Considerable debate has focused on whether sampling of molecular dynamics trajectories restrained by crystallographic data can be used to develop realistic ensemble models for proteins in their natural, solution state. For the SARS-CoV-2 main protease, Mpro, we evaluated agreement between solution residual dipolar couplings (RDCs) and various recently reported multi-conformer and dynamic-ensemble crystallographic models. Although Phenix-derived ensemble models showed only small improvements in crystallographic Rfree, substantially improved RDC agreement over fits to a conventionally refined 1.2-Å X-ray structure was observed, in particular for residues with above average disorder in the ensemble. For a set of six lower resolution (1.55-2.19 Å) Mpro X-ray ensembles, obtained at temperatures ranging from 100 to 310 K, no significant improvement over conventional two-conformer representations was found. At the residue level, large differences in motions were observed among these ensembles, suggesting high uncertainties in the X-ray derived dynamics. Indeed, combining the six ensembles from the temperature series with the two 1.2-Å X-ray ensembles into a single 381-member "super ensemble" averaged these uncertainties and substantially improved agreement with RDCs. However, all ensembles showed excursions that were too large for the most dynamic fraction of residues. Our results suggest that further improvements to X-ray ensemble refinement are feasible, and that RDCs provide a sensitive benchmark in such endeavors. Remarkably, a weighted ensemble of 350 PDB Mpro X-ray structures provided slightly better cross-validated agreement with RDCs than any individual ensemble refinement, implying that differences in lattice confinement also limit the fit of RDCs to X-ray coordinates.

The identification of amino acid substitutions that both enhance the stability and function of a protein is a key challenge in protein engineering. Technological advances have enabled assaying thousands of protein variants in a single high-throughput experiment, and more recent studies use such data in protein engineering. We present a Global Multi-Mutant Analysis (GMMA) that exploits the presence of multiply-substituted variants to identify individual amino acid substitutions that are beneficial for the stability and function across a large library of protein variants. We have applied GMMA to a previously published experiment reporting on >54,000 variants of green fluorescent protein (GFP), each with known fluorescence output, and each carrying 1-15 amino acid substitutions (Sarkisyan et al., 2016). The GMMA method achieves a good fit to this dataset while being analytically transparent. We show experimentally that the six top-ranking substitutions progressively enhance GFP. More broadly, using only a single experiment as input our analysis recovers nearly all the substitutions previously reported to be beneficial for GFP folding and function. In conclusion, we suggest that large libraries of multiply-substituted variants may provide a unique source of information for protein engineering.

The Zα domain of ADARp150 is critical for proper Z-RNA substrate binding and is a key factor in the type-I interferon response pathway. Two point-mutations in this domain (N173S and P193A), which cause neurodegenerative disorders, are linked to decreased A-to-I editing in disease models. To understand this phenomenon at the molecular level, we biophysically and structurally characterized these two mutated domains, revealing that they bind Z-RNA with a decreased affinity. Less efficient binding to Z-RNA can be explained by structural changes in beta-wing, part of the Z-RNA-protein interface, and alteration of conformational dynamics of the proteins.

The nuclear protein 1 (NUPR1) is an intrinsically disordered protein involved in stress-mediated cellular conditions. Its paralogue nuclear protein 1-like (NUPR1L) is p53-regulated, and its expression down-regulates that of the NUPR1 gene. Peptidyl-arginine deiminase 4 (PADI4) is an isoform of a family of enzymes catalyzing arginine to citrulline conversion; it is also involved in stress-mediated cellular conditions. We characterized the interaction between NUPR1 and PADI4 in vitro, in silico, and in cellulo. The interaction of NUPR1 and PADI4 occurred with a dissociation constant of 18 ± 6 μM. The binding region of NUPR1, mapped by NMR, was a hydrophobic polypeptide patch surrounding the key residue Ala33, as pinpointed by: (i) computational results; and, (ii) site-directed mutagenesis of residues of NUPR1. The association between PADI4 and wild-type NUPR1 was also assessed in cellulo by using proximity ligation assays (PLAs) and immunofluorescence (IF), and it occurred mainly in the nucleus. Moreover, binding between NUPR1L and PADI4 also occurred in vitro with an affinity similar to that of NUPR1. Molecular modelling provided information on the binding hot spot for PADI4. This is an example of a disordered partner of PADI4, whereas its other known interacting proteins are well-folded. Altogether, our results suggest that the NUPR1/PADI4 complex could have crucial functions in modulating DNA-repair, favoring metastasis, or facilitating citrullination of other proteins.

Human WASP and N-WASP are homologous proteins that require the binding of multiple regulators, including the acidic lipid PIP2 and the small GTPase Cdc42, to relieve autoinhibition before they can stimulate the initiation of actin polymerization. Autoinhibition involves intramolecular binding of the C-terminal acidic and central motifs to an upstream basic region and GTPase binding domain. Little is known about how a single intrinsically disordered protein, WASP or N-WASP, binds multiple regulators to achieve full activation. Here we used molecular dynamics simulations to characterize the binding of WASP and N-WASP with PIP2 and Cdc42. In the absence of Cdc42, both WASP and N-WASP strongly associate with PIP2-containing membranes, through their basic region and also possibly through a tail portion of the N-terminal WH1 domain. The basic region also participates in Cdc42 binding, especially for WASP; consequently Cdc42 binding significantly compromises the ability of the basic region in WASP, but not N-WASP, to bind PIP2. PIP2 binding to the WASP basic region is restored only when Cdc42 is prenylated at the C-terminus and tethered to the membrane. This distinction in the activation of WASP and N-WASP may contribute to their different functional roles.

The human ATP-binding cassette (ABC) transporter ABCA1 plays a critical role in lipid homeostasis as it extracts sterols and phospholipids from the plasma membrane for excretion to the extracellular apolipoprotein A-I and subsequent formation of high-density lipoprotein (HDL) particles. Deleterious mutations of ABCA1 lead to sterol accumulation and are associated with atherosclerosis, poor cardiovascular outcomes, cancer, and Alzheimer's disease. The mechanism by which ABCA1 drives lipid movement is poorly understood, and a unified platform to produce active ABCA1 protein for both functional and structural studies has been missing. In this work, we established a stable expression system for both a human cell-based sterol export assay and protein purification for in vitro biochemical and structural studies. ABCA1 produced in this system was active in sterol export and displayed enhanced ATPase activity after reconstitution into a lipid bilayer. Our single-particle cryo-EM study of ABCA1 in nanodiscs showed protein induced membrane curvature, revealed multiple distinct conformations, and generated a structure of nanodisc-embedded ABCA1 at 4.0-Å resolution representing a previously unknown conformation. Comparison of different ABCA1 structures and molecular dynamics simulations demonstrates both concerted domain movements and conformational variations within each domain. Taken together, our platform for producing and characterizing ABCA1 in a lipid membrane enabled us to gain important mechanistic and structural insights and paves the way for investigating modulators that target the functions of ABCA1.

The detection of structural chromosomal abnormalities (SCA) is crucial for diagnosis, prognosis and management of many genetic diseases and cancers. This detection, done by highly qualified medical experts, is tedious and time-consuming. We propose a highly performing and intelligent method to assist cytogeneticists to screen for SCA. Each chromosome is present in two copies that make up a pair of chromosomes. Usually, SCA are present in only one copy of the pair. Convolutional neural networks (CNN) with Siamese architecture are particularly relevant for evaluating similarities between two images, which is why we used this method to detect abnormalities between both chromosomes of a given pair. As a proof-of-concept, we first focused on a deletion occurring on chromosome 5 (del(5q)) observed in hematological malignancies. Using our dataset, we conducted several experiments without and with data augmentation on seven popular CNN models. Overall, performances obtained were very relevant for detecting deletions, particularly with Xception and InceptionResNetV2 models achieving 97.50% and 97.01% of F1-score, respectively. We additionally demonstrated that these models successfully recognized another SCA, inversion inv(3), which is one of the most difficult SCA to detect. The performance improved when the training was applied on inversion inv(3) dataset, achieving 94.82% of F1-score. The technique that we propose in this paper is the first highly performing method based on Siamese architecture that allows the detection of SCA. Our code is publicly available at: https://github.com/MEABECHAR/ChromosomeSiameseAD.

The biological function of macromolecular complexes depends not only on large-scale transitions between conformations, but also on small-scale conformational fluctuations at equilibrium. Information on the equilibrium dynamics of biomolecular complexes could, in principle, be obtained from local resolution (LR) data in cryo-electron microscopy (cryo-EM) maps. However, this possibility had not been validated by comparing, for a same biomolecular complex, LR data with quantitative information on equilibrium dynamics obtained by an established solution technique. In this study we determined the cryo-EM structure of the minute virus of mice (MVM) capsid as a model biomolecular complex. The LR values obtained correlated with crystallographic B factors and with hydrogen/deuterium exchange (HDX) rates obtained by mass spectrometry (HDX-MS), a gold standard for determining equilibrium dynamics in solution. This result validated a LR-based cryo-EM approach to investigate, with high spatial resolution, the equilibrium dynamics of biomolecular complexes. As an application of this approach, we determined the cryo-EM structure of two mutant MVM capsids and compared their equilibrium dynamics with that of the wild-type MVM capsid. The results supported a previously suggested linkage between mechanical stiffening and impaired equilibrium dynamics of a virus particle. Cryo-EM is emerging as a powerful approach for simultaneously acquiring information on the atomic structure and local equilibrium dynamics of biomolecular complexes.

SHP2 is a phosphatase/adaptor protein that plays an important role in various signaling pathways. Its mutations are associated with cancers and developmental diseases. SHP2 contains a protein tyrosine phosphatase (PTP) and two SH2 domains. Selective inhibition of these domains has been challenging due to the multitude of homologous proteins in the proteome. Here, we developed a monobody, synthetic binding protein, that bound to and inhibited the SHP2 PTP domain. It was selective to SHP2 PTP over close homologs. A crystal structure of the monobody-PTP complex revealed that the monobody bound both highly conserved residues in the active site and less conserved residues in the periphery, rationalizing its high selectivity. Its epitope overlapped with the interface between the PTP and N-terminal SH2 domains that is formed in auto-inhibited SHP2. By using the monobody as a probe for the accessibility of the PTP active site, we developed a simple, nonenzymatic assay for the allosteric regulation of SHP2. The assay showed that, in the absence of an activating phospho-Tyr ligand, wild-type SHP2 and the "PTP-dead" C459E mutant were predominantly in the closed state in which the PTP active site is inaccessible, whereas the E76K and C459S mutants were in the open, active state. It also revealed that previously developed monobodies to the SH2 domains, ligands lacking a phospho-Tyr, weakly favored the open state. These results provide corroboration for a conformational equilibrium underlying allosteric regulation of SHP2, provide powerful tools for characterizing and controlling SHP2 functions, and inform drug discovery against SHP2.

All life forms sense and respond to mechanical stimuli. Throughout evolution, organisms develop diverse mechanosensing and mechanotransduction pathways, leading to fast and sustained mechanoresponses. Memory and plasticity characteristics of mechanoresponses are thought to be stored in the form of epigenetic modifications, including chromatin structure alterations. These mechanoresponses in the chromatin context share conserved principles across species, such as lateral inhibition during organogenesis and development. However, it remains unclear how mechanotransduction mechanisms alter chromatin structure for specific cellular functions, and if altered chromatin structure can mechanically affect the environment. In this review, we discuss how chromatin structure is altered by environmental forces via an outside-in pathway for cellular functions, and the emerging concept of how chromatin structure alterations can mechanically affect nuclear, cellular, and extracellular environments. This bidirectional mechanical feedback between chromatin of the cell and the environment can potentially have important physiological implications, such as in centromeric chromatin regulation of mechanobiology in mitosis, or in tumor-stroma interactions. Finally, we highlight the current challenges and open questions in the field and provide perspectives for future research.

The discovery of diverse bacterial CRISPR-Cas systems has reignited interest in understanding bacterial defense pathways while yielding exciting new tools for genome editing. CRISPR-Cas systems are widely distributed in prokaryotes, found in 40% of bacteria and 90% of archaea, where they function as adaptive immune systems against bacterial viruses (phage) and other mobile genetic elements. In turn, phage have evolved inhibitors, called anti-CRISPR proteins, to prevent targeting. Type V CRISPR-Cas12 systems have emerged as a particularly exciting arena in this co-evolutionary arms race. Type V anti-CRISPRs have highly diverse and novel mechanisms of action, some of which appear to be unusually potent or widespread. In this review, we discuss the discovery and mechanism of these anti-CRISPRs as well as future areas for exploration.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) systems provide bacteria and archaea with an adaptive immune response against invasion by mobile genetic elements like phages, plasmids, and transposons. These systems have been repurposed as very powerful biotechnological tools for gene editing applications in both bacterial and eukaryotic systems. The discovery of natural off-switches for CRISPR-Cas systems, known as anti-CRISPR proteins, provided a mechanism for controlling CRISPR-Cas activity and opened avenues for the development of more precise editing tools. In this review, we focus on the inhibitory mechanisms of anti-CRISPRs that are active against type II CRISPR-Cas systems and briefly discuss their biotechnological applications.

Numerous viruses infecting bacteria and archaea encode CRISPR-Cas system inhibitors, known as anti-CRISPR proteins (Acr). The Acrs typically are highly specific for particular CRISPR variants, resulting in remarkable sequence and structural diversity and complicating accurate prediction and identification of Acrs. In addition to their intrinsic interest for understanding the coevolution of defense and counter-defense systems in prokaryotes, Acrs could be natural, potent on-off switches for CRISPR-based biotechnological tools, so their discovery, characterization and application are of major importance. Here we discuss the computational approaches for Acr prediction. Due to the enormous diversity and likely multiple origins of the Acrs, sequence similarity searches are of limited use. However, multiple features of protein and gene organization have been successfully harnessed to this end including small protein size and distinct amino acid compositions of the Acrs, association of acr genes in virus genomes with genes encoding helix-turn-helix proteins that regulate Acr expression (Acr-associated proteins, Aca), and presence of self-targeting CRISPR spacers in bacterial and archaeal genomes containing Acr-encoding proviruses. Productive approaches for Acr prediction also involve genome comparison of closely related viruses, of which one is resistant and the other one is sensitive to a particular CRISPR variant, and "guilt by association" whereby genes adjacent to a homolog of a known Aca are identified as candidate Acrs. The distinctive features of Acrs are employed for Acr prediction both by developing dedicated search algorithms and through machine learning. New approaches will be needed to identify novel types of Acrs that are likely to exist.

The assembly of an HIV-1 particle begins with the construction of a spherical lattice composed of hexamer subunits of the Gag polyprotein. The cellular metabolite inositol hexakisphosphate (IP6) binds and stabilizes the immature Gag lattice via an interaction with the six-helix bundle (6HB), a crucial structural feature of Gag hexamers that modulates both virus assembly and infectivity. The 6HB must be stable enough to promote immature Gag lattice formation, but also flexible enough to be accessible to the viral protease, which cleaves the 6HB during particle maturation. 6HB cleavage liberates the capsid (CA) domain of Gag from the adjacent spacer peptide 1 (SP1) and IP6 from its binding site. This pool of IP6 molecules then promotes the assembly of CA into the mature conical capsid that is required for infection. Depletion of IP6 in virus-producer cells results in severe defects in assembly and infectivity of wild-type (WT) virions. Here we show that in an SP1 double mutant (M4L/T8I) with a hyperstable 6HB, IP6 can block virion infectivity by preventing CA-SP1 processing. Thus, depletion of IP6 in virus-producer cells markedly increases M4L/T8I CA-SP1 processing and infectivity. We also show that the introduction of the M4L/T8I mutations partially rescues the assembly and infectivity defects induced by IP6 depletion on WT virions, likely by increasing the affinity of the immature lattice for limiting IP6. These findings reinforce the importance of the 6HB in virus assembly, maturation, and infection and highlight the ability of IP6 to modulate 6HB stability.