High Conformational Flexibility Research Articles

Implications of the Conformationally Flexible, Macrocyclic Structure of the First-Generation, Direct-Acting Anti-Viral Paritaprevir on Its Solid Form Complexity and Chameleonic Behavior.

Direct-acting antiviral regimens have transformed therapeutic management of hepatitis C across all prevalent genotypes. Most of the chemical matter in these regimens comprises molecules well outside the traditional drug development chemical space and presents significant challenges. Herein, the implications of high conformational flexibility and the presence of a 15-membered macrocyclic ring in paritaprevir are studied through a combination of advanced computational and experimental methods with focus on molecular chameleonicity and crystal form complexity. The ability of the molecule to toggle between high and low 3D polar surface area (PSA) conformations is underpinned by intramolecular hydrogen bonding (IMHB) interactions and intramolecular steric effects. Computational studies consequently show a very significant difference of over 75 Å2 in 3D PSA between polar and apolar environments and provide the structural basis for the perplexingly favorable passive permeability of the molecule. Crystal packing and protein binding resulting in strong intermolecular interactions disrupt these intramolecular interactions. Crystalline Form I benefits from strong intermolecular interactions, whereas the weaker intermolecular interactions in Form II are partially compensated by the energetic advantage of an IMHB. Like Form I, no IMHB is observed within the receptor-bound conformation; instead, an intermolecular H-bond contributes to the potency of the molecule. The choice of metastable Form II is derisked through strategies accounting for crystal surface and packing features to manage higher form specific solid-state chemical reactivity and specific processing requirements. Overall, the results show an unambiguous link between structural features and derived properties from crystallization to dissolution, permeation, and docking into the protein pocket.

Targeted Redesign of Suramin Analogs for Novel Antimicrobial Lead Development.

The emergence of new viral infections and drug-resistant bacteria urgently necessitates expedient therapeutic development. Repurposing and redesign of existing drugs against different targets are one potential way in which to accelerate this process. Suramin was initially developed as a successful antiparasitic drug but has also shown promising antiviral and antibacterial activities. However, due to its high conformational flexibility and negative charge, suramin is considered quite promiscuous toward positively charged sites within nucleic acid binding proteins. Although some suramin analogs have been developed against specific targets, only limited structure-activity relationship studies were performed, and virtual screening has yet to be used to identify more specific inhibitor(s) based on its scaffold. Using available structures, we investigated suramin's target diversity, confirming that suramin preferentially binds to protein pockets that are both positively charged and enriched in aromatic or leucine residues. Further, suramin's high conformational flexibility allows adaptation to structurally diverse binding surfaces. From this platform, we developed a framework for structure- and docking-guided elaboration of suramin analog scaffolds using virtual screening of suramin and heparin analogs against a panel of diverse therapeutically relevant viral and bacterial protein targets. Use of this new framework to design potentially specific suramin analogs is exemplified using the SARS-CoV-2 RNA-dependent RNA polymerase and nucleocapsid protein, identifying leads that might inhibit a wide range of coronaviruses. The approach presented here establishes a computational framework for designing suramin analogs against different bacterial and viral targets and repurposing existing drugs for more specific inhibitory activity.

Molecular “Hozo”: Thermally Stable Yet Conformationally Flexible Self-Assemblies Driven by Tight Molecular Meshing

Abstract Various noncovalent molecular interactions have been employed as driving forces to construct well-defined discrete self-assemblies. Among them, coordination and hydrogen bonds are widely used due to their high directionality and appropriate bond strength. However, the utilization of nondirectional, week molecular interactions for this purpose still presents a key challenge in supramolecular self-assembly. To tackle this critical issue, we presented a novel design concept, molecular “Hozo”, that the components with large, indented complementary hydrophobic surfaces tightly mesh with each other driven by the hydrophobic effect in water. Based on this concept, we developed a series of water-soluble cube-shaped molecular assemblies, i.e., nanocubes, composed of six molecules of identical gear-shaped amphiphiles (GSAs) with the aid of van der Waals (vdW) and cation-π interactions as well as the hydrophobic effect. The nanocubes exhibit unique properties derived from molecular meshing of the building blocks, such as high thermal stability yet as high conformational flexibility as biological molecules and emission whose intensity is affected by the structural change of the nanocube.

High Conformational Flexibility of the E2F1/DP1/DNA Complex

The E2F1 transcription factor is a master regulator of cell-cycle progression whose uncontrolled activation contributes to tumor cells growth. E2F1 binds DNA as a heterodimer with DP partners, resulting in a multi-domain quaternary-structure complex composed of DNA binding domains, a coiled coil domain and a marked box domain separated by short linkers. Building on the 3D knowledge of the single domains of E2F and DPs, we characterized the structure and dynamics of the complete E2F1/DP1/DNA complex by a combination of small-angle X-ray scattering and molecular dynamics simulations. It shows an asymmetric contribution of the dynamics of the two proteins. Namely, the coiled-coil domain leans toward the DP1 side of the complex; the DP1 loop between α2 and α3 of the DBD partially populates a helical structure leaning far from the DNA and in the same direction of the coiled-coil domain; and the N-terminal disordered region of DP1, rich in basic residues, contributes to DNA binding stabilization. Intriguingly, tumor mutations in the flexible regions of the complex suggest that perturbation of protein dynamics could affect protein function in a context-dependent way. Our data suggest fundamental contributions of DP proteins in distinct aspects of E2F biology.

Mechanistic basis for ubiquitin modulation of a protein energy landscape

Ubiquitin is a common posttranslational modification canonically associated with targeting proteins to the 26S proteasome for degradation and also plays a role in numerous other nondegradative cellular processes. Ubiquitination at certain sites destabilizes the substrate protein, with consequences for proteasomal processing, while ubiquitination at other sites has little energetic effect. How this site specificity-and, by extension, the myriad effects of ubiquitination on substrate proteins-arises remains unknown. Here, we systematically characterize the atomic-level effects of ubiquitination at various sites on a model protein, barstar, using a combination of NMR, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics simulation. We find that, regardless of the site of modification, ubiquitination does not induce large structural rearrangements in the substrate. Destabilizing modifications, however, increase fluctuations from the native state resulting in exposure of the substrate's C terminus. Both of the sites occur in regions of barstar with relatively high conformational flexibility. Nevertheless, destabilization appears to occur through different thermodynamic mechanisms, involving a reduction in entropy in one case and a loss in enthalpy in another. By contrast, ubiquitination at a nondestabilizing site protects the substrate C terminus through intermittent formation of a structural motif with the last three residues of ubiquitin. Thus, the biophysical effects of ubiquitination at a given site depend greatly on local context. Taken together, our results reveal how a single posttranslational modification can generate a broad array of distinct effects, providing a framework to guide the design of proteins and therapeutics with desired degradation and quality control properties.

Bcl-xL: A Focus on Melanoma Pathobiology.

Apoptosis is the main mechanism by which multicellular organisms eliminate damaged or unwanted cells. To regulate this process, a balance between pro-survival and pro-apoptotic proteins is necessary in order to avoid impaired apoptosis, which is the cause of several pathologies, including cancer. Among the anti-apoptotic proteins, Bcl-xL exhibits a high conformational flexibility, whose regulation is strictly controlled by alternative splicing and post-transcriptional regulation mediated by transcription factors or microRNAs. It shows relevant functions in different forms of cancer, including melanoma. In melanoma, Bcl-xL contributes to both canonical roles, such as pro-survival, protection from apoptosis and induction of drug resistance, and non-canonical functions, including promotion of cell migration and invasion, and angiogenesis. Growing evidence indicates that Bcl-xL inhibition can be helpful for cancer patients, but at present, effective and safe therapies targeting Bcl-xL are lacking due to toxicity to platelets. In this review, we summarized findings describing the mechanisms of Bcl-xL regulation, and the role that Bcl-xL plays in melanoma pathobiology and response to therapy. From these findings, it emerged that even if Bcl-xL plays a crucial role in melanoma pathobiology, we need further studies aimed at evaluating the involvement of Bcl-xL and other members of the Bcl-2 family in the progression of melanoma and at identifying new non-toxic Bcl-xL inhibitors.

Biophysical and Computational Studies of Human Disease Related Proteins with a Single-Pass Transmembrane Helix

Single-pass transmembrane receptors (SPTMRs) are involved in essential processes of biophysical and pathological nature in the human. This membrane protein family includes receptor tyrosine kinases, integrins, and immunoreceptors, which play an important role in metabolism, growth, proliferation, and apoptosis. SPTMR consists of several distinct domains including the extracellular domain (ECD), the transmembrane domain (TMD), and the intracellular domain (ICD) and exists as a monomer, homo- and/or heterodimer. Upon a ligand ligation through ECD, homo- or heterodimerization of SPTMR forms, followed by consequent modification of the ICDs, leading to the initiation of cellular signaling events. This activation requires interactions between TMD helices whose role in receptor activation becomes important. TMD is further highlighted by the discovery of mutations in the TMD or juxtamembrane domain (JMD) that are associated with human diseases. However, the details of cross-membrane signal transduction via SPTMRs have to be elucidated. Due to the high conformational flexibility of SPTMRs with their diverse structural composition, it is hard to characterize SPTMRs structurally. This drives us to work with only TMD helices of SPTMRs and focus on their interactions in the lipid bilayer environment. Our approach is the use of not only experimental data but also computational MD simulations to understand how TMD helices interact and how mutants associated with diseases affect the dimerization of TMD helices.

Macrocyclization strategies for cyclic peptides and peptidomimetics.

Peptides are a growing therapeutic class due to their unique spatial characteristics that can target traditionally “undruggable” protein–protein interactions and surfaces. Despite their advantages, peptides must overcome several key shortcomings to be considered as drug leads, including their high conformational flexibility and susceptibility to proteolytic cleavage. As a general approach for overcoming these challenges, macrocyclization of a linear peptide can usually improve these characteristics. Their synthetic accessibility makes peptide macrocycles very attractive, though traditional synthetic methods for macrocyclization can be challenging for peptides, especially for head-to-tail cyclization. This review provides an updated summary of the available macrocyclization chemistries, such as traditional lactam formation, azide–alkyne cycloadditions, ring-closing metathesis as well as unconventional cyclization reactions, and it is structured according to the obtained functional groups. Keeping peptide chemistry and screening in mind, the focus is given to reactions applicable in solution, on solid supports, and compatible with contemporary screening methods.

Ligand-based optimization and biological evaluation of N-(2,2,2-trichloro-1-(3-phenylthioureido)ethyl)acetamide derivatives as potent intrinsically disordered protein c-Myc inhibitors

The transcription factor c-Myc is a well-known onco-protein and an intrinsically disordered protein (IDP). As its aberrant expression is frequently observed in various human cancers, c-Myc is considered as a key drug target. However, due to its high conformational flexibility, directly targeting c-Myc remains difficult. Here we explored the structure–activity relationships (SAR) of N-(2,2,2-trichloro-1-(3-phenylthioureido)ethyl)acetamide compounds and reported sixteen novel active compounds. Among them, compound PKUMDL-CLM-32 (hereafter, 32) showed the best anti-proliferation activity in cells with an EC50 of 3.3 μM. We demonstrated that 32 directly disrupts c-Myc/Max interaction and induces the degradation of c-Myc protein in cells. We showed that 32 induces cell cycle arrest at S phase and promotes apoptosis of HL-60 cells. This study provides an example of using ligand-based analysis to optimize IDP ligands.

Endolysins from Antarctic Pseudomonas Display Lysozyme Activity at Low Temperature.

Organisms specialized to thrive in cold environments (so-called psychrophiles) produce enzymes with the remarkable ability to catalyze chemical reactions at low temperature. Cold activity relies on adaptive changes in the proteins’ sequence and structural organization that result in high conformational flexibility. As a consequence of flexibility, several such enzymes are inherently heat sensitive. Cold-active enzymes are of interest for application in a number of bioprocesses, where cold activity coupled with easy thermal inactivation can be of advantage. We describe the biochemical and functional properties of two glycosyl hydrolases (named LYS177 and LYS188) of family 19 (GH19), identified in the genome of an Antarctic marine Pseudomonas. Molecular evolutionary analysis placed them in a group of characterized GH19 endolysins active on lysozyme substrates, such as peptidoglycan. Enzyme activity peaks at about 25–35 °C and 40% residual activity is retained at 5 °C. LYS177 and LYS188 are thermolabile, with Tm of 52 and 45 °C and half-lives of 48 and 12 h at 37 °C, respectively. Bioinformatics analyses suggest that low heat stability may be associated to temperature-driven increases in local flexibility occurring mainly in a specific region of the polypeptide that is predicted to contain hot spots for aggregation.

Ligand-bound glutamine binding protein assumes multiple metastable binding sites with different binding affinities

Protein dynamics plays key roles in ligand binding. However, the microscopic description of conformational dynamics-coupled ligand binding remains a challenge. In this study, we integrate molecular dynamics simulations, Markov state model (MSM) analysis and experimental methods to characterize the conformational dynamics of ligand-bound glutamine binding protein (GlnBP). We show that ligand-bound GlnBP has high conformational flexibility and additional metastable binding sites, presenting a more complex energy landscape than the scenario in the absence of ligand. The diverse conformations of GlnBP demonstrate different binding affinities and entail complex transition kinetics, implicating a concerted ligand binding mechanism. Single molecule fluorescence resonance energy transfer measurements and mutagenesis experiments are performed to validate our MSM-derived structure ensemble as well as the binding mechanism. Collectively, our study provides deeper insights into the protein dynamics-coupled ligand binding, revealing an intricate regulatory network underlying the apparent binding affinity.

The bacterial metalloprotease NleD selectively cleaves mitogen-activated protein kinases that have high flexibility in their activation loop

Microbial pathogens often target the host mitogen-activated protein kinase (MAPK) network to suppress host immune responses. We previously identified a bacterial type III secretion system effector, termed NleD, a metalloprotease that inactivates MAPKs by specifically cleaving their activation loop. Here, we show that NleDs form a growing family of virulence factors harbored by human and plant pathogens as well as insect symbionts. These NleDs disable specifically Jun N-terminal kinases (JNKs) and p38s that are required for host immune response, whereas extracellular signal-regulated kinase (ERK), which is essential for host cell viability, remains intact. We investigated the mechanism that makes ERK resistant to NleD cleavage. Biochemical and structural analyses revealed that NleD exclusively targets activation loops with high conformational flexibility. Accordingly, NleD cleaved the flexible loops of JNK and p38 but not the rigid loop of ERK. Our findings elucidate a compelling mechanism of native substrate proteolysis that is promoted by entropy-driven specificity. We propose that such entropy-based selectivity is a general attribute of proteolytic enzymes.

Computational investigations on the dynamic binding effect of molecular tweezer CLR01 toward intrinsically disordered HIV-1 Nef.

Intrinsically disordered proteins (IDPs) are highly flexible molecules that undergo disorder to order transition through their interaction with other molecules. IDPs play a vital role in several biological processes ranging from molecular recognition to several human diseases through the protein-protein interaction. The dynamic flexibility of IDPs and their implications in several human diseases enable these molecules to serve as novel therapeutic targets. However, the challenging task is to develop novel drugs against IDPs because of their lack of stable structures and the nature of high conformational flexibility. In this study, we have calculated the dynamic binding effect of the supramolecular tweezer CLR01 against the intrinsically disordered HIV-1 Nef by employing molecular docking and dynamics simulation approaches. From docking results, we predicted the strong binding affinity of the tweezer with the target residues of Nef. The docking results were further validated from the molecular dynamics simulation studies confirming the conformational stability of Nef upon tweezer binding. These findings provide useful insights into the development of potent inhibitors for targeting Nef protein functions.

Drosophila OTK Is a Glycosaminoglycan-Binding Protein with High Conformational Flexibility

SummaryThe transmembrane protein OTK plays an essential role in plexin and Wnt signaling during Drosophila development. We have determined a crystal structure of the last three domains of the OTK ectodomain and found that OTK shows high conformational flexibility resulting from mobility at the interdomain interfaces. We failed to detect direct binding between Drosophila Plexin A (PlexA) and OTK, which was suggested previously. We found that, instead of PlexA, OTK directly binds semaphorin 1a. Our binding analyses further revealed that glycosaminoglycans, heparin and heparan sulfate, are ligands for OTK and thus may play a role in the Sema1a-PlexA axon guidance system.

Crystal structure of the Csm5 subunit of the type III-A CRISPR-Cas system

The Csm complex eliminates foreign RNA and DNA in the microbial defense CRISPR-Cas system. Csm5, one of the five subunits in the complex, facilitates crRNA maturation and target RNA binding in the type III system. However, the exact functional mechanism of Csm5 has remained elusive. Here, we report the crystal structure of the apo form of the Csm5 subunit at a resolution of 2.6 Å. Structural comparison of amino acids in the complex bound to RNA exhibits notable conformational changes in the crRNA and the target RNA binding sites. Shifts in the β-hairpin motif (β5-β6), α13 helix (resides 352-383), and G-rich loop (residues 335-337) in the C-terminal domain indicate an induced movement by crRNA binding. The positively charged residues (Lys 92, Arg 95 and Lys 96) located in the β-α4 loop of the target RNA interface show high conformational flexibility, while three-helix bundles (α1-α3) of the N-domain involved in Csm2 binding exhibit a rotational shift. The altered architecture of the Csm5 subunit demonstrates remarkable versatility of the ferredoxin-like fold in the RNA binding protein and provides a structural basis for the mechanism for crRNA and target RNA binding in the type III-A Crispr-Cas system.

Proteolytic and nonproteolytic activation mechanisms result in conformationally and functionally different forms of coagulation factor XIII A.

Factor XIIIA (FXIIIA) is a transglutaminase that cross-links intra- and extracellular protein substrates. FXIIIA is expressed as an inactive zymogen, and during blood coagulation, it is activated by removal of an activation peptide by the protease thrombin. No such proteolytic FXIIIA activation is known to occur in other tissues or the intracellular form of FXIIIA. For those locations, FXIIIA is assumed instead to undergo activation by Ca2+ ions. Previously, we demonstrated a monomeric state for active FXIIIA. Current analytical ultracentrifugation and kinetic experiments revealed that thrombin-activated FXIIIA has a higher conformational flexibility and a stronger affinity toward glutamine substrate than does nonproteolytically activated FXIIIA. The proteolytic activation of FXIIIA was further investigated in a context of fibrin clotting. In a series of fibrin cross-linking assays and scanning electron microscopy studies of plasma clots, the activation rates of FXIIIA V34X variants were correlated with the extent of fibrin cross-linking and incorporation of nonfibrous protein into the clot. Overall, the results suggest conformational and functional differences between active FXIIIA forms, thus expanding the understanding of FXIIIA function. Those differences may serve as a basis for developing therapeutic strategies to target FXIIIA in different physiological environments. ENZYMES: Factor XIIIA ( EC 2.3.2.13).

Intrinsically Disordered Proteins and Their “Mysterious” (Meta)Physics

Recognition of the natural abundance and functional importance of intrinsically disordered proteins (IDPs) and hybrid proteins containing ordered and intrinsically disordered protein regions (IDPRs) is changing protein science. IDPs and IDPRs; i.e., functional proteins and protein regions without unique structures, are commonly found in all organisms, where they have crucial roles in various biological processes. Disorder-based functionality complements functions of ordered proteins and domains. However, by virtue of their existence, IDPs/IDPRs, which are characterized by the remarkable conformational flexibility and structural plasticity, break multiple rules elaborated over the years to explain structure, folding, and functionality of well-folded proteins with unique structures. Despite the general believe, which dominated in protein science for more than a century, that unique biological functions of proteins require unique 3D-structures, structure-less IDPs/IDPRs are functional, being able to perform impossible tricks and to be engaged in biological activities, which are improbable for ordered proteins. With their exceptional spatio-temporal heterogeneity and high conformational flexibility, IDPs/IDPRs represent complex systems that act at the edge of chaos and are specifically tunable by various means. In this article, some of the wanders of intrinsic disorder are discussed as illustrations of their ‘mysterious’ (meta)physics.

Helical Structure of Recombinant Melittin.

Melittin is an extensively studied, 26-residue toxic peptide from honey bee venom. Because of its versatility in adopting a variety of secondary (helix or coil) and quaternary (monomer or tetramer) structures in various environments, melittin has been the focus of numerous investigations as a model peptide in protein folding studies as well as in studies involving binding to proteins, lipids, and polysaccharides. A significant body of evidence supports the view that melittin binds to these macromolecules in a predominantly helical conformation, but detailed structural knowledge of this conformation is lacking. In this report, we present nuclear magnetic resonance (NMR)-based structural insights into the helix formation of recombinant melittin in the presence of trifluoroethanol (TFE): a known secondary structure inducer in peptides. These studies were performed at neutral pH, with micromolar amounts of the peptide. Using nuclear Overhauser effect (NOE)-derived distance restraints from three-dimensional NMR spectra, we determined the atomic resolution solution NMR structure of recombinant melittin bearing a TFE-stabilized helix. To circumvent the complications with structure determination of small peptides with high conformational flexibility, we developed a workflow for enhancing proton NOEs by increasing the viscosity of the medium. In the TFE-containing medium, recombinant monomeric melittin forms a long, continuous helical structure, which consists of the N- and C-terminal α-helices and the noncanonical 310-helix in the middle. The noncanonical 310-helix is missing in the previously solved X-ray structure of tetrameric melittin and the NMR structure of melittin in methanol. Melittin's structure in TFE-containing medium provides insights into melittin's conformational transitions, which are relevant to the peptide's interactions with its biological targets.

Phylogenetic, molecular evolution and structural analyses of the WFDC1/prostate stromal protein 20 (ps20)

The WFDC1 gene is frequently down-regulated or lost in prostate cancer, and the encoded protein, ps20, has been implicated in epithelial cell behaviour and angiogenesis. However, ps20 remains largely uncharacterised with respect to its structure and interacting partners. This study characterised the evolution, functionality and structural characteristics of WFDC1/ps20 using phylogenetic reconstruction and other computational approaches. Bayesian phylogenetic analyses suggested that ps20 appeared in a common ancestor of deuterostomes-protostomes. The rate of evolutionary change within the coding regions of vertebrate WFDC1 genes and the synteny conservation in mammals differed from that of other vertebrate clades, indicating a possible functional diversity of ps20 homologues. A gene set enrichment analysis of the genes around WFDC1 (conserved synteny) showed functional relationships between the WFDC1, CDH13, CRISPLD2, IRF8 and TFPI2 genes. The molecular evolution of ps20 has been driven by purifying selection, particularly in the segments corresponding to exons 3 and 4, which encode the most conserved regions of the protein. A co-evolution analysis showed that residues within these regions co-vary with each other during the evolution of ps20. These results show that the regions corresponding to exons 3 and 4 are ps20-specific structure-function modules. Homology modelling of the exon 2-encoded polypeptide and subsequent dynamics calculus using a Gaussian network model showed that residues with high conformational flexibility are part of a loop region involved in protein-protein recognition, given the similarity with other serine protease inhibitors. Residues C96, R94, L105, and C66 are critical for the integrity and functionality of this ps20 region.

Conformational states of Zika virus non-structural protein 3 determined by molecular dynamics simulations with small-angle X-Ray scattering data

Zika virus (ZIKV) has become a great public health emergency. Its non-structural protein 3 (NS3) is a key enzyme in viral replication and has been considered as a potential therapeutic target. A conformational characterization of ZIKV NS3 is critical for a comprehensive understanding of its molecular interactions and functions. However, the high conformational flexibility of solution NS3 obstacles the structural characterization of NS3 solely from the experimental observable that averages over its heterogeneous conformations. Here, we employed replica exchange with solute tempering (REST) method to simulate the di-domain protein ZIKV NS3. Three independent MD simulations identified a conserved conformational ensemble of NS3, consisting of a major conformational state and several minor states from compact to loose conformations. The major state agrees well with the scattering profile from small-angle X-ray scattering (SAXS) experiments. Moreover, the simulated ensemble is supported by a direct data-fitting result that requires both short- and long-range structural contacts to recover the experimental data. We discussed the interplay between simulation and experiment in ensemble construction of flexible biomolecules and shed light on the physically derived conformational ensembles.