Bridging the maytansine and vinca sites: Cryptophycins target ß-tubulin's T5-loop.

Cryptophycins are microtubule-targeting agents (MTAs) that belong to the most potent antimitotic compounds known to date; however, their exact molecular mechanism of action remains unclear. Here, we present the 2.2 Å resolution X-ray crystal structure of a potent cryptophycin derivative bound to the αß-tubulin heterodimer. The structure addresses conformational issues present in a previous 3.3 Å resolution cryo-electron microscopy structure of cryptophycin-52 bound to the maytansine site of ß-tubulin. It further provides atomic details on interactions of cryptophycins, which had not been described previously, including ones that are in line with structure-activity relationship studies. Interestingly, we discovered a second cryptophycin-binding site that involves the T5-loop of ß-tubulin, a critical secondary structure element involved in the exchange of the guanosine nucleotide and in the formation of longitudinal tubulin contacts in microtubules. Cryptophycins are the first natural ligands found to bind to this new "ßT5-loop site" that bridges the maytansine and vinca sites. Our results offer unique avenues to rationally design novel MTAs with the capacity to modulate T5-loop dynamics and to simultaneously engage multiple ß-tubulin binding sites.

Filling of a water-free void explains the allosteric regulation of the ß1-adrenergic receptor by cholesterol.

Recent high-pressure NMR results indicate that the preactive conformation of the ß1-adrenergic receptor (ß1AR) harbours completely empty cavities of ~100 Å3 volume, which disappear in the active conformation of the receptor. Here we have localized these cavities using X-ray crystallography of xenon-derivatized ß1AR crystals. One of the cavities is in direct contact with the cholesterol-binding pocket. Solution NMR shows that addition of the cholesterol analogue cholesteryl hemisuccinate impedes the formation of the active conformation of detergent-solubilized ß1AR by blocking conserved G protein-coupled receptor microswitches, concomitant with an affinity reduction of both isoprenaline and G protein-mimicking nanobody Nb80 for ß1AR detected by isothermal titration calorimetry. This wedge-like action explains the function of cholesterol as a negative allosteric modulator of ß1AR. A detailed understanding of G protein-coupled receptor regulation by cholesterol by filling of a dry void and the easy scouting for such voids by xenon may provide new routes for the development of allosteric drugs.

Chimeric single α-helical domains as rigid fusion protein connections for protein nanotechnology and structural biology.

Chimeric fusion proteins are essential tools for protein nanotechnology. Non-optimized protein-protein connections are usually flexible and therefore unsuitable as structural building blocks. Here we show that the ER/K motif, a single α-helical domain (SAH), can be seamlessly fused to terminal helices of proteins, forming an extended, partially free-standing rigid helix. This enables the connection of two domains at a defined distance and orientation. We designed three constructs termed YFPnano, T4Lnano, and MoStoNano. Analysis of experimentally determined structures and molecular dynamics simulations reveals a certain degree of plasticity in the connections that allows the adaptation to crystal contact opportunities. Our data show that SAHs can be stably integrated into designed structural elements, enabling new possibilities for protein nanotechnology, for example, to improve the exposure of epitopes on nanoparticles (structural vaccinology), to engineer crystal contacts with minimal impact on construct flexibility (for the study of protein dynamics), and to design novel biomaterials.

Poly-l-lysine Glycoconjugates Inhibit DC-SIGN-mediated Attachment of Pandemic Viruses.

The C-type lectin receptor DC-SIGN mediates interactions with envelope glycoproteins of many viruses such as SARS-CoV-2, ebola, and HIV and contributes to virus internalization and dissemination. In the context of the recent SARS-CoV-2 pandemic, involvement of DC-SIGN has been linked to severe cases of COVID-19. Inhibition of the interaction between DC-SIGN and viral glycoproteins has the potential to generate broad spectrum antiviral agents. Here, we demonstrate that mannose-functionalized poly-l-lysine glycoconjugates efficiently inhibit the attachment of viral glycoproteins to DC-SIGN-presenting cells with picomolar affinity. Treatment of these cells leads to prolonged receptor internalization and inhibition of virus binding for up to 6 h. Furthermore, the polymers are fully bio-compatible and readily cleared by target cells. The thermodynamic analysis of the multivalent interactions reveals enhanced enthalpy-driven affinities and promising perspectives for the future development of multivalent therapeutics.

A Familiar Protein-Ligand Interaction Revisited with Multiple Methods.

The interaction of hen egg white lysozyme with the trisaccharide tri-N-acetyl glucosamine has been well-characterized by biophysical methods and structural biology. In this chapter, we present a series of experiments designed to detect and quantify that interaction using several commonly available biophysical methods: thermal shift assay, fluorescence intensity, microscale thermophoresis, isothermal titration calorimetry, and surface plasmon resonance.These experiments have been used for teaching and troubleshooting in a core facility. By taking a set of representative data from several years of practical courses, we are able to demonstrate the robustness of the protocols, calculate confidence intervals for the dissociation constant from each method, and illustrate the degree of consistency between those methods when applied to a simple system in a single location by different experimenters.

A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death.

BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionizing radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.

Regulation of chaperone function by coupled folding and oligomerization.

The homotrimeric molecular chaperone Skp of Gram-negative bacteria facilitates the transport of outer membrane proteins across the periplasm. It has been unclear how its activity is modulated during its functional cycle. Here, we report an atomic-resolution characterization of the Escherichia coli Skp monomer-trimer transition. We find that the monomeric state of Skp is intrinsically disordered and that formation of the oligomerization interface initiates folding of the α-helical coiled-coil arms via a unique "stapling" mechanism, resulting in the formation of active trimeric Skp. Native client proteins contact all three Skp subunits simultaneously, and accordingly, their binding shifts the Skp population toward the active trimer. This activation mechanism is shown to be essential for Salmonella fitness in a mouse infection model. The coupled mechanism is a unique example of how an ATP-independent chaperone can modulate its activity as a function of the presence of client proteins.

A high-resolution description of ß1-adrenergic receptor functional dynamics and allosteric coupling from backbone NMR.

Signal transmission and regulation of G-protein-coupled receptors (GPCRs) by extra- and intracellular ligands occurs via modulation of complex conformational equilibria, but their exact kinetic details and underlying atomic mechanisms are unknown. Here we quantified these dynamic equilibria in the ß1-adrenergic receptor in its apo form and seven ligand complexes using 1H/15N NMR spectroscopy. We observe three major exchanging conformations: an inactive conformation (Ci), a preactive conformation (Cp) and an active conformation (Ca), which becomes fully populated in a ternary complex with a G protein mimicking nanobody. The Ci ↔ Cp exchange occurs on the microsecond scale, the Cp ↔ Ca exchange is slower than ~5 ms and only occurs in the presence of two highly conserved tyrosines (Y5.58, Y7.53), which stabilize the active conformation of TM6. The Cp→Ca chemical shift changes indicate a pivoting motion of the entire TM6 that couples the effector site to the orthosteric ligand pocket.

Homodimerization of coronin A through the C-terminal coiled-coil domain is essential for multicellular differentiation of Dictyostelium discoideum.

Coronin proteins are widely expressed among eukaryotic organisms. Most coronins consist of a WD-repeat domain followed by a C-terminal coiled coil. Dictyostelium discoideum expresses a single short coronin coronin A, which has been implicated in both actin modulation and multicellular differentiation. Whether coronin A's coiled coil is important for functionality, as well as the oligomeric state of coronin A is not known. Here, we show that the coiled-coil domain in Dictyostelium coronin A functions in homodimerization, is dispensable for coronin A stability and localization but essential for multicellular differentiation. These results allow a better understanding of the role for the coiled-coil domain of coronin A in oligomerization and demonstrate that its presence is essential for multicellular differentiation.

The electrostatic core of the outer membrane protein X from E. coli.

Electrostatic side chain contacts can contribute substantial interaction energy terms to the stability of proteins. The impact of electrostatic interactions on the structure and architecture of outer membrane proteins is however not well studied compared to soluble proteins. Here, we report the results of a systematic study of all charged side chains of the E. coli outer membrane protein X (OmpX). The data identify three distinct salt-bridge clusters in the core of OmpX that contribute significantly to protein stability in dodecylphosphocholine detergent micelles. The three clusters form an "electrostatic core" of the membrane protein OmpX, corresponding in its architectural role to the hydrophobic core of soluble proteins. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.

Author Correction: Chimeric peptidomimetic antibiotics against Gram-negative bacteria.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Chimeric peptidomimetic antibiotics against Gram-negative bacteria.

There is an urgent need for new antibiotics against Gram-negative pathogens that are resistant to carbapenem and third-generation cephalosporins, against which antibiotics of last resort have lost most of their efficacy. Here we describe a class of synthetic antibiotics inspired by scaffolds derived from natural products. These chimeric antibiotics contain a ß-hairpin peptide macrocycle linked to the macrocycle found in the polymyxin and colistin family of natural products. They are bactericidal and have a mechanism of action that involves binding to both lipopolysaccharide and the main component (BamA) of the ß-barrel folding complex (BAM) that is required for the folding and insertion of ß-barrel proteins into the outer membrane of Gram-negative bacteria. Extensively optimized derivatives show potent activity against multidrug-resistant pathogens, including all of the Gram-negative members of the ESKAPE pathogens1. These derivatives also show favourable drug properties and overcome colistin resistance, both in vitro and in vivo. The lead candidate is currently in preclinical toxicology studies that-if successful-will allow progress into clinical studies that have the potential to address life-threatening infections by the Gram-negative pathogens, and thus to resolve a considerable unmet medical need.

A DNA-Encoded Chemical Library Incorporating Elements of Natural Macrocycles.

Here we show a seven-step chemical synthesis of a DNA-encoded macrocycle library (DEML) on DNA. Inspired by polyketide and mixed peptide-polyketide natural products, the library was designed to incorporate rich backbone diversity. Achieving this diversity, however, comes at the cost of the custom synthesis of bifunctional building block libraries. This study outlines the importance of careful retrosynthetic design in DNA-encoded libraries, while revealing areas where new DNA synthetic methods are needed.

Two-State Folding of the Outer Membrane Protein X into a Lipid Bilayer Membrane.

Folding and insertion of ß-barrel membrane proteins into native membranes is efficiently catalyzed by ß-barrel assembly machineries. Understanding this catalysis requires a detailed description of the corresponding uncatalyzed folding mechanisms, which however have so far remained largely unclear. Herein, the folding and membrane insertion of the E. coli outer membrane protein X (OmpX) into 1,2-didecanoyl-sn-glycero-3-phosphocholine (PC10:0) membranes is resolved at the atomic level. By combining four different experimental techniques, global folding kinetics were correlated with global and local hydrogen bond-formation kinetics. Under a well-defined reaction condition, these processes follow single-exponential velocity laws, with rate constants identical within experimental error. The data thus establish, at atomic resolution, that OmpX folds and inserts into the lipid bilayer of PC10:0 liposomes by a two-state mechanism.

KinITC-One Method Supports both Thermodynamic and Kinetic SARs as Exemplified on FimH Antagonists.

Affinity data, such as dissociation constants (KD ) or inhibitory concentrations (IC50 ), are widely used in drug discovery. However, these parameters describe an equilibrium state, which is often not established in vivo due to pharmacokinetic effects and they are therefore not necessarily sufficient for evaluating drug efficacy. More accurate indicators for pharmacological activity are the kinetics of binding processes, as they shed light on the rate of formation of protein-ligand complexes and their half-life. Nonetheless, although highly desirable for medicinal chemistry programs, studies on structure-kinetic relationships (SKR) are still rare. With the recently introduced analytical tool kinITC this situation may change, since not only thermodynamic but also kinetic information of the binding process can be deduced from isothermal titration calorimetry (ITC) experiments. Using kinITC, ITC data of 29 mannosides binding to the bacterial adhesin FimH were re-analyzed to make their binding kinetics accessible. To validate these kinetic data, surface plasmon resonance (SPR) experiments were conducted. The kinetic analysis by kinITC revealed that the nanomolar affinities of the FimH antagonists arise from both (i) an optimized interaction between protein and ligand in the bound state (reduced off-rate constant koff ) and (ii) a stabilization of the transition state or a destabilization of the unbound state (increased on-rate constant kon ). Based on congeneric ligand modifications and structural input from co-crystal structures, a strong relationship between the formed hydrogen-bond network and koff could be concluded, whereas electrostatic interactions and conformational restrictions upon binding were found to have mainly an impact on kon .

Mutant Variants of the Substrate-Binding Protein DppA from Escherichia coli Enhance Growth on Nonstandard γ-Glutamyl Amide-Containing Peptides.

The import of nonnatural molecules is a recurring problem in fundamental and applied aspects of microbiology. The dipeptide permease (Dpp) of Escherichia coli is an ABC-type multicomponent transporter system located in the cytoplasmic membrane, which is capable of transporting a wide range of di- and tripeptides with structurally and chemically diverse amino acid side chains into the cell. Given this low degree of specificity, Dpp was previously used as an entry gate to deliver natural and nonnatural cargo molecules into the cell by attaching them to amino acid side chains of peptides, in particular, the γ-carboxyl group of glutamate residues. However, the binding affinity of the substrate-binding protein dipeptide permease A (DppA), which is responsible for the initial binding of peptides in the periplasmic space, is significantly higher for peptides consisting of standard amino acids than for peptides containing side-chain modifications. Here, we used adaptive laboratory evolution to identify strains that utilize dipeptides containing γ-substituted glutamate residues more efficiently and linked this phenotype to different mutations in DppA. In vitro characterization of these mutants by thermal denaturation midpoint shift assays and isothermal titration calorimetry revealed significantly higher binding affinities of these variants toward peptides containing γ-glutamyl amides, presumably resulting in improved uptake and therefore faster growth in media supplemented with these nonstandard peptides.IMPORTANCE Fundamental and synthetic biology frequently suffer from insufficient delivery of unnatural building blocks or substrates for metabolic pathways into bacterial cells. The use of peptide-based transport vectors represents an established strategy to enable the uptake of such molecules as a cargo. We expand the scope of peptide-based uptake and characterize in detail the obtained DppA mutant variants. Furthermore, we highlight the potential of adaptive laboratory evolution to identify beneficial insertion mutations that are unlikely to be identified with existing directed evolution strategies.

CDKL Family Kinases Have Evolved Distinct Structural Features and Ciliary Function.

Various kinases, including a cyclin-dependent kinase (CDK) family member, regulate the growth and functions of primary cilia, which perform essential roles in signaling and development. Neurological disorders linked to CDK-Like (CDKL) proteins suggest that these underexplored kinases may have similar functions. Here, we present the crystal structures of human CDKL1, CDKL2, CDKL3, and CDKL5, revealing their evolutionary divergence from CDK and mitogen-activated protein kinases (MAPKs), including an unusual ?J helix important for CDKL2 and CDKL3 activity. C. elegans CDKL-1, most closely related to CDKL1-4 and localized to neuronal cilia transition zones, modulates cilium length; this depends on its kinase activity and ?J helix-containing C terminus. Human CDKL5, linked to Rett syndrome, also localizes to cilia, and it impairs ciliogenesis when overexpressed. CDKL5 patient mutations modeled in CDKL-1 cause localization and/or cilium length defects. Together, our studies establish a disease model system suggesting cilium length defects as a pathomechanism for neurological disorders, including epilepsy.

The dynamic dimer structure of the chaperone Trigger Factor.

The chaperone Trigger Factor (TF) from Escherichia coli forms a dimer at cellular concentrations. While the monomer structure of TF is well known, the spatial arrangement of this dimeric chaperone storage form has remained unclear. Here, we determine its structure by a combination of high-resolution NMR spectroscopy and biophysical methods. TF forms a symmetric head-to-tail dimer, where the ribosome binding domain is in contact with the substrate binding domain, while the peptidyl-prolyl isomerase domain contributes only slightly to the dimer affinity. The dimer structure is highly dynamic, with the two ribosome binding domains populating a conformational ensemble in the center. These dynamics result from intermolecular in trans interactions of the TF client-binding site with the ribosome binding domain, which is conformationally frustrated in the absence of the ribosome. The avidity in the dimer structure explains how the dimeric state of TF can be monomerized also by weakly interacting clients.

Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16.

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has a unique consensus substrate phosphorylation motif compared with conventional CDKs. To elucidate the structure and inhibitor-binding properties of this atypical CDK, we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multitargeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity, suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts.

Syrosingopine sensitizes cancer cells to killing by metformin.

We report that the anticancer activity of the widely used diabetic drug metformin is strongly potentiated by syrosingopine. Synthetic lethality elicited by combining the two drugs is synergistic and specific to transformed cells. This effect is unrelated to syrosingopine's known role as an inhibitor of the vesicular monoamine transporters. Syrosingopine binds to the glycolytic enzyme α-enolase in vitro, and the expression of the γ-enolase isoform correlates with nonresponsiveness to the drug combination. Syrosingopine sensitized cancer cells to metformin and its more potent derivative phenformin far below the individual toxic threshold of each compound. Thus, combining syrosingopine and codrugs is a promising therapeutic strategy for clinical application for the treatment of cancer.