Local Structural Flexibility Research Articles

PLDDT Values in AlphaFold2 Protein Models Are Unrelated to Globular Protein Local Flexibility

Two non-redundant, high-quality sets of protein X-ray crystal structures from the Protein Data Bank (room temperature, 288–298 K, and low temperature, 95–105 K) were compared to structural predictions conducted using ColabFold/AlphaFold2. In particular, the relationship between B-factors and pLDDT values, which estimate the degree of prediction confidence, was investigated. It was observed that there is basically no correlation between these two quantities and, consequently, that the level of confidence in predictions does not provide information about the degree of local structural flexibility of globular proteins.

Binding selectivity-dependent molecular mechanism of inhibitors towards CDK2 and CDK6 investigated by multiple short molecular dynamics and free energy landscapes

Understanding selectivity-dependent molecular mechanism of inhibitors towards CDK2 over CDK6 is prominent for improving drug design towards the CDK family. Multiple short molecular dynamics (MD) simulations combined with MM-GBSA approach are adopted to investigate molecular mechanism on binding selectivity of inhibitors X64, X3A, and 4 AU to CDK2 and CDK6. The RMSF analysis and calculations of molecular surface areas indicate that local structural and global flexibility of CDK6 are stronger than that of CDK2. Based on dynamics cross-correlation maps (DCCMs), motion modes of CDK2 and CDK6 produce difference due to associations of X64, X3A, and 4 AU. The calculated binding free energies (BFEs) demonstrate that the compensation between binding enthalpy and entropy of X64, X34, and 4 AU is a key force driving selectivity of inhibitors towards CDK2 over CDK6. This work provides valuable information for designing highly selective inhibitors towards CDK2 and CDK6 and further promotes identification of efficient anticancer drugs in the future.

Direct electron transfer of fructose dehydrogenase immobilized on thiol-gold electrodes

Direct electrochemical electron transfer (DET) of oxidoreductases has attracted increasing attention in pure and applied bioelectrochemistry (e.g. biosensors and biofuel cells) over the last decades. We report here a systematic study of DET-type bioelectrocatalysis of the membrane-bound redox enzyme fructose dehydrogenase (FDH, Gluconobacter sp.), on variable-length and variably terminated thiol self-assembled monolayers (SAMs) both on Au(111) and nanoporous gold (NPG) electrodes. FDH on Au(111) modified by short-chain moderately hydrophilic 2-mercaptoethanol (BME) SAMs exhibits the highest DET activities and largest DET-capable fraction. Fitting of theoretical polarization curves to the data and homology modeling/docking of FDH offer further mechanistic insight. The dependence of the DET efficiency of FDH on the length and differently terminated carbon chain is systematically presented. The decreased DET rate with increasing chain length is associated with increasingly unfavourable long-range electron tunnelling, and not with lowered enzyme loading. The porous structure of NPG is favorable for FDH bioelectrocatalysis by improving both efficient enzyme orientation and operational stability. Overall, our study maps systematically the controlled local environmental structural flexibility of the Au/SAM/enzyme/solution interface, a paradigm for thiol modified surfaces in biosensors and bioelectronics.

Frontispiece: Amorphous Oxide Nanostructures for Advanced Electrocatalysis

Amorphous oxides have unique local structural flexibility and attractive electrocatalytic properties, and have been used for a range of electrocatalytic applications, from hydrogen evolution and oxygen evolution to oxygen reduction, CO2 electroreduction and nitrogen electroreduction. They even outperform their crystalline counterparts in terms of electrocatalytic activity and stability. This Minireview by X. Huang and co-workers, on page 3943 ff., summarizes the achievements and challenges around electrocatalysts based on amorphous oxides.

Nucleosomal DNA Dynamics Mediate Oct4 Pioneer Factor Binding

Transcription factor (TF) proteins bind to DNA to regulate gene expression. Normally, accessibility to DNA is required for their function. However, in the nucleus, the DNA is often inaccessible, wrapped around histone proteins in nucleosomes forming the chromatin. Pioneer TFs are thought to induce chromatin opening by recognizing their DNA binding sites on nucleosomes. For example, Oct4, a master regulator and inducer of stem cell pluripotency, binds to DNA in nucleosomes in a sequence-specific manner. Here, we reveal the structural dynamics of nucleosomes that mediate Oct4 binding from molecular dynamics simulations. Nucleosome flexibility and the amplitude of nucleosome motions such as breathing and twisting are enhanced in nucleosomes with multiple TF binding sites. Moreover, the regions around the binding sites display higher local structural flexibility. Probing different structures of Oct4-nucleosome complexes, we show that alternative configurations in which Oct4 recognizes partial binding sites display stable TF-DNA interactions similar to those observed in complexes with free DNA and compatible with the DNA curvature and DNA-histone interactions. Therefore, we propose a structural basis for nucleosome recognition by a pioneer TF that is essential for understanding how chromatin is unraveled during cell fate conversions.

Regioselective Chemical Modification of Cysteine Residues on Protein Surfaces Focusing on Local Environment around the Conjugation Site.

For chemical modification of cysteines in a protein, the regioselectivity among cysteine residues on the protein surface is an issue to be considered. To elucidate the determinants of cysteine reactivities on protein surfaces, we have investigated the chemical modification of the adenylate kinase A55C/C77S/V169C mutant as an experimental model. Although Cys55 and Cys169 are commonly located on the protein surface, Cys55 showed the ca. 3-6-fold higher reactivity compared to Cys169 in a reaction with a pyrene derivative. By a further conjugation of a phenanthroline derivative into the vacant Cys thiol, fluorescence quenching was attained by a pyrene-phenanthroline interaction that occurred by the conformational change of the protein. The K50A mutation further enhanced the regioselectivity of pyrene conjugation in Cys55, which is attributed to the effects of structural flexibility in the vicinity of Cys55 on its reactivity. To regioselectively conjugate different types of synthetic molecules onto the surface of a protein, perturbation in the local structural flexibility around the conjugation sites will be a useful strategy.

Amorphous Oxide Nanostructures for Advanced Electrocatalysis.

Amorphous oxides have attracted special attention as advanced electrocatalysts owing to their unique local structural flexibility and attractive electrocatalytic properties. With abundant randomly oriented bonds and surface-exposed defects (e.g., oxygen vacancies) as active catalytic sites, the adsorption/desorption of reactants can be optimized, leading to superior catalytic activities. Amorphous oxide materials have found wide electrocatalytic applications ranging from hydrogen evolution and oxygen evolution to oxygen reduction, CO2 electroreduction and nitrogen electroreduction. The amorphous oxide electrocatalysts even outperform their crystalline counterparts in terms of electrocatalytic activity and stability. Despite of the merits and achievements for amorphous oxide electrocatalysts, there are still issues and challenges existing for amorphous oxide electrocatalysts. There are rarely reviews specifically focusing on amorphous oxide electrocatalysts and therefore it is imperative to have a comprehensive overview of the research progress and to better understand the achievements and issues with amorphous oxide electrocatalysts. In this minireview, several general preparation methods for amorphous oxides are first introduced. Then, the achievements in amorphous oxides for several important electrocatalytic reactions are summarized. Finally, the challenges and perspectives for the development of amorphous oxide electrocatalysts are outlined.

Deciphering complex dynamics of water counteraction around secondary structural elements of allosteric protein complex: Case study of SAP-SLAM system in signal transduction cascade.

The first hydration shell of a protein exhibits heterogeneous behavior owing to several attributes, majorly local polarity and structural flexibility as revealed by solvation dynamics of secondary structural elements. We attempt to recognize the change in complex water counteraction generated due to substantial alteration in flexibility during protein complex formation. The investigation is carried out with the signaling lymphocytic activation molecule (SLAM) family of receptors, expressed by an array of immune cells, and interacting with SLAM-associated protein (SAP), composed of one SH2 domain. All atom molecular dynamics simulations are employed to the aqueous solutions of free SAP and SLAM-peptide bound SAP. We observed that water dynamics around different secondary structural elements became highly affected as well as nicely correlated with the SLAM-peptide induced change in structural rigidity obtained by thermodynamic quantification. A few instances of contradictory dynamic features of water to the change in structural flexibility are explained by means of occluded polar residues by the peptide. For βD, EFloop, and BGloop, both structural flexibility and solvent accessibility of the residues confirm the obvious contribution. Most importantly, we have quantified enhanced restriction in water dynamics around the second Fyn-binding site of the SAP due to SAP-SLAM complexation, even prior to the presence of Fyn. This observation leads to a novel argument that SLAM induced more restricted water molecules could offer more water entropic contribution during the subsequent Fyn binding and provide enhanced stability to the SAP-Fyn complex in the signaling cascade. Finally, SLAM induced water counteraction around the second binding site of the SAP sheds light on the allosteric property of the SAP, which becomes an integral part of the underlying signal transduction mechanism.

Structural Versatility of Hepatitis C Virus Proteins: Implications for the Design of Novel Anti-HCV Intervention Strategies.

HCV-linked pathologies represent worldwide health threats. Over the years, an enormous number of independent studies have been devoted to the understanding of the molecular bases of HCV infection. A significant amount of these investigations has been focused on the structural characterization of the virus proteins with the aim of developing structure-based innovative therapeutic approaches. An analysis of the current Protein Data Bank content unravels that the structural biology of the virus has hitherto covered a large fraction of the HCV proteins (75%). The present review recapitulates the state-of-the-art of structural characterizations of HCV individual proteins with a specific focus on their structural versatility/flexibility. This survey indicates there is accumulating evidence that structural flexibility is a common feature among HCV proteins. This versatility can be detected at different structural level i.e. occurrence of alternative oligomeric states and/or of local and global flexibility. Somewhat surprisingly, some disordered or highly flexible regions of HCV proteins, such as the core and the antigenic fragment 412-423 of E2, present highly conserved sequences among the virus genotypes. The overall versatility of HCV proteins plays an important role in host protein recognition, drug resistance mechanisms, and virus escape from the host immunogenic system. Of particular relevance is the emerging idea that HCV uses local structural flexibility as an alternative tool to sequence variability to evade the immune response of the host organism. We believe that concepts emerged from this survey will be important for the development of anti-HCV vaccines that are eagerly needed.

Human Neuronal Calcium Sensor-1 Protein Avoids Histidine Residues To Decrease pH Sensitivity.

pH is highly regulated in mammalian central nervous systems. Neuronal calcium sensor-1 (NCS-1) can interact with numerous target proteins. Compared to that in the NCS-1 protein of Caenorhabditis elegans, evolution has avoided the placement of histidine residues at positions 102 and 83 in the NCS-1 protein of humans and Xenopus laevis, possibly to decrease the conformational sensitivity to pH gradients in synaptic processes. We used all-atom molecular dynamics simulations to investigate the effects of amino acid substitutions between species on human NCS-1 by substituting Arg102 and Ser83 for histidine at neutral (R102H and S83H) and acidic pHs (R102Hp and S83Hp). Our cumulative 5 μs simulations revealed that the R102H mutation slightly increases the structural flexibility of loop L2 and the R102Hp mutation decreases protein stability. Community network analysis illustrates that the R102H and S83H mutations weaken the interdomain and strengthen the intradomain communications. Secondary structure contents in the S83H and S83Hp mutants are similar to those in the wild type, whereas the global structural stabilities and salt-bridge probabilities decrease. This study highlights the conformational dynamics effects of the R102H and S83H mutations on the local structural flexibility and global stability of NCS-1, whereas protonated histidine decreases the stability of NCS-1. Thus, histidines at positions 102 and 83 may not be compatible with the function of NCS-1 whether in the neutral or protonated state.

Controlled Growth of Ceria Nanoarrays on Anatase Titania Powder: A Bottom-up Physical Picture.

The leading edge of catalysis research motivates physical understanding of the growth of nanoscale oxide structures on different supporting oxide materials that are themselves also nanostructured. This research opens up for consideration a diverse range of facets on the support material, versus the single facet typically involved in wide-area growth of thin films. Here, we study the growth of ceria nanoarchitectures on practical anatase titania powders as a showcase inspired by recent experiments. Density functional theory (DFT)-based methods are employed to characterize and rationalize the broad array of low energy nanostructures that emerge. Using a bottom-up approach, we are able to identify and characterize the underlying mechanisms for the facet-dependent growth of various ceria motifs on anatase titania based on formation energy. These motifs include 0D clusters, 1D chains, 2D plates, and 3D nanoparticles. The ceria growth mode and morphology are determined by the interplay of several factors including the role of the common cation valence, the interface template effect for different facets of the anatase support, enhanced ionic binding for more compact ceria motifs, and the local structural flexibility of oxygen ions in bridging the interface between anatase and ceria structures.

Molecular Origin of Ultrafast Water-Protein Coupled Interactions.

The fluctuations of hydration water and the protein are coupled together at the protein surface and often such water-protein dynamic interactions are controlled presumably by hydration water motions. However, direct evidence is scarce and it requires measuring the dynamics of hydration water and protein side chain simultaneously. Here, we use a unique protein with a single tryptophan to directly probe interfacial water and related side chain relaxations with temperature dependence. With systematic mutations to change local chemical identity and structural flexibility, we found that the side chain relaxations are always slower than hydration water motions and the two dynamic processes are linearly correlated with the same energy barriers, indicating the same origin of both relaxations. The charge mutations change the rates of hydration water relaxations but not the relaxation barriers. These results convincingly show that the water-protein relaxations are strongly coupled and the hydration water molecules govern such fluctuations on the picosecond time scales.

Structure features of GH10 xylanase from <italic>Caldicellulosiruptor bescii</italic>: implication for its thermophilic adaption and substrate binding preference

Caldicellulosiruptor bescii is the most thermophilic cellulolytic species of organisms known to date. In our previous study, GH10 xylanase CbXyn10B from C. bescii displayed outstanding hydrolytic activity toward various xylans at high temperatures. To understand the structural basis for this protein's catalysis and thermostability, we solved the crystal structures of CbXyn10B and its complexes with xylooligosaccharides. These structural models were used to guide comparison with its mesophilic counterpart PbXyn10B. A distinctive structural feature is that thermophilic CbXyn10B presents a relatively stable interaction between the extended loops L7 and L8 in the catalytic cleft by an extensive hydrogen bonding network, which is mediated by Lys306, Arg314 and three well-ordered water molecules. Moreover, a unique aromatic cluster consisting of Try17, Phe20, Phe21, and Phe337 may enhance the interaction between the N- and C- terminus. Targeted mutagenesis demonstrated that these interactions substantially contribute to enzyme stabilization, as indicated by a considerable decrease in the melting temperature (Tm) of CbXyn10B by substituting critical residues with Ala. Therefore, it was shown that not only the aromatic interaction connecting protein termini but also the extensive hydrogen bonding network formed between surface loops could restrict the local structural flexibility and contribute significantly to the overall stability of enzymes. Furthermore, the xylooligosaccharides were found to tightly bind to the glycone subsites of xylanase, indicating higher affinities at these subsites and reflecting its substrate binding preference. Our results suggest that CbXyn10B is stabilized with distinct rigidity at the catalytic cleft as well as the terminal regions, which provides insights into the evolutionary strategy for accommodating the functional needs of GH10 enzymes to high temperature.

Sequence, structure, and cooperativity in folding of elementary protein structural motifs

Residue-level unfolding of two helix-turn-helix proteins--one naturally occurring and one de novo designed--is reconstructed from multiple sets of site-specific (13)C isotopically edited infrared (IR) and circular dichroism (CD) data using Ising-like statistical-mechanical models. Several model variants are parameterized to test the importance of sequence-specific interactions (approximated by Miyazawa-Jernigan statistical potentials), local structural flexibility (derived from the ensemble of NMR structures), interhelical hydrogen bonds, and native contacts separated by intervening disordered regions (through the Wako-Saitô-Muñoz-Eaton scheme, which disallows such configurations). The models are optimized by directly simulating experimental observables: CD ellipticity at 222 nm for model proteins and their fragments and (13)C-amide I' bands for multiple isotopologues of each protein. We find that data can be quantitatively reproduced by the model that allows two interacting segments flanking a disordered loop (double sequence approximation) and incorporates flexibility in the native contact maps, but neither sequence-specific interactions nor hydrogen bonds are required. The near-identical free energy profiles as a function of the global order parameter are consistent with expected similar folding kinetics for nearly identical structures. However, the predicted folding mechanism for the two motifs is different, reflecting the order of local stability. We introduce free energy profiles for "experimental" reaction coordinates--namely, the degree of local folding as sensed by site-specific (13)C-edited IR, which highlight folding heterogeneity and contrast its overall, average description with the detailed, local picture.

Structural Similarity of Secretins from Type II and Type III Secretion Systems

Secretins, the outer membrane components of several secretion systems in Gram-negative bacteria, assemble into channels that allow exoproteins to traverse the membrane. The membrane-inserted, multimeric regions of PscC, the Pseudomonas aeruginosa type III secretion system secretin, and PulD, the Klebsiella oxytoca type II secretion system secretin, were purified after cell-free synthesis and their structures analyzed by single particle cryoelectron microscopy. Both homomultimeric, barrel-like structures display a "cup and saucer" architecture. The "saucer" region of both secretins is composed of two distinct rings, with that of PulD being less segmented than that of PscC. Both secretins have a central chamber that is occluded by a plug linked to the chamber walls through hairpin-like structures. Comparisons with published structures from other bacterial systems reveal that secretins have regions of local structural flexibility, probably reflecting their evolved functions in protein secretion and needle assembly.

Size and Shape Effects of Pd@Pt Core–Shell Nanoparticles: Unique Role of Surface Contraction and Local Structural Flexibility

In this article, we present a density functional theory (DFT) study of nanoparticles (NPs) using a more realistic particle model, which allows us to model Pd@Pt core–shell NPs in size of 1–3 nm (number of atoms: 35–405) and shape [tetrahedron (TH); sphere-like truncated octahedron (SP)] precisely. Our results show that the size and shape have significant effects on the stability and activity of a Pd@Pt NP toward the oxygen reduction reaction (ORR). More importantly it is found for the first time that the variation in activity with particle size is shape-dependent. In addition, under the ORR conditions the adsorbate-driven structural changes on the terraces of nanoparticles can occur, which is relevant for understanding the observed activity and stability. According to our DFT calculations, the catalytic behaviors of Pd@Pt nanoparticles are associated with the surface contraction (compressive strain) and the local structural flexibility, which are strongly size- and shape-dependent. Our study demonstrates ...

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Specific Changes in the Local Flexibility of Plasminogen Activator Inhibitor 1 upon Binding to the Somatomedin B Domain of Vitronectin

The native fold of plasminogen activator inhibitor 1 (PAI-1) represents an active metastable conformation that spontaneously converts to an inactive latent form. Binding of the somatomedin B domain (SMB) of the endogenous cofactor vitronectin to PAI-1 delays the transition to the latent state and increases the thermal stability of the protein dramatically. We have used hydrogen/deuterium exchange mass spectrometry to assess the inherent structural flexibility of PAI-1 and to monitor the changes induced by SMB binding. Our data show that the PAI-1 core consisting of β-sheet B is rather protected against exchange with the solvent, while the remainder of the molecule is more dynamic. SMB binding causes a pronounced and widespread stabilization of PAI-1 that is not confined to the binding interface with SMB. We further explored the local structural flexibility in a mutationally stabilized PAI-1 variant (14-1B) as well as the effect of stabilizing antibody Mab-1 on wild-type PAI-1. The three modes of stabilizing PAI-1 (SMB, Mab-1, and the mutations in 14-1B) all cause a delayed latency transition, and this effect was accompanied by unique signatures on the flexibility of PAI-1. Reduced flexibility in the region around helices B, C, and I was seen in all three cases, which suggests an involvement of this region in mediating structural flexibility necessary for the latency transition. These data therefore add considerable depth to our current understanding of the local structural flexibility in PAI-1 and provide novel indications of regions that may affect the functional stability of PAI-1.

Local Flexibility Facilitates Oxidization of Buried Methionine Residues

In proteins, all amino acid residues are susceptible to oxidation by various reactive oxygen species (ROS), with methionine and cysteine residues being particularly sensitive to oxidation. Methionine oxidation is known to lead to destabilization and inactivation of proteins, and oxidatively modified proteins can accumulate during aging, oxidative stress, and in various age-related diseases. Although the efficiency of a given methionine oxidation can depend on its solvent accessibility (evaluated from a protein structure as the accessible surface area of the corresponding methionine residue), many experimental results on oxidation rate and oxidation sites cannot be unequivocally explained by the methionine solvent accessible surface area alone. In order to explore other possible mechanisms, we analyzed a set of seventy-one oxidized methionines contained in thirty-one proteins by various bioinformatics tools. In which, 41% of the methionines are exposed, 15% are buried but with various degree of flexibility, and the rest 44% are buried and structured. Buried but highly flexible methionines can be oxidized. Buried and less flexible methionines can acquire additional local structural flexibility from flanking regions to facilitate the oxidation. Oxidation of buried and structured methionine can also be promoted by the oxidation of neighboring methionine that is more exposed and/or flexible. Our data are consistent with the hypothesis that protein structural flexibility represents another important factor favoring the oxidation process.

Probing the α‐Helical Structural Stability of Stapled p53 Peptides: Molecular Dynamics Simulations and Analysis

Reactivation of the p53 cell apoptosis pathway through inhibition of the p53-hDM2 interaction is a viable approach to suppress tumor growth in many human cancers and stabilization of the helical structure of synthetic p53 analogs via a hydrocarbon cross-link (staple) has been found to lead to increased potency and inhibition of protein-protein binding (J. Am. Chem. Soc. 129: 5298). However, details of the structure and dynamic stability of the stapled peptides are not well understood. Here, we use extensive all-atom molecular dynamics simulations to study a series of stapled alpha-helical peptides over a range of temperatures in solution. The peptides are found to exhibit substantial variations in predicted alpha-helical propensities that are in good agreement with the experimental observations. In addition, we find significant variation in local structural flexibility of the peptides with the position of the linker, which appears to be more closely related to the observed differences in activity than the absolute alpha-helical stability. These simulations provide new insights into the design of alpha-helical stapled peptides and the development of potent inhibitors of alpha-helical protein-protein interfaces.

Simulation Study of Stapled Alpha-Helical P53 Peptide Analogs:probing the Relationship between Structural Stability and Biological Potency

Reactivation of the p53 cell apoptosis pathway through inhibition of the p53-hDM2 interaction is known to be a viable approach to suppressing tumor growth in many human cancers and stabilization of the helical structure of p53 analogs via a hydrocarbon cross-link (staple) has been found to lead to increased potency and inhibition of protein-protein binding. However, details of the structure and dynamic stability of the stapled peptides and their relationship to the nature and location of hydrocarbon linker are not well understood. Here, we use extensive molecular dynamics simulations to study a series of stapled α−helical peptides over a range of temperatures in solution. The peptides are found to exhibit substantial variations in predicted helicities that are in good agreement with the experimental values. In addition, we find significant variation in local structural flexibility of the peptides with the position of the linker, which appears to be more closely related to the observed differences in activity than the absolute helical stability. These simulations provide new insights into the design of α−helical stapled peptides and could aid in the development of potent inhibitors of protein interfaces.