High resolution analysis of proteolytic substrate processing.

Members of the widely conserved HtrA family of serine proteases are involved in multiple aspects of protein quality control. In this context, they have been shown to efficiently degrade misfolded proteins or protein fragments. However, recent reports suggest that folded proteins can also be native substrates. To gain a deeper understanding of how folded proteins are initially processed and subsequently degraded into short peptides by human HTRA1, we established an integrated and quantitative approach using time-resolved mass spectrometry, circular dichroism spectroscopy and bioinformatics. The resulting data provide high-resolution information on up to 178 individual proteolytic sites within folded ANXA1 (consisting of 346 amino acids), the relative frequency of cuts at each proteolytic site, the preferences of the protease for the amino acid sequence surrounding the scissile bond, as well as the degrees of sequential structural relaxation and unfolding of the substrate that occur during progressive degradation. Our workflow provides precise molecular insights into protease-substrate interactions, which could be readily adapted to address other post-translational modifications such as phosphorylation in dynamic protein complexes.

An allosteric HTRA1-calpain 2 complex with restricted activation profile.

SignificanceClassic serine proteases are synthesized as inactive precursors that are proteolytically processed, resulting in irreversible activation. We report an alternative and reversible mechanism of activation that is executed by an inactive protease. This mechanism involves a protein complex between the serine protease HTRA1 and the cysteine protease calpain 2. Surprisingly, activation is restricted as it improves the proteolysis of soluble tau protein but not the dissociation and degradation of its amyloid fibrils, a task that free HTRA1 is efficiently performing. These data exemplify a challenge for protein quality control proteases in the clearing of pathogenic fibrils and suggest a potential for unexpected side effects of chemical modulators targeting PDZ or other domains located at a distance to the active site.

Supramolecular Mechanism of Viral Envelope Disruption by Molecular Tweezers.

Broad-spectrum antivirals are powerful weapons against dangerous viruses where no specific therapy exists, as in the case of the ongoing SARS-CoV-2 pandemic. We discovered that a lysine- and arginine-specific supramolecular ligand (CLR01) destroys enveloped viruses, including HIV, Ebola, and Zika virus, and remodels amyloid fibrils in semen that promote viral infection. Yet, it is unknown how CLR01 exerts these two distinct therapeutic activities. Here, we delineate a novel mechanism of antiviral activity by studying the activity of tweezer variants: the "phosphate tweezer" CLR01, a "carboxylate tweezer" CLR05, and a "phosphate clip" PC. Lysine complexation inside the tweezer cavity is needed to antagonize amyloidogenesis and is only achieved by CLR01. Importantly, CLR01 and CLR05 but not PC form closed inclusion complexes with lipid head groups of viral membranes, thereby altering lipid orientation and increasing surface tension. This process disrupts viral envelopes and diminishes infectivity but leaves cellular membranes intact. Consequently, CLR01 and CLR05 display broad antiviral activity against all enveloped viruses tested, including herpesviruses, Measles virus, influenza, and SARS-CoV-2. Based on our mechanistic insights, we potentiated the antiviral, membrane-disrupting activity of CLR01 by introducing aliphatic ester arms into each phosphate group to act as lipid anchors that promote membrane targeting. The most potent ester modifications harbored unbranched C4 units, which engendered tweezers that were approximately one order of magnitude more effective than CLR01 and nontoxic. Thus, we establish the mechanistic basis of viral envelope disruption by specific tweezers and establish a new class of potential broad-spectrum antivirals with enhanced activity.

Utilities for Mass Spectrometry Analysis of Proteins (UMSAP): Fast post-processing of mass spectrometry data.

RATIONALE: Mass spectrometry (MS) is an invaluable tool for the analysis of proteins. However, the sheer amount of data generated in MS studies demands dedicated data-processing tools that are efficient and require minimal user intervention. METHODS: Utilities for Mass Spectrometry Analysis of Proteins (UMSAP) is a graphical user interface designed for efficient post-processing of MS result files. The software is written in Tcl/Tk and can be used in Windows, OS X or Linux. No third party programs or libraries are required. Currently, UMSAP can process data obtained from proteolytic degradation experiments and generates graphical outputs allowing a straightforward interpretation of statistically relevant results. RESULTS: UMSAP is used here to analyze the proteolytic degradation of glycerophosphoryl diester phosphodiesterase GlpQ by the protein quality control protease DegP. Mass spectrometry was used to monitor proteolysis over time in the absence and presence of a peptidic allosteric activator of DegP. The software's output clearly shows the increased proteolytic activity of DegP in the presence of the activating peptide, identifies statistically significant products of the proteolysis and offers valuable insights into substrate specificity. CONCLUSIONS: Utilities for Mass Spectrometry Analysis of Proteins is an open-source software designed for efficient post-processing of large datasets obtained by MS analyses of proteins. In addition, the modular architecture of the software allows easy incorporation of new modules to analyze various experimental mass spectrometry setups.

The Molecular Tweezer CLR01 Stabilizes a Disordered Protein-Protein Interface.

Protein regions that are involved in protein-protein interactions (PPIs) very often display a high degree of intrinsic disorder, which is reduced during the recognition process. A prime example is binding of the rigid 14-3-3 adapter proteins to their numerous partner proteins, whose recognition motifs undergo an extensive disorder-to-order transition. In this context, it is highly desirable to control this entropy-costly process using tailored stabilizing agents. This study reveals how the molecular tweezer CLR01 tunes the 14-3-3/Cdc25CpS216 protein-protein interaction. Protein crystallography, biophysical affinity determination and biomolecular simulations unanimously deliver a remarkable finding: a supramolecular "Janus" ligand can bind simultaneously to a flexible peptidic PPI recognition motif and to a well-structured adapter protein. This binding fills a gap in the protein-protein interface, "freezes" one of the conformational states of the intrinsically disordered Cdc25C protein partner and enhances the apparent affinity of the interaction. This is the first structural and functional proof of a supramolecular ligand targeting a PPI interface and stabilizing the binding of an intrinsically disordered recognition motif to a rigid partner protein.

Inhibition of Huntingtin Exon-1 Aggregation by the Molecular Tweezer CLR01.

Huntington's disease is a neurodegenerative disorder associated with the expansion of the polyglutamine tract in the exon-1 domain of the huntingtin protein (htte1). Above a threshold of 37 glutamine residues, htte1 starts to aggregate in a nucleation-dependent manner. A 17-residue N-terminal fragment of htte1 (N17) has been suggested to play a crucial role in modulating the aggregation propensity and toxicity of htte1. Here we identify N17 as a potential target for novel therapeutic intervention using the molecular tweezer CLR01. A combination of biochemical experiments and computer simulations shows that binding of CLR01 induces structural rearrangements within the htte1 monomer and inhibits htte1 aggregation, underpinning the key role of N17 in modulating htte1 toxicity.

Predicted incorporation of non-native substrates by a polyketide synthase yields bioactive natural product derivatives.

The polyether ionophore monensin is biosynthesized by a polyketide synthase that delivers a mixture of monensins A and B by the incorporation of ethyl- or methyl-malonyl-CoA at its fifth module. Here we present the first computational model of the fifth acyltransferase domain (AT5mon ) of this polyketide synthase, thus affording an investigation of the basis of the relaxed specificity in AT5mon , insights into the activation for the nucleophilic attack on the substrate, and prediction of the incorporation of synthetic malonic acid building blocks by this enzyme. Our predictions are supported by experimental studies, including the isolation of a predicted derivative of the monensin precursor premonensin. The incorporation of non-native building blocks was found to alter the ratio of premonensins A and B. The bioactivity of the natural product derivatives was investigated and revealed binding to prenyl-binding protein. We thus show the potential of engineered biosynthetic polyketides as a source of ligands for biological macromolecules.

The benzylperoxyl radical as a source of hydroxyl and phenyl radicals.

The benzyl radical (1) is a key intermediate in the combustion and tropospheric oxidation of toluene. Because of its relevance, the reaction of 1 with molecular oxygen was investigated by matrix-isolation IR and EPR spectroscopy as well as computational methods. The primary reaction product of 1 and O2 is the benzylperoxyl radical (2), which exists in several conformers that can easily interconvert even at cryogenic temperatures. Photolysis of radical 2 at 365 nm results in a formal [1,3]-H migration and subsequent cleavage of the O-O bond to produce a hydrogen-bonded complex between the hydroxyl radical and benzaldehyde (4). Prolonged photolysis produces the benzoyl radical (5) and water, which finally yield the phenyl radical (7), CO, and H2O. Thus, via a sequence of exothermic reactions 1 is transformed into radicals of even higher reactivity, such as OH and 7. Our results have implications for the development of models for the highly complicated process of combustion of aromatic compounds.

Solvent-induced conformational changes in cyclic peptides: a vibrational circular dichroism study.

The three-dimensional structure of a peptide is strongly influenced by its solvent environment. In the present study, we study three cyclic tetrapeptides which serve as model peptides for ß-turns. They are of the general structure cyclo(Boc-Cys-Pro-X-Cys-OMe) with the amino acid X being either glycine (1), or L- or D-leucine (L- or D-2). Using vibrational circular dichroism (VCD) spectroscopy, we confirm previous NMR results which showed that D-2 adopts predominantly a ßII turn structure in apolar and polar solvents. Our results for L-2 indicate a preference for a ßI structure over ßII. With increasing solvent polarity, the preference for 1 is shifted from ßII towards ßI. This conformational change goes along with the breaking of an intramolecular hydrogen bond which stabilizes the ßII conformation. Instead, a hydrogen bond with a solvent molecule can stabilize the ßI turn conformation.

Rational correction of pathogenic conformational defects in HTRA1.

Loss-of-function mutations in the homotrimeric serine protease HTRA1 cause cerebral vasculopathy. Here, we establish independent approaches to achieve the functional correction of trimer assembly defects. Focusing on the prototypical R274Q mutation, we identify an HTRA1 variant that promotes trimer formation thus restoring enzymatic activity in vitro. Genetic experiments in Htra1R274Q mice further demonstrate that expression of this protein-based corrector in trans is sufficient to stabilize HtrA1-R274Q and restore the proteomic signature of the brain vasculature. An alternative approach employs supramolecular chemical ligands that shift the monomer-trimer equilibrium towards proteolytically active trimers. Moreover, we identify a peptidic ligand that activates HTRA1 monomers. Our findings open perspectives for tailored protein repair strategies.

Interactions of aromatic radicals with water.

The interactions of the benzyl radical (1), the anilinyl radical (2), and the phenoxyl radical (3) with water are investigated using density functional theory (DFT). In addition, we report dispersion-corrected DFT-D molecular dynamics simulations on these three systems and a matrix isolation study on 1-water. The radicals 1-3 form an interesting series with the number of lone pairs increasing from none to two. The anilinyl and benzyl radicals can act as Lewis base through their unpaired electrons, the lone pairs of the heteroatoms, or the doubly occupied π orbitals of the aromatic system. Matrix isolation experiments provide evidence for the formation of a π complex between 1 and water. By combining computational and experimental techniques we identify the possible interactions between the aromatic radicals 1-3 and water, predict the structure and vibrational spectra of the resulting complexes, and analyze the effects of substitution and temperature.

Molecular tweezers with varying anions: a comparative study.

Selective binding of the phosphate-substituted molecular tweezer 1a to protein lysine residues was suggested to explain the inhibition of certain enzymes and the aberrant aggregation of amyloid petide Aß42 or α-synuclein, which are assumed to be responsible for Alzheimer's and Parkinson's disease, respectively. In this work we systematically investigated the binding of four water-soluble tweezers 1a-d (substituted by phosphate, methanephosphonate, sulfate, or O-methylenecarboxylate groups) to amino acids and peptides containing lysine or arginine residues by using fluorescence spectroscopy, NMR spectroscopy, and isothermal titration calorimetry (ITC). The comparison of the experimental results with theoretical data obtained by a combination of QM/MM and ab initio(1)H NMR shift calculations provides clear evidence that the tweezers 1a-c bind the amino acid or peptide guest molecules by threading the lysine or arginine side chain through the tweezers' cavity, whereas in the case of 1d the guest molecule is preferentially positioned outside the tweezer's cavity. Attractive ionic, CH-π, and hydrophobic interactions are here the major binding forces. The combination of experiment and theory provides deep insight into the host-guest binding modes, a prerequisite to understanding the exciting influence of these tweezers on the aggregation of proteins and the activity of enzymes.

The role of DNA nanostructures in the catalytic properties of an allosterically regulated protease.

DNA-scaffolded enzymes typically show altered kinetic properties; however, the mechanism behind this phenomenon is still poorly understood. We address this question using thrombin, a model of allosterically regulated serine proteases, encaged into DNA origami cavities with distinct structural and electrostatic features. We compare the hydrolysis of substrates that differ only in their net charge due to a terminal residue far from the cleavage site and presumably involved in the allosteric activation of thrombin. Our data show that the reaction rate is affected by DNA/substrate electrostatic interactions, proportionally to the degree of DNA/enzyme tethering. For substrates of opposite net charge, this leads to an inversion of the catalytic response of the DNA-scaffolded thrombin when compared to its freely diffusing counterpart. Hence, by altering the electrostatic environment nearby the encaged enzyme, DNA nanostructures interfere with charge-dependent mechanisms of enzyme-substrate recognition and may offer an alternative tool to regulate allosteric processes through spatial confinement.

Adoption of a Turn Conformation Drives the Binding Affinity of p53 C-Terminal Domain Peptides to 14-3-3σ.

The interaction between the adapter protein 14-3-3σ and transcription factor p53 is important for preserving the tumor-suppressor functions of p53 in the cell. A phosphorylated motif within the C-terminal domain (CTD) of p53 is key for binding to the amphipathic groove of 14-3-3. This motif is unique among 14-3-3 binding partners, and the precise dynamics of the interaction is not yet fully understood. Here, we investigate this interaction at the molecular level by analyzing the binding of different length p53 CTD peptides to 14-3-3σ using ITC, SPR, NMR, and MD simulations. We observed that the propensity of the p53 peptide to adopt turn-like conformation plays an important role in the binding to the 14-3-3σ protein. Our study contributes to elucidate the molecular mechanism of the 14-3-3-p53 binding and provides useful insight into how conformation properties of a ligand influence protein binding.

Theoretical study of imidazole...NO complexes.

A set of weak complexes between imidazole (Imi) and nitric oxide (NO) were calculated with UMP2/6-31++G(d,p) and UMP2/6-311++G(2d,2p) levels of theory. Planar and nonplanar geometries were considered. Complexes of NO and protonated imidazole (ImiH(+)) were also studied due to the biological important effect of Imi protonation. It was found that ring protonation increases the stability of planar complexes and does not affect significantly nonplanar complexes. The Z-H...XY (Z = C, N and X, Y = N, O) interactions resulted as hydrogen bonds according to Koch and Popelier criteria based on AIM theory. Charge transfer was also found very important for complex stabilization within our theoretical framework. Planar NO...ImiH(+) complexes are more stable than those obtained with neutral Imi.

Chemical Validation of DegS As a Target for the Development of Antibiotics with a Novel Mode of Action.

Despite the availability of hundreds of antibiotic drugs, infectious diseases continue to remain one of the most notorious health issues. In addition, the disparity between the spread of multidrug-resistant pathogens and the development of novel classes of antibiotics exemplify an important unmet medical need that can only be addressed by identifying novel targets. Herein we demonstrate, by the development of the first in vivo active DegS inhibitors based on a pyrazolo[1,5-a]-1,3,5-triazine scaffold, that the serine protease DegS and the cell envelope stress-response pathway σE represent a target for generating antibiotics with a novel mode of action. Moreover, DegS inhibition is synergistic with well-established membrane-perturbing antibiotics, thereby opening promising avenues for rational antibiotic drug design.

Mechanism of Inhibition of Beta Amyloid Toxicity by Supramolecular Tweezers.

The Aß1-42 dimer is the smallest oligomer of the 42-residue Aß peptide (Aß1-42), which is involved in Alzheimer's disease. The molecular tweezer CLR01 is a synthetic molecule that selectively binds lysine and arginine residues to inhibit toxic aggregation of amyloidogenic peptides. Here, we performed replica exchange molecular dynamics simulations of Aß1-42 in explicit water to study, at the molecular level, the effect of CLR01 binding to the lysine and arginine residues in the dimer. We found that CLR01 molecules encapsulate both lysine residues of each Aß1-42 monomer while only establishing labile interactions with the arginine residues. This encapsulation leads to the noncovalent disruption of inter- and intramolecular interactions involving the monomers. Additionally, the total ß-sheet content in the Aß1-42 dimer decreases because of this binding. With CLR03, a negative control molecule that shares the charged core of CLR01 but does not form inclusion complexes, Aß1-42 dimer formation is observed, similar to the reference system without ligands. Our work allows establishing a molecular mechanism for the modulation of protein-protein interactions in Aß1-42 by CLR01. This mechanism is characterized by an aggregation pathway driven by the encapsulation of lysine residues as well as by the secondary interactions of tweezers with the peptide units and with other CLR01 molecules.

Self-assembly of Mutant Huntingtin Exon-1 Fragments into Large Complex Fibrillar Structures Involves Nucleated Branching.

Huntingtin (HTT) fragments with extended polyglutamine tracts self-assemble into amyloid-like fibrillar aggregates. Elucidating the fibril formation mechanism is critical for understanding Huntington's disease pathology and for developing novel therapeutic strategies. Here, we performed systematic experimental and theoretical studies to examine the self-assembly of an aggregation-prone N-terminal HTT exon-1 fragment with 49 glutamines (Ex1Q49). Using high-resolution imaging techniques such as electron microscopy and atomic force microscopy, we show that Ex1Q49 fragments in cell-free assays spontaneously convert into large, highly complex bundles of amyloid fibrils with multiple ends and fibril branching points. Furthermore, we present experimental evidence that two nucleation mechanisms control spontaneous Ex1Q49 fibrillogenesis: (1) a relatively slow primary fibril-independent nucleation process, which involves the spontaneous formation of aggregation-competent fibrillary structures, and (2) a fast secondary fibril-dependent nucleation process, which involves nucleated branching and promotes the rapid assembly of highly complex fibril bundles with multiple ends. The proposed aggregation mechanism is supported by studies with the small molecule O4, which perturbs early events in the aggregation cascade and delays Ex1Q49 fibril assembly, comprehensive mathematical and computational modeling studies, and seeding experiments with small, preformed fibrillar Ex1Q49 aggregates that promote the assembly of amyloid fibrils. Together, our results suggest that nucleated branching in vitro plays a critical role in the formation of complex fibrillar HTT exon-1 aggregates with multiple ends.

Tailored protein encapsulation into a DNA host using geometrically organized supramolecular interactions.

The self-organizational properties of DNA have been used to realize synthetic hosts for protein encapsulation. However, current strategies of DNA-protein conjugation still limit true emulation of natural host-guest systems, whose formation relies on non-covalent bonds between geometrically matching interfaces. Here we report one of the largest DNA-protein complexes of semisynthetic origin held in place exclusively by spatially defined supramolecular interactions. Our approach is based on the decoration of the inner surface of a DNA origami hollow structure with multiple ligands converging to their corresponding binding sites on the protein surface with programmable symmetry and range-of-action. Our results demonstrate specific host-guest recognition in a 1:1 stoichiometry and selectivity for the guest whose size guarantees sufficient molecular diffusion preserving short intermolecular distances. DNA nanocontainers can be thus rationally designed to trap single guest molecules in their native form, mimicking natural strategies of molecular recognition and anticipating a new method of protein caging.

Quantitative interaction mapping reveals an extended UBX domain in ASPL that disrupts functional p97 hexamers.

Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity.