Backbone Conformational Changes Research Articles

An Infrared Nanospectroscopy Technique for the Study of Electric-Field-Induced Molecular Dynamics.

Static electric fields play a considerable role in a variety of molecular nanosystems as diverse as single-molecule junctions, molecules supporting electrostatic catalysis, and biological cell membranes incorporating proteins. External electric fields can be applied to nanoscale samples with a conductive atomic force microscopy (AFM) probe in contact mode, but typically, no structural information is retrieved. Here we combine photothermal expansion infrared (IR) nanospectroscopy with electrostatic AFM probes to measure nanometric volumes where the IR field enhancement and the static electric field overlap spatially. We leverage the vibrational Stark effect in the polymer poly(methyl methacrylate) for calibrating the local electric field strength. In the relevant case of membrane protein bacteriorhodopsin, we observe electric-field-induced changes of the protein backbone conformation and residue protonation state. The proposed technique also has the potential to measure DC currents and IR spectra simultaneously, insofar enabling the monitoring of the possible interplay between charge transport and other effects.

Encounter Complexes Between the N-terminal of Neurotensin with the Extracellular Loop 2 of the Neurotensin Receptor 1 Steer Neurotensin to the Orthosteric Binding Pocket

Neurotensin (NT) is a linear disordered peptide that activates two different class A GPCRs, neurotensin receptor 1 (NTS1) and NTS2. Resolved structures of the complex of the C-terminal fragment of NT, NT8-13, with NTS1 shows the peptide takes a well-defined structure in the bound state. However, the mechanisms underlying NT recognition of NTS1, and the conformational transition of NT upon binding NTS1 is an open question that if answered may aid discovery of highly selective drugs and reveal potential secondary binding sites on the surface of the receptor. Herein we investigated the interactions guiding NT to the orthosteric binding pocket of NTS1 by combining NMR experiments with kinetic analysis of the binding pathway using stopped-flow fluorescence and mutagenesis on both NT and NTS1. We show the presence of transient structures in the middle part of NT that kinetically regulate the binding of NT to NTS1. Moreover, our results indicate that the binding pathway of NT onto NTS1 is mediated via electrostatic interactions between the N-terminal region of NT with the extracellular loop 2 of NTS1. These interactions induce backbone conformational changes in neurotensin similar to the bound-state neurotensin, suggesting that the N-terminal region of NT and these interactions should be considered for development of selective drugs against NTS1.

Colorimetric Transition of Polydiacetylene/Cyclodextrin Supramolecular Assemblies and Implications as Colorimetric Sensors for Food Phenolic Antioxidants

AbstractMolecular self‐assembly has significant potential in the field of sensing. Polydiacetylenes (PDAs) are conjugated polymers possessing peculiar optical properties obtained by photopolymerization of self‐assembled diacetylene monomers. Herein, the blue‐to‐red phase transition upon either thermal stimulus or interaction with cyclodextrins (CDs) of two PDAs, bearing either carboxylic (PCDA) or amino (PCDA‐NH2) polar heads, is investigated to develop a colorimetric sensor for food phenolic antioxidants. The change in the PDA polar head does not affect significantly thermo‐chromatic transition. Upon thermal stimulus, in both PDAs, color transition occurs straightforward between two distinct stable states and does not involve the disordering of the PDA crystal phase, as revealed by UV–vis spectroscopy and SAXS analysis. Contrarily, PDA/α‐CD interaction is influenced by intermolecular forces among PDA polar heads and is more efficient for PCDA. α‐CDs presumably cause changes in both PDA backbone conformation and local environment surrounding the individual PDA chains. The PCDA/α‐CD assemblies are investigated as colorimetric sensors for the detection of Tyrosol (Ty) and caffeic acid (CAF), by using the principle of competitive inclusion complex formation. The system results to be more sensitive to CAF than Ty and may permit the determination of CAF in concentration ranges suitable for different food products.

Ion Binding to the bcl-2 G-Quadruplex from Polarizable Simulations with the Drude Force Field

G-quadruplexes (GQs) are noncanonical nucleic acid structures that can form in guanine-rich sequences and can adopt many diverse conformations depending on their constituent nucleotides. GQs are commonly present in DNA promoter regions, so alterations to their structures can contribute to diseases such as cancer. The B-cell lymphoma 2 (bcl-2) proto-oncogene codes for a protein that inhibits cell apoptosis and overexpression of bcl-2 is associated with cancer progression. Transcription of bcl-2 is controlled by two promoters, one of which contains a GQ-forming sequence. Understanding the stabilizing forces in this structure may lead to novel chemotherapeutic design. There are gaps in our current understanding of under what conditions GQs fold, which is crucial information that can give insight into the equilibrium of GQ conformations. To advance our understanding of the dominant bcl-2 promoter GQ, we performed molecular dynamics (MD) simulations using the Drude-2017 polarizable force field. Our simulation outcomes highlight two distinct changes within the GQ that coincide with ion binding. The first is the recruitment of a bulk K+ ion to the solvent-exposed face of the tetrad stem, which led to changes in base dipole moments and the K+ interaction energies of the guanines within the tetrad core. The second is the emergence of an “electronegative pocket” between the tetrad core and the long loop below the tetrad core. Ion binding in the electronegative pocket induces a backbone conformational change that may be relevant for inducing local conformational changes at K+ concentrations similar to those found in the cell. These results suggest two different locations within the bcl-2 GQ that may be targeted by small molecules to induce conformational changes.

Solid-State Conformational Flexibility at Work: Energetic Landscape of a Single Crystal-to-Single Crystal Transformation in a Cyclic Hexapeptoid.

We describe the energetic landscape beyond the solid-state dynamic behavior of a cyclic hexapeptoid decorated with four propargyl and two methoxyethyl side chains, namely, cyclo-(Nme-Npa2)2, Nme = N-(methoxyethyl)glycine, Npa = N-(propargyl)glycine. By increasing the temperature above 40 °C, the acetonitrile solvate form 1A starts to release acetonitrile molecules and undergoes a reversible single crystal-to-single crystal transformation into crystal form 1B with a remarkable conformational change in the macrocycle: two propargyl side chains move by 113° to form an unprecedented “CH-π zipper”. Then, upon acetonitrile adsorption, the “CH-π zipper” opens and the crystal form 1B transforms back to 1A. By conformational energy and lattice energy calculations, we demonstrate that the dramatic side-chain movement is a peculiar feature of the solid-state assembly and is determined by a backbone conformational change that leads to stabilizing CH···OC backbone-to-backbone interactions tightening the framework upon acetonitrile release. Weak interactions as CH···OC and CH-π bonds with the guest molecules are able to reverse the transformation, providing the energy contribution to unzip the framework. We believe that the underlined mechanism could be used as a model system to understand how external stimuli (as temperature, humidity, or volatile compounds) could determine conformational changes in the solid state.

Ion Binding Properties and Dynamics of the bcl-2 G-Quadruplex Using a Polarizable Force Field.

G-quadruplexes (GQs) are topologically diverse, highly thermostable noncanonical nucleic acid structures that form in guanine-rich sequences in DNA and RNA. GQs are implicated in transcriptional and translational regulation and genome maintenance, and deleterious alterations to their structures contribute to diseases such as cancer. The expression of the B-cell lymphoma 2 (Bcl-2) antiapoptotic protein, for example, is under transcriptional control of a GQ in the promoter of the bcl-2 gene. Modulation of the bcl-2 GQ by small molecules is of interest for chemotherapeutic development but doing so requires knowledge of the factors driving GQ folding and stabilization. To develop a greater understanding of the electrostatic properties of the bcl-2 promoter GQ, we performed molecular dynamics simulations using the Drude-2017 polarizable force field and compared relevant outcomes to the nonpolarizable CHARMM36 force field. Our simulation outcomes highlight the importance of dipole-dipole interactions in the bcl-2 GQ, particularly during the recruitment of a bulk K+ ion to the solvent-exposed face of the tetrad stem. We also predict and characterize an "electronegative pocket" at the tetrad-long loop junction that induces local backbone conformational change and may induce local conformational changes at cellular concentrations of K+. These outcomes suggest that moieties within the bcl-2 GQ can be targeted by small molecules to modulate bcl-2 GQ stability.

Inhibition of the Activity of Cyclophilin A Impedes Prolactin Receptor-Mediated Signaling, Mammary Tumorigenesis, and Metastases

SummaryProlactin (PRL) and its receptor (PRLr) play important roles in the pathogenesis of breast cancer. Cyclophilin A (CypA) is a cis-trans peptidyl-prolyl isomerase (PPI) that is constitutively associated with the PRLr and facilitates the activation of the tyrosine kinase Jak2. Treatment with the non-immunosuppressive prolyl isomerase inhibitor NIM811 or CypA short hairpin RNA inhibited PRL-stimulated signaling, breast cancer cell growth, and migration. Transcriptomic analysis revealed that NIM811 inhibited two-thirds of the top 50 PRL-induced genes and a reduction in gene pathways associated with cancer cell signaling. In vivo treatment of NIM811 in a TNBC xenograft lessened primary tumor growth and induced central tumor necrosis. Deletion of CypA in the MMTV-PyMT mouse model demonstrated inhibition of tumorigenesis with significant reduction in lung and lymph node metastasis. The regulation of PRLr/Jak2-mediated biology by NIM811 demonstrates that a non-immunosuppressive prolyl isomerase inhibitor can function as a potential breast cancer therapeutic.

Orientation and dynamics of Cu2+ based DNA labels from force field parameterized MD elucidates the relationship between EPR distance constraints and DNA backbone distances.

Pulsed electron paramagnetic resonance (EPR) based distance measurements using the recently developed Cu2+-DPA label present a promising strategy for measuring DNA backbone distance constraints. Herein we develop force field parameters for Cu2+-DPA in order to understand the features of this label at an atomic level. We perform molecular dynamics (MD) simulations using the force field parameters of Cu2+-DPA on four different DNA duplexes. The distance between the Cu2+ centers, extracted from the 2 μs MD trajectories, agrees well with the experimental distance for all the duplexes. Further analyses of the trajectory provide insight into the orientation of the Cu2+-DPA inside the duplex that leads to such agreement with experiments. The MD results also illustrate the ability of the Cu2+-DPA to report on the DNA backbone distance constraints. Furthermore, measurement of fluctuations of individual residues showed that the flexibility of Cu2+-DPA in a DNA depends on the position of the label in the duplex, and a 2 μs MD simulation is not sufficient to fully capture the experimental distribution in some cases. Finally, the MD trajectories were utilized to understand the key aspects of the double electron electron resonance (DEER) results. The lack of orientational selectivity effects of the Cu2+-DPA at Q-band frequency is rationalized in terms of fluctuations in the Cu2+ coordination environment and rotameric fluctuations of the label linker. Overall, a combination of EPR and MD simulations based on the Cu2+-DPA labelling strategy can contribute towards understanding changes in DNA backbone conformations during protein-DNA interactions.

Broad-Band Ultraviolet CD Spectroscopy of Ultrafast Peptide Backbone Conformational Dynamics.

The far-UV spectral window widely used for the conformational analysis of biomolecules is not easily covered with broad-band lasers. This has made it difficult to use circular dichroism (CD) spectroscopy to directly follow fast structure changes. By combining transient CD spectroscopy in the deep-UV with thioamide substitution, we demonstrate a method to overcome this difficulty. We investigated a dipeptide whose two carbonyl oxygen atoms were replaced by sulfur, red-shifting the strong lowest-lying ππ* transitions into the more accessible 250-370 nm spectral window. Coupling of the two thioamide units cannot be resolved by achiral 2D-UV spectroscopy, but it gives rise to a pronounced bisignate CD spectrum. The transient CD spectra reveal weakening of this coupling in the electronically excited state, where conformational constraints are released. Our results show that direct local probing of fast backbone conformational change via CD spectroscopy is possible in combination with site-selective thio substitution in peptides and proteins.

Ternary crystal structure of human ROR\u03b3 ligand-binding-domain, an inhibitor and corepressor peptide provides a new insight into corepressor interaction

Retinoic acid-related orphan receptor gamma (RORγ) plays pivotal roles in autoimmune diseases by controlling the lineage of interleukin 17 (IL-17)-producing CD4+ T cells (Th17 cells). Structure-based drug design has proven fruitful in the development of inhibitors targeting the ligand binding domain (LBD) of RORγ. Here, we present the crystal structure of a novel RORγ inhibitor co-complex, in the presence of a corepressor (CoR) peptide. This ternary complex with compound T reveals the structural basis for an inhibitory mechanism different from the previously reported inverse agonist. Compared to the inverse agonist, compound T induces about 2 Å shift of helix 5 (H5) backbone and side-chain conformational changes of Met365 on H5. These conformational changes correlate to reduced CoR peptide binding to RORγ-LBD in the presence of compound T, which suggests that the shift of H5 is responsible. This crystal structure analysis will provide useful information for the development of novel and efficacious drugs for autoimmune disorders.

Unique mechanism of target recognition by PfoI restriction endonuclease of the CCGG-family

Restriction endonucleases (REs) of the CCGG-family recognize a set of 4–8 bp target sequences that share a common CCGG or CCNGG core and possess PD…D/ExK nuclease fold. REs that interact with 5 bp sequence 5′-CCNGG flip the central N nucleotides and ‘compress’ the bound DNA to stack the inner base pairs to mimic the CCGG sequence. PfoI belongs to the CCGG-family and cleaves the 7 bp sequence 5′-T|CCNGGA ("|" designates cleavage position). We present here crystal structures of PfoI in free and DNA-bound forms that show unique active site arrangement and mechanism of sequence recognition. Structures and mutagenesis indicate that PfoI features a permuted E…ExD…K active site that differs from the consensus motif characteristic to other family members. Although PfoI also flips the central N nucleotides of the target sequence it does not ‘compress’ the bound DNA. Instead, PfoI induces a drastic change in DNA backbone conformation that shortens the distance between scissile phosphates to match that in the unperturbed CCGG sequence. Our data demonstrate the diversity and versatility of structural mechanisms employed by restriction enzymes for recognition of related DNA sequences.

Applying Pose Clustering and MD Simulations To Eliminate False Positives in Molecular Docking.

In this work, we developed a computational protocol that employs multiple molecular docking experiments, followed by pose clustering, molecular dynamic simulations (10 ns), and energy rescoring to produce reliable 3D models of antibody-carbohydrate complexes. The protocol was applied to 10 antibody-carbohydrate co-complexes and three unliganded (apo) antibodies. Pose clustering significantly reduced the number of potential poses. For each system, 15 or fewer clusters out of 100 initial poses were generated and chosen for further analysis. Molecular dynamics (MD) simulations allowed the docked poses to either converge or disperse, and rescoring increased the likelihood that the best-ranked pose was an acceptable pose. This approach is amenable to automation and can be a valuable aid in determining the structure of antibody-carbohydrate complexes provided there is no major side chain rearrangement or backbone conformational change in the H3 loop of the CDR regions. Further, the basic protocol of docking a small ligand to a known binding site, clustering the results, and performing MD with a suitable force field is applicable to any protein ligand system.

Improving the thermal stability of cellobiohydrolase Cel7A from Hypocrea jecorina by directed evolution

Secreted mixtures of Hypocrea jecorina cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. H. jecorina Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 °C. Enhanced thermal stability is desirable to enable the use of higher processing temperatures and to improve the economic feasibility of industrial biomass conversion. Here, we enhanced the thermal stability of Cel7A through directed evolution. Sites with increased thermal stability properties were combined, and a Cel7A variant (FCA398) was obtained, which exhibited a 10.4 °C increase in Tm and a 44-fold greater half-life compared with the wild-type enzyme. This Cel7A variant contains 18 mutated sites and is active under application conditions up to at least 75 °C. The X-ray crystal structure of the catalytic domain was determined at 2.1 Å resolution and showed that the effects of the mutations are local and do not introduce major backbone conformational changes. Molecular dynamics simulations revealed that the catalytic domain of wild-type Cel7A and the FCA398 variant exhibit similar behavior at 300 K, whereas at elevated temperature (475 and 525 K), the FCA398 variant fluctuates less and maintains more native contacts over time. Combining the structural and dynamic investigations, rationales were developed for the stabilizing effect at many of the mutated sites.

Formation of Annular Protofibrillar Assembly by Cysteine Tripeptide: Unraveling the Interactions with NMR, FTIR, and Molecular Dynamics.

Both hydrogen-bonding and hydrophobic interactions play a significant role in molecular assembly, including self-assembly of proteins and peptides. In this study, we report the formation of annular protofibrillar structure (diameter ∼500 nm) made of a newly synthesized s-benzyl-protected cysteine tripeptide, which was primarily stabilized by hydrogen-bonding and hydrophobic interactions. Atomic force microscopy and field emission scanning electron microscopy analyses found small oligomers (diameter ∼60 nm) to bigger annular (outer diameter ∼300 nm; inner diameter, 100 nm) and protofibrillar structures after 1-2 days of incubation. Rotating-frame Overhauser spectroscopic (ROESY) analysis revealed the presence of several nonbonded proton-proton interactions among the residues, such as amide protons with methylene group, aromatic protons with tertiary butyl group, and methylene protons with tertiary butyl group. These added significant stability to bring the peptides closer to form a well-ordered assembled structure. Hydrogen-deuterium exchange NMR measurement further suggested that two individual amide protons among the three amide groups were strongly engaged with the adjacent tripeptide via H-bond interaction. However, the remaining amide proton was found to be exposed to solvent and remained noninteracting with other tripeptide molecules. In addition to chemical shift values, a significant change in amide bond vibrations of the tripeptide was found due to the formation of the self-assembled structure. The amide I mode of vibrations involving two amide linkages appeared at 1641 and 1695 cm-1 in the solid state. However, in the assembled state, the stretching band at 1695 cm-1 became broad and slightly shifted to ∼1689 cm-1. On the contrary, the band at 1641 cm-1 shifted to 1659 cm-1 and indicated that the -C═O bond associated with this vibration became stronger in the assembled state. These changes in Fourier transform infrared spectroscopy frequency clearly indicated changes in the amide backbone conformation and the associated hydrogen-bonding pattern due to the formation of the assembled structure. In addition to hydrogen bonding, molecular dynamics simulation indicated that the number of π-π interactions also increased with increasing number of tripeptides participated in the self-assembly process. Combined results envisaged a cross β-sheet assembly unit consisting of four intermolecular hydrogen bonds. Such noncovalent peptide assemblies glued by hydrogen-bonding and other weak forces may be useful in developing nanocapsule and related materials.

Thermodynamic contribution of backbone conformational entropy in the binding between SH3 domain and proline-rich motif

Biological functions of intrinsically disordered proteins (IDPs), and proteins containing intrinsically disordered regions (IDRs) are often mediated by short linear motifs, like proline-rich motifs (PRMs). Upon binding to their target proteins, IDPs undergo a disorder-to-order transition which is accompanied by a large conformational entropy penalty. Hence, the molecular mechanisms underlying control of conformational entropy are critical for understanding the binding affinity and selectivity of IDPs-mediated protein-protein interactions (PPIs). Here, we investigated the backbone conformational entropy change accompanied by binding of the N-terminal SH3 domain (nSH3) of CrkII and PRM derived from guanine nucleotide exchange factor 1 (C3G). In particular, we focused on the estimation of conformational entropy change of disordered PRM upon binding to the nSH3 domain. Quantitative characterization of conformational dynamics of disordered peptides like PRMs is limited. Hence, we combined various methods, including NMR model-free analysis, δ2D, DynaMine, and structure-based calculation of entropy loss. This study demonstrates that the contribution of backbone conformational entropy change is significant in the PPIs mediated by IDPs/IDRs.

Pushing the Backbone in Protein-Protein Docking

Conformational changes of proteins that occur upon binding typically confound computational docking algorithms. In this study, we test computational methods to capture protein backbone conformational change related to binding. To address how well existing algorithms can sample bound-like backbones, wequery seven techniques including Monte Carlo-based sampling, molecular dynamics, and normal mode analysis. All methods tested rarely sample near-bound states from the unbound conformation. Nevertheless, the direction of the predicted motions overlapwith the actual conformational change. We next forced the backbone from the unbound toward the bound conformation to create a family of dockingenergy landscapes. Seventy percent of docking targets succeed when the unbound backbones is pushed to within 0.6Å of the bound. Current methods can capture an average of 22% of unbound-bound transitions through conformer selection methods and another 57% through induced-fit methodologies, delineating a stubborn gap (21%) in backbone motion not covered by any current approach.

Direct Observation of Aggregation‐Induced Backbone Conformational Changes in Tau Peptides

AbstractIn tau proteins, the hexapeptides in the R2 and R3 repeats are known to initiate tau fibril formation, which causes a class of neurodegenerative diseases called the taupathies. We show that in R3, in addition to the presence of the hexapeptides, the correct turn conformation upstream to it is also essential for producing prion‐like fibrils that are capable of propagation. A time‐dependent NMR aggregation assay of a slow fibril forming R3‐S316P peptide revealed a trans to cis equilibrium shift in the peptide‐bond conformation preceding P316 during the growth phase of the aggregation process. S316 was identified as the key residue in the turn that confers templating capacity on R3 fibrils to accelerate the aggregation of the R3‐S316P peptide. These results on the specific interactions and conformational changes responsible for tau aggregation could prove useful for developing an efficient therapeutic intervention in Alzheimer's disease.

Direct Observation of Aggregation-Induced Backbone Conformational Changes in Tau Peptides.

In tau proteins, the hexapeptides in the R2 and R3 repeats are known to initiate tau fibril formation, which causes a class of neurodegenerative diseases called the taupathies. We show that in R3, in addition to the presence of the hexapeptides, the correct turn conformation upstream to it is also essential for producing prion-like fibrils that are capable of propagation. A time-dependent NMR aggregation assay of a slow fibril forming R3-S316P peptide revealed a trans to cis equilibrium shift in the peptide-bond conformation preceding P316 during the growth phase of the aggregation process. S316 was identified as the key residue in the turn that confers templating capacity on R3 fibrils to accelerate the aggregation of the R3-S316P peptide. These results on the specific interactions and conformational changes responsible for tau aggregation could prove useful for developing an efficient therapeutic intervention in Alzheimer's disease.

NQO1 rs1800566 polymorph is more prone to NOx induced lung injury: Endorsing deleterious functionality through informatics approach

Gene-environment interaction studies have led to the identification of genetic mutations in individuals with increased susceptibility to pollution related diseases. rs1800566 polymorphism of NQO1, leading to P187S missense mutation in the transcribed antioxidant protein, causes individuals carrying this mutation more prone to NO2 induced lung inflammatory injury. Here, we report significant structural and functional changes incurred by NQO1 antioxidant protein as a result of alteration in its nucleotide (C609T) and hence, protein sequence. Detailed insights were obtained regarding the prospective impact of this mutation on the structural stability of normal and mutated NQO1 protein, using a myriad of bioinformatic tools and webservers. Structure analysis showed no significant change at secondary level. A change in the native backbone conformation was observed due to formation of a hydrogen bond. Hydrophobicity and phosphorylation properties, decisive factors for functioning and stability of NQO1 were considerably influenced by P187S mutation. Computational study of the properties of a polymorph linked with NOx induced lung injury sheds light on the molecular basis of this polymorphism and endorses previous findings, reported by the scientists working in this domain.

Indirect DNA Sequence Recognition and Its Impact on Nuclease Cleavage Activity

LAGLIDADG meganucleases are DNA cleaving enzymes used for genome engineering. While their cleavage specificity can be altered using several protein engineering and selection strategies, their overall targetability is limited by highly specific indirect recognition of the central four base pairs within their recognition sites. In order to examine the physical basis of indirect sequence recognition and to expand the number of such nucleases available for genome engineering, we have determined the target sites, DNA-bound structures, and central four cleavage fidelities of nine related enzymes. Subsequent crystallographic analyses of a meganuclease bound to two noncleavable target sites, each containing a single inactivating base pair substitution at its center, indicates that a localized slip of the mutated base pair causes a small change in the DNA backbone conformation that results in a loss of metal occupancy at one binding site, eliminating cleavage activity.