Multiple parameters shape the 3D chromatin structure of single nuclei

The spatial organization of chromatin at the scale of topologically associating domains (TADs) and below displays large cell-to-cell variations. Up until now, how this heterogeneity in chromatin conformation is shaped by chromatin condensation, TAD insulation, and transcription has remained mostly elusive. Here, we used Hi-M, a multiplexed DNA-FISH imaging technique providing developmental timing and transcriptional status, to show that the emergence of TADs at the ensemble level partially segregates the conformational space explored by single nuclei during the early development of Drosophila embryos. Surprisingly, a substantial fraction of nuclei displayed strong insulation even before TADs emerged. Moreover, active transcription within a TAD led to minor changes to the local inter- and intra-TAD chromatin conformation in single nuclei and only weakly affected insulation to the neighboring TAD. Overall, our results indicate that multiple parameters contribute to shaping the chromatin architecture of single nuclei at the TAD scale.

Redirecting RiPP biosynthetic enzymes to proteins and backbone-modified substrates

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are peptide-derived natural products that include the FDA-approved analgesic ziconotide1,2 as well as compounds with potent antibiotic, antiviral, and anticancer properties.3 RiPP enzymes known as cyclodehydratases and dehydrogenases represent an exceptionally well-studied enzyme class.3 These enzymes work together to catalyze intramolecular, interresidue condensation3,4 and aromatization reactions that install oxazoline/oxazole and thiazoline/thiazole heterocycles within ribosomally produced polypeptide chains. Here we show that the previously reported enzymes MicD-F and ArtGox accept backbone-modified monomers, including aramids and beta-amino acids, within leader-free polypeptides, even at positions immediately preceding or following the site of cyclization/dehydrogenation. The products are sequence-defined chemical polymers with multiple, diverse, non-alpha-amino acid subunits. We show further that MicD-F and ArtGox can install heterocyclic backbones within protein loops and linkers without disrupting the native tertiary fold. Calculations reveal the extent to which these heterocycles restrict conformational space; they also eliminate a peptide bond. Both features could improve the stability or add function to linker sequences now commonplace in emerging biotherapeutics. Moreover, as thiazoles and thiazoline heterocycles are replete in natural products,5,6,7 small molecule drugs,8,9 and peptide-mimetic therapeutics,10 their installation in protein-based biotherapeutics could improve or augment performance, activity, stability, and/or selectivity. This work represents a general strategy to expand the chemical diversity of the proteome beyond and in synergy with what can now be accomplished by expanding the genetic code.

In-Depth Molecular Dynamics Study of All Possible Chondroitin Sulfate Disaccharides Reveals Key Insight into Structural Heterogeneity and Dynamism

GAGs exhibit a high level of conformational and configurational diversity, which remains untapped in terms of the recognition and modulation of proteins. Although GAGs are suggested to bind to more than 800 biologically important proteins, very few therapeutics have been designed or discovered so far. A key challenge is the inability to identify, understand and predict distinct topologies accessed by GAGs, which may help design novel protein-binding GAG sequences. Recent studies on chondroitin sulfate (CS), a key member of the GAG family, pinpointing its role in multiple biological functions led us to study the conformational dynamism of CS building blocks using molecular dynamics (MD). In the present study, we used the all-atom GLYCAM06 force field for the first time to explore the conformational space of all possible CS building blocks. Each of the 16 disaccharides was solvated in a TIP3P water box with an appropriate number of counter ions followed by equilibration and a production run. We analyzed the MD trajectories for torsional space, inter- and intra-molecular H-bonding, bridging water, conformational spread and energy landscapes. An in-house phi and psi probability density analysis showed that 1→3-linked sequences were more flexible than 1→4-linked sequences. More specifically, phi and psi regions for 1→4-linked sequences were held within a narrower range because of intra-molecular H-bonding between the GalNAc O5 atom and GlcA O3 atom, irrespective of sulfation pattern. In contrast, no such intra-molecular interaction arose for 1→3-linked sequences. Further, the stability of 1→4-linked sequences also arose from inter-molecular interactions involving bridged water molecules. The energy landscape for both classes of CS disaccharides demonstrated increased ruggedness as the level of sulfation increased. The results show that CS building blocks present distinct conformational dynamism that offers the high possibility of unique electrostatic surfaces for protein recognition. The fundamental results presented here will support the development of algorithms that help to design longer CS chains for protein recognition.

Modeling the Interplay of Conformational and Electronic Structure in Conjugated Polyelectrolytes

Conjugated polyelectrolytes (CPEs) combine ionic, electronic, and optical functionality with the mechanical and thermodynamic properties of semiflexible, amphiphilic polyelectrolytes. Critical to CPE design is the coupling between macromolecular conformations, ionic interactions, and electronic transport, the combination of which spans electronic to mesoscopic length scales, rendering coherent theoretical analysis challenging. Here, we utilize a recently developed anisotropic CG model in combination with a phenomenological tight-binding Hamiltonian to explore the interplay of single-chain conformational and electronic structure in CPEs. Accessible single chain conformations are explored as a function of solvent conditions and chain stiffness, reproducing a rich landscape of rod-like, racquet, pearl necklace, and helical conformations observed in previous works. The electronic structure of each conformational archtype is further analyzed, incorporating through-bond coupling, through-space coupling, and electrostatic contributions to the Hamiltonian. Electrostatics is observed to influence electronic structure primarily by modifying the accessible conformational space, and only minimally by direct modulation of on-site energies. Electron transport in CPEs is most efficient in helical and racquet conformations, which is attributed to the flattening of dihedrals and through-space coupling within collapsed conformations. Relatedly, kink formation within racquets does not significantly deteriorate electronic conjugation within CPEs - an insight critical to understanding transport within locally ordered aggregates. These conclusions provide unprecedented computational insight into structure function relationships defining emerging classes of CPEs.

Structural Aspects of LIMK Regulation and Pharmacology

Malfunction of the actin cytoskeleton is linked to numerous human diseases including neurological disorders and cancer. LIMK1 (LIM domain kinase 1) and its paralogue LIMK2 are two closely related kinases that control actin cytoskeleton dynamics. Consequently, they are potential therapeutic targets for the treatment of such diseases. In the present review, we describe the LIMK conformational space and its dependence on ligand binding. Furthermore, we explain the unique catalytic mechanism of the kinase, shedding light on substrate recognition and how LIMK activity is regulated. The structural features are evaluated for implications on the drug discovery process. Finally, potential future directions for targeting LIMKs pharmacologically, also beyond just inhibiting the kinase domain, are discussed.

Point Mutations at a Key Site Alter the Cytochrome P450 OleP Structural Dynamics

Substrate binding to the cytochrome P450 OleP is coupled to a large open-to-closed transition that remodels the active site, minimizing its exposure to the external solvent. When the aglycone substrate binds, a small empty cavity is formed between the I and G helices, the BC loop, and the substrate itself, where solvent molecules accumulate mediating substrate-enzyme interactions. Herein, we analyzed the role of this cavity in substrate binding to OleP by producing three mutants (E89Y, G92W, and S240Y) to decrease its volume. The crystal structures of the OleP mutants in the closed state bound to the aglycone 6DEB showed that G92W and S240Y occupied the cavity, providing additional contact points with the substrate. Conversely, mutation E89Y induces a flipped-out conformation of this amino acid side chain, that points towards the bulk, increasing the empty volume. Equilibrium titrations and molecular dynamic simulations indicate that the presence of a bulky residue within the cavity impacts the binding properties of the enzyme, perturbing the conformational space explored by the complexes. Our data highlight the relevance of this region in OleP substrate binding and suggest that it represents a key substrate-protein contact site to consider in the perspective of redirecting its activity towards alternative compounds.

Enhanced Conformational Sampling of Nanobody CDR H3 Loop by Generalized Replica-Exchange with Solute Tempering

The variable domains of heavy-chain antibodies, known as nanobodies, are potential substitutes for IgG antibodies. They have similar affinities to antigens as antibodies, but are more heat resistant. Their small size allows us to exploit computational approaches for structural modeling or design. Here, we investigate the applicability of an enhanced sampling method, a generalized replica-exchange with solute tempering (gREST) for sampling CDR-H3 loop structures of nanobodies. In the conventional replica-exchange methods, temperatures of only a whole system or scaling parameters of a solute molecule are selected for temperature or parameter exchange. In gREST, we can flexibly select a part of a solute molecule and a part of the potential energy terms as a parameter exchange region. We selected the CDR-H3 loop and investigated which potential energy term should be selected for the efficient sampling of the loop structures. We found that the gREST with dihedral terms can explore a global conformational space, but the relaxation to the global equilibrium is slow. On the other hand, gREST with all the potential energy terms can sample the equilibrium distribution, but the structural exploration is slower than with dihedral terms. The lessons learned from this study can be applied to future studies of loop modeling.

Conformational Sampling for Transition State Searches on a Computational Budget

Transition state searches are the basis for characterizing reaction mechanisms and activation energies, and are thus central to myriad chemical applications. Nevertheless, common search algorithms are sensitive to molecular conformation and the conformational space of even medium-sized reacting systems are too complex to explore with brute force. Here we show that it is possible to train a classifier to learn the features of conformers that conduce successful transition state searches, such that optimal conformers can be down-selected before incurring the cost of a high-level transition state search. To this end, we have benchmarked the use of a modern conformational generation algorithm with our reaction prediction methodology, Yet Another Reaction Program (YARP), for reaction prediction tasks. We demonstrate that neglecting conformer contributions leads to qualitatively incorrect activation energy estimations for a broad range of reactions, whereas a simple random forest classifier can be used to reliably down-select low-barrier conformers. We also compare the relative advantage of performing conformational sampling on reactant, product, and putative transition state geometries. The robust performance of this relatively simple machine learning classifier mitigates cost as a factor when implementing conformational sampling into contemporary reaction prediction workflows.

Validity of Anti-PSMA ScFvD2B as a Theranostic Tool: A Narrative-Focused Review

Prostate cancer (PCa) is the second leading cause of cancer among men, and its diagnosis and adequate staging are fundamental. Among the biomarkers identified in recent years for PCa management, prostate-specific-membrane-antigen (PSMA), physiologically expressed at a low level on healthy prostate and in other normal tissues and highly overexpressed in PCa, represents a reliable marker ideal for imaging and therapy. The development of anti-PSMA antibodies, such as D2B, demonstrated slow clearance of intact antibodies compared with fragments resulting in low tumor-to-blood ratios; however, the modular structural and functional nature of antibodies allowed the generation of smaller fragments, such as scFvs. In this review of the anti-PSMA antibody fragment scFvD2B, we combined further characterization of its biomolecular and tissue cross-reactivity characteristics with a comprehensive summary of what has already been performed in preclinical models to evaluate imaging and therapeutic activities. A molecular dynamics study was performed, and ScFvD2B occupied a limited conformational space, characterized by low-energy conformational basins, confirming the high stability of the protein structure. In the cross-reactivity study, the weak/absent immunoreactivity in non-tumor tissues was comparable to the PSMA expression reported in the literature. Biodistribution studies and therapeutic treatments were conducted in different animal models obtained by subcutaneous or locoregional injection of PSMA-positive-versus-negative xenografts. The maximum tumor uptake was observed for 123I(SPECT), 124I(PET), and optical imaging, which avoids kidney accumulation (compared with radiometals) and leads to an optimal tumor-to-kidney and tumor-to-background ratios. Regarding its possible use in therapy, experimental data suggested a strong and specific antitumor activity, in vitro and in vivo, obtained using CAR-T or NK-92/CAR cells expressing scFvD2B. Based on presented/reviewed data, we consider that scFvD2B, due to its versatility and robustness, seems to: (i) overcome some problems observed in other studied scFvs, very often relatively unstable and prone to form aggregates; (ii) have sufficient tumor-to-background ratios for targeting and imaging PSMA-expressing cancer; (iii) significantly redirect immune killing cells to PSMA-positive tumors when inserted in second-generation CAR-T or NK-92/CAR cells. These data suggest that our product can be considered the right reagent to fill the gap that still exists in PCa diagnosis and treatment.