall mechanical properties. Although great advances have been made in our biochemical understanding of the remodeling of the actin cytoskeleton, the structural basis of this process is still being deciphered. In this review, we summarize our current understanding of this process. We outline how ABPs control the nucleation and disassembly, and how these processes are affected by the nucleotide state of the filaments. In addition, we highlight recent advances in the understanding of actomyosin force generation, and describe recent advances brought forward by the developments of electron cryomicroscopy.

Profilin and formin constitute a pacemaker system for robust actin filament growth.

The actin cytoskeleton drives many essential biological processes, from cell morphogenesis to motility. Assembly of functional actin networks requires control over the speed at which actin filaments grow. How this can be achieved at the high and variable levels of soluble actin subunits found in cells is unclear. Here we reconstitute assembly of mammalian, non-muscle actin filaments from physiological concentrations of profilin-actin. We discover that under these conditions, filament growth is limited by profilin dissociating from the filament end and the speed of elongation becomes insensitive to the concentration of soluble subunits. Profilin release can be directly promoted by formin actin polymerases even at saturating profilin-actin concentrations. We demonstrate that mammalian cells indeed operate at the limit to actin filament growth imposed by profilin and formins. Our results reveal how synergy between profilin and formins generates robust filament growth rates that are resilient to changes in the soluble subunit concentration.

Cryo-EM structure of the ClpXP protein degradation machinery.

The ClpXP machinery is a two-component protease complex that performs targeted protein degradation in bacteria and mitochondria. The complex consists of the AAA+ chaperone ClpX and the peptidase ClpP. The hexameric ClpX utilizes the energy of ATP binding and hydrolysis to engage, unfold and translocate substrates into the catalytic chamber of tetradecameric ClpP, where they are degraded. Formation of the complex involves a symmetry mismatch, because hexameric AAA+ rings bind axially to the opposing stacked heptameric rings of the tetradecameric ClpP. Here we present the cryo-EM structure of ClpXP from Listeria monocytogenes. We unravel the heptamer-hexamer binding interface and provide novel insight into the ClpX-ClpP cross-talk and activation mechanism. Comparison with available crystal structures of ClpP and ClpX in different states allows us to understand important aspects of the complex mode of action of ClpXP and provides a structural framework for future pharmacological applications.

SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM.

Selecting particles from digital micrographs is an essential step in single-particle electron cryomicroscopy (cryo-EM). As manual selection of complete datasets-typically comprising thousands of particles-is a tedious and time-consuming process, numerous automatic particle pickers have been developed. However, non-ideal datasets pose a challenge to particle picking. Here we present the particle picking software crYOLO which is based on the deep-learning object detection system You Only Look Once (YOLO). After training the network with 200-2500 particles per dataset it automatically recognizes particles with high recall and precision while reaching a speed of up to five micrographs per second. Further, we present a general crYOLO network able to pick from previously unseen datasets, allowing for completely automated on-the-fly cryo-EM data preprocessing during data acquisition. crYOLO is available as a standalone program under http://sphire.mpg.de/ and is distributed as part of the image processing workflow in SPHIRE.

Tc toxin activation requires unfolding and refolding of a ß-propeller.

Tc toxins secrete toxic enzymes into host cells using a unique syringe-like injection mechanism. They are composed of three subunits, TcA, TcB and TcC. TcA forms the translocation channel and the TcB-TcC heterodimer functions as a cocoon that shields the toxic enzyme. Binding of the cocoon to the channel triggers opening of the cocoon and translocation of the toxic enzyme into the channel. Here we show in atomic detail how the assembly of the three components activates the toxin. We find that part of the cocoon completely unfolds and refolds into an alternative conformation upon binding. The presence of the toxic enzyme inside the cocoon is essential for its subnanomolar binding affinity for the TcA subunit. The enzyme passes through a narrow negatively charged constriction site inside the cocoon, probably acting as an extruder that releases the unfolded protein with its C terminus first into the translocation channel.

Protein topology determines substrate-binding mechanism in homologous enzymes.

During evolution, some homologs proteins appear with different connectivity between secondary structures (different topology) but conserving the tridimensional arrangement of them (same architecture). These events can produce two types of arrangements; circular permutation or non-cyclic permutations. The first one results in the N and C terminus transferring to a different position on a protein sequence while the second refers to a more complex arrangement of the structural elements. In ribokinase superfamily, two different topologies can be identified, which are related to each other as a non-cyclic permutation occurred during the evolution. Interestingly, this change in topology is correlated with the nucleotide specificity of its members. Thereby, the connectivity of the secondary elements allows us to distinguish an ATP-dependent and an ADP-dependent topology. Here we address the impact of introducing the topology of a homologous ATP-dependent kinase in an ADP-dependent kinase (Thermococcus litoralis glucokinase) in the structure, nucleotide specificity, and substrate binding order of the engineered enzyme. Structural evidence demonstrates that rewiring the topology of TlGK leads to an active and soluble enzyme without modifications on its three-dimensional architecture. The permuted enzyme (PerGK) retains the nucleotide preference of the parent TlGK enzyme but shows a change in the substrate binding order. Our results illustrate how the rearrangement of the protein folding topology during the evolution of the ribokinase superfamily enzymes may have dictated the substrate-binding order in homologous enzymes of this superfamily.

Structural transitions of F-actin upon ATP hydrolysis at near-atomic resolution revealed by cryo-EM.

The function of actin is coupled to the nucleotide bound to its active site. ATP hydrolysis is activated during polymerization; a delay between hydrolysis and inorganic phosphate (Pi) release results in a gradient of ATP, ADP-Pi and ADP along actin filaments (F-actin). Actin-binding proteins can recognize F-actin's nucleotide state, using it as a local 'age' tag. The underlying mechanism is complex and poorly understood. Here we report six high-resolution cryo-EM structures of F-actin from rabbit skeletal muscle in different nucleotide states. The structures reveal that actin polymerization repositions the proposed catalytic base, His161, closer to the γ-phosphate. Nucleotide hydrolysis and Pi release modulate the conformational ensemble at the periphery of the filament, thus resulting in open and closed states, which can be sensed by coronin-1B. The drug-like toxin jasplakinolide locks F-actin in an open state. Our results demonstrate in detail how ATP hydrolysis links to F-actin's conformational dynamics and protein interaction.

Electron cryo-microscopy structure of the canonical TRPC4 ion channel.

Canonical transient receptor channels (TRPC) are non-selective cation channels. They are involved in receptor-operated Ca2+ signaling and have been proposed to act as store-operated channels (SOC). Their malfunction is related to cardiomyopathies and their modulation by small molecules has been shown to be effective against renal cancer cells. The molecular mechanism underlying the complex activation and regulation is poorly understood. Here, we report the electron cryo-microscopy structure of zebrafish TRPC4 in its unliganded (apo), closed state at an overall resolution of 3.6 Å. The structure reveals the molecular architecture of the cation conducting pore, including the selectivity filter and lower gate. The cytoplasmic domain contains two key hubs that have been shown to interact with modulating proteins. Structural comparisons with other TRP channels give novel insights into the general architecture and domain organization of this superfamily of channels and help to understand their function and pharmacology.