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Proteins capable of switching between distinct active states in response to biochemical cues are ideal for sensing and controlling biological processes. Activatable CRISPR‐Cas systems are significant in precise genetic manipulation and sensitive molecular diagnostics, yet directly controlling Cas protein function remains challenging. Herein, we explore anti‐CRISPR (Acr) proteins as modules to create synthetic Cas protein switches (CasPSs) based on computational chemistry‐directed rational protein interface engineering. Guided by molecular fingerprint analysis, electrostatic potential mapping, and binding free energy calculations, we rationally engineer the molecular interaction interface between Cas12a and its cognate Acr proteins (AcrVA4 and AcrVA5) to generate a series of orthogonal protease‐responsive CasPSs. These CasPSs enable the conversion of specific proteolytic events into activation of Cas12a function with high switching ratios (up to 34.3‐fold). These advancements enable specific proteolysis‐inducible genome editing in mammalian cells and sensitive detection of viral protease activities during virus infection. This work provides a promising strategy for developing CRISPR‐Cas tools for controllable gene manipulation and regulation and clinical diagnostics. [ABSTRACT FROM AUTHOR]

The enzyme complex IκB kinase (IKK) is an essential activator of NF-κB signaling pathway involved in propagating the cellular response to inflammation. The complex contains two functional subunits IKKαand IKKβ, which are structurally conserved kinases and selective inhibition of them would result in distinct biological effects. However, most existing IKK inhibitors show moderate or high promiscuity for the two homologous kinases. Understanding of the molecular mechanism and biological implication underlying the specific interactions in IKK–ligand recognition is thus fundamentally important for the rational design of selective IKK inhibitors. In the current work, we integrated molecular docking, quantum mechanics/molecular mechanics calculation and Poisson–Boltzmann/surface area analysis to investigate the structural basis and energetic property of the selective binding of small-molecule ligands to IKKαand IKKβ. It was found that the selectivity is primarily determined by the size and topology difference in ATP-binding pocket of IKKαand IKKβkinase domains; bulky inhibitor molecules commonly have, respectively, low and appropriate affinities towards IKKαand IKKβ, and thus exhibit relatively high selectivity for IKKβover IKKα, whereas small ligands can only bind weakly to both the two kinases with low selectivity. In addition, the conformation, arrangement and distribution of residues in IKK pockets are also responsible for constituting the exquisite specificity of ligand binding to KKαand IKKβ. Next, a novel quantitative structure–selectivity relationship model was developed to characterize the relative contribution of each kinase residue to inhibitor selectivity and to predict the selectivity and specificity for a number of known IKK inhibitors. Results showed that the active-site residues contribute significantly to the selectivity by directly interacting with inhibitor ligands, while those protein portions far away from the kinase active sites may also play an important role in determining the selectivity through long-range non-bonded forces and indirect allosteric effect. [ABSTRACT FROM PUBLISHER]

Selective modulation of ligand-receptor interaction is essential in targeted therapy. In this study, we design an intelligent "scan and unlock" DNA automaton (SUDA) system to equip a native protein-ligand with cell-identity recognition and receptor-mediated signaling in a cell-type-specific manner. Using embedded DNA-based chemical reaction networks (CRNs) on the cell surface, SUDA scans and evaluates molecular profiles of cell-surface proteins via Boolean logic circuits. Therefore, it achieves cell-specific signal modulation by quickly unlocking the protein-ligand in proximity to the target cell-surface to activate its cognate receptor. As a proof of concept, we non-genetically engineered hepatic growth factor (HGF) with distinct logic SUDAs to elicit target cell-specific HGF signaling and wound healing behaviors in multiple heterogeneous cell types. Furthermore, the versatility of the SUDA strategy was shown by engineering tumor necrotic factor-α (TNFα) to induce programmed cell death of target cell subpopulations through cell-specific modulation of TNFR1 signaling., (© 2020 Wiley-VCH GmbH.)

A click-type protein-DNA conjugation, named as MnDDC (Mn 2+ -activated DCV-DNA conjunction), is presented, where DCV (rep protein of duck circovirus) and its target DNA work as the modular blocks to rapidly and effectively generate Mn 2+ -dependent and site-specific protein-DNA linkage. On the basis of MnDCC, a fluorescent Mn 2+ biosensor composed of DCV and a molecular beacon, was developed for rapid sensing of Mn 2+ within 2 min with nanomolar sensitivity. Using the proposed biosensor, not only analysis of Mn 2+ in real samples (e.g., serum and food), but also wash-free fluorescent imaging of Mn 2+ in extracellular environment and cytoplasm have been achieved. Moreover, the MnDDC-based sensor was proved to be a powerful tool for visualization of Mn 2+ during exploration of the associated cytotoxicity in living neural cells, which is helpful to reveal the cellular responses toward the disordered homeostasis of Mn 2+ in both extracellular and intracellular microenvironments.

The comprehensive understanding of the mechanisms underlying the interaction of cells with their membrane microenvironment is of great value for fundamental biological research; however, tracking biomolecules on cell surfaces with high temporal and spatial resolution remains a challenge. Herein, a modular strategy is presented for the construction of cell surface DNA-based sensors by engineering DNA motifs and synthetic cofactors. In this strategy, a stimuli-reactive organic molecule is employed as the cofactor for the DNA motif, and the self-assembly of them forms a FRET-based holo DNA-based sensor. With the use of the DNA-based sensors, the versatility of this modular strategy has been demonstrated in the ratiometric imaging of the cellular extrusion process of endogenous signaling molecules, including sulfur dioxide derivatives and nitric oxide., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The enzyme complex IκB kinase (IKK) is an essential activator of NF-κB signaling pathway involved in propagating the cellular response to inflammation. The complex contains two functional subunits IKKα and IKKβ, which are structurally conserved kinases and selective inhibition of them would result in distinct biological effects. However, most existing IKK inhibitors show moderate or high promiscuity for the two homologous kinases. Understanding of the molecular mechanism and biological implication underlying the specific interactions in IKK-ligand recognition is thus fundamentally important for the rational design of selective IKK inhibitors. In the current work, we integrated molecular docking, quantum mechanics/molecular mechanics calculation and Poisson-Boltzmann/surface area analysis to investigate the structural basis and energetic property of the selective binding of small-molecule ligands to IKKα and IKKβ. It was found that the selectivity is primarily determined by the size and topology difference in ATP-binding pocket of IKKα and IKKβ kinase domains; bulky inhibitor molecules commonly have, respectively, low and appropriate affinities towards IKKα and IKKβ, and thus exhibit relatively high selectivity for IKKβ over IKKα, whereas small ligands can only bind weakly to both the two kinases with low selectivity. In addition, the conformation, arrangement and distribution of residues in IKK pockets are also responsible for constituting the exquisite specificity of ligand binding to KKα and IKKβ. Next, a novel quantitative structure-selectivity relationship model was developed to characterize the relative contribution of each kinase residue to inhibitor selectivity and to predict the selectivity and specificity for a number of known IKK inhibitors. Results showed that the active-site residues contribute significantly to the selectivity by directly interacting with inhibitor ligands, while those protein portions far away from the kinase active sites may also play an important role in determining the selectivity through long-range non-bonded forces and indirect allosteric effect.