Molecular Binding Sites Research Articles

Introduction of Protonated Sites on Exfoliated, Large-Area Sheets of Hexagonal Boron Nitride.

Hexagonal boron nitride (h-BN) sheets possess an exclusive set of properties, including wide energy band gap, high optical transparency, high dielectric breakdown strength, high thermal conductivity, UV cathodoluminescence, and pronounced thermochemical stability. However, functionalization of large h-BN layers has remained a challenge due to their chemical resistance and unavailable molecular-binding sites. Here we report on the protonation of h-BN via treatment with chlorosulfonic acid that not only exfoliates "large" h-BNs (up to 10 000 μm2) at high yields (∼23%) but also results in their covalent functionalization by introducing four forms of aminated nitrogen (N) sites within the h-BN lattice: sp2-delocalized and sp3-quaternary protonation on internal N sites (>N+═ and >NH+-) and pyridinic-like protonation on the edge N sites (═NH+- and -NH-). The presence of these groups transforms the chemically passive h-BN sheets to their chemically active form, which as demonstrated here can be used as scaffolds for forming composites with plasmonic gold nanoparticles and organic dye molecules. The dispersion of h-BNs exhibits an optical energy band gap of 5.74 eV and a zeta potential of ζ = +36.25 mV at pH = 6.1 (ζmax = +150 mV), confirming high dispersion stability. We envision that these two-dimensional nanomaterials with an atomically packed honeycomb lattice and high-energy band gap will evolve next-generation applications in controlled-UV emission, atomic-tunneling-barrier devices, ultrathin controlled-permeability membranes, and thermochemically resistive transparent coatings.

Quercetin: the bioactive compound from Allium cepa L. as anti-inflammation based on in silico screening

Inflammation is a tissue injury that occurs due to physical trauma or microbiological substances that involve the activities of many cell types. Inflammation can be prevented using the natural medicines from Allium cepa L. Quercetin is one of the bioactive compounds found in Allium cepa L and has been reported to have anti-inflammatory activity. The natural medicines have been used to minimize non-steroidal anti-inflammatory drugs. This study aims to investigated the modeling structures and the protein receptor from quecertin in inflammation mechanism and their optimization of the effectiveness in the human body. The bioinformatics tools used in this study are the database of quercetin compounds, Pubchem and Swis Target Prediction protein prediction databases, PyRx 0.8 molecular docking software, ligand docking, and binding site analysis with PyMOL and LigPlus software. The results from in silico show that quercetin compounds can interact with Muscleblind-like protein 1 target protein with a Binding Affinity minus value which is not much different from the dexamethasone compound. Dexamethason is a standart because it is a corticosteroid drug that can be used as an anti-inflammatory to reduce inflammation, allergic reactions, arthritis and other inflammatory diseases.

New Targets for Old Drugs: Cardiac Glycosides Inhibit Atrial-Specific K2P3.1 (TASK-1) Channels.

Cardiac glycosides have been used in the treatment of arrhythmias for more than 200 years. Two-pore-domain (K2P) potassium channels regulate cardiac action potential repolarization. Recently, K2P3.1 [tandem of P domains in a weak inward rectifying K+ channel (TWIK)-related acid-sensitive K+ channel (TASK)-1] has been implicated in atrial fibrillation pathophysiology and was suggested as an atrial-selective antiarrhythmic drug target. We hypothesized that blockade of cardiac K2P channels contributes to the mechanism of action of digitoxin and digoxin. All functional human K2P channels were screened for interactions with cardiac glycosides. Human K2P channel subunits were expressed in Xenopus laevis oocytes, and voltage clamp electrophysiology was used to record K+ currents. Digitoxin significantly inhibited K2P3.1 and K2P16.1 channels. By contrast, digoxin displayed isolated inhibitory effects on K2P3.1. K2P3.1 outward currents were reduced by 80% (digitoxin, 1 Hz) and 78% (digoxin, 1 Hz). Digitoxin inhibited K2P3.1 currents with an IC50 value of 7.4 µM. Outward rectification properties of the channel were not affected. Mutagenesis studies revealed that amino acid residues located at the cytoplasmic site of the K2P3.1 channel pore form parts of a molecular binding site for cardiac glycosides. In conclusion, cardiac glycosides target human K2P channels. The antiarrhythmic significance of repolarizing atrial K2P3.1 current block by digoxin and digitoxin requires validation in translational and clinical studies.

Conformational Plasticity in Tyrosine Kinase Inhibitor-Kinase Interactions Revealed with Fluorescence Spectroscopy and Theoretical Calculations.

To understand drug-protein dynamics, it is necessary to account for drug molecular flexibility and binding site plasticity. Herein, we exploit fluorescence from a tyrosine kinase inhibitor, AG1478, as a reporter of its conformation and binding site environment when complexed with its cognate kinase. Water-soluble kinases, aminoglycoside phosphotransferase APH(3')-Ia and mitogen-activated protein kinase 14 (MAPK14), were chosen for this study. On the basis of our prior work, the AG1478 conformation (planar or twisted) was inferred from the fluorescence excitation spectrum and the polarity of the AG1478-binding site was deduced from the fluorescence emission spectrum, while red-edge excitation shift (REES) probed the heterogeneity of the binding site (protein conformation and hydration) distributions in the protein conformational ensemble. In the AG1478-APH(3')-Ia complex, both twisted (or partially twisted) and planar AG1478 conformations were evidenced from emission wavelength-dependent excitation spectra. The binding site environment provided by APH(3')-Ia was moderately polar (λmax = 480 nm) with evidence for considerable heterogeneity (REES = 34 nm). In contrast, in the AG1478-MAPK14 complex, AG1478 was in a predominantly planar conformation with a lower degree of conformational heterogeneity. The binding site environment provided by the MAPK14 protein was of relatively low polarity (λmax = 430 nm) with a smaller degree of heterogeneity (REES = 11 nm). The results are compared with the available X-ray data and discussed in the context of our current understanding of tyrosine kinase inhibitor conformation and protein conformational ensembles.

Designing Selective Protein Binding Sites

Molecular recognition is a critical process for many biological functions and consists in non-covalent binding of different molecules, such as protein-protein, antigen-antibody and many others. The host-guest molecules involved often show a shape complementarity, and one of the main specification for molecular recognition is that the interaction must be selective, i.e. the host should bind strongly to one selected guest and poorly, if at all, to all other biomolecules. Our work focuses on the role played by the chemical heterogeneity and the steric compatibility on the selectivity power of the binding site between two proteins. We developed a computational design procedure, based on the caterpillar coarse-grained model, for a reference system where we fashioned the protein binding site as a protein-like surface which perfectly shapes a portion of the partner protein. The investigated range of surface area for the artificial binding sites falls in typical natural sizes (750 −1500 Å2). On the protein-like surface, we decorated anchoring points whose chemical functionalization is chosen so to optimise the binding with the protein. Using highly-efficient Monte Carlo simulations methods we explore the binding and folding properties of our artificial proteins. A significant result is that, despite the fact that protein and surface chemical sequences are interdependent and simultaneously generated to stabilise the bound folded structure, the protein is stable in the folded conformation even in the absence of the protein-like partner for all investigated systems. Moreover, we observe that an increase of the surface area results in a decrease of specificity for the binding, therefore imposing an upper limit for molecular recognition binding sites at the typical physiological temperature range.

Photochemical Identification of Molecular Binding Sites on the Surface of Amyloid-β Fibrillar Aggregates

Summary The aggregation of amyloid-β (Aβ) into insoluble fibrils has been associated with the development of Alzheimer's disease. The study of Aβ aggregation with [Re(CO) 3 (dppz)(Py)] + (dppz = dipyrido[3,2-a:2′,3′-c]phenazine; Py = pyridine) has led to the observation of an irradiation-induced light-switching response accompanied by the oxidation of the Aβ fibril. Here, we used the photophysical and photochemical properties of this complex, as well as spectroscopic and computational methods, to elucidate molecular binding sites on Aβ fibrils. [Re(CO) 3 (dppz)(Py)] + binds to Aβ fibrils with a dissociation constant of 4.2 μM and a binding stoichiometry 2.8:1 (Aβ/complex). Molecular dynamics (MD) simulations predicted binding of [Re(CO) 3 (dppz)(Py)] + through a hydrophobic cleft on the fibril axis between Val18 and Phe20. Tandem mass spectrometry analysis indicated that oxidation occurred at Met35, footprinting the place of binding, which is close to the site predicted by the MD simulations. Finding binding sites in Aβ is of great importance for the design of Aβ-binding drugs.

Molecular Dynamics Simulations Support Multiple Binding Sites for Phospholamban on SERCA

Ion‐motive ATPases use the energy of ATP hydrolysis to transport ions across membrane bilayer against a concentration gradient. Here we investigate the functional regulation of the sarco(endo)plasmic reticulum calcium ATPase (SERCA), which plays a key role in cellular Ca handling. In the heart, the activity of the SERCA is regulated by its inhibitory partner phospholamban (PLB). PLB has been shown to inhibit the transporter by binding to the SERCA M6 helix, but this site is not available when SERCA is bound to Ca. Our previous fluorescence resonance energy transfer (FRET) experiments suggested that SERCA Ca binding causes PLB to translocate from the known binding site to a putative secondary site. Based on an analogous interaction of the sodium/potassium ATPase (NKA) with a regulatory partner (FXYD10), we hypothesized that PLB could interact with the M9 helix on SERCA after leaving the canonical binding site. In the present study we performed molecular dynamics simulations to compare the energetics of binding of PLB to candidate sites on SERCA. We created models of the regulatory complex using structural information from available crystal structures of the PLB‐SERCA and FXYD10‐NKA complexes. Quantification of the interaction energies of these models suggested PLB can form a stable complex with both the canonical (−856 kJ/mol) and the proposed secondary site (−510 kJ/mol). In contrast, docking of PLB to the M3 helix on the opposite side of SERCA shows a much less favorable interaction energy (−108 kJ/mol). We also used umbrella sampling to quantify the free energy of PLB bound to several candidate binding sites on SERCA transmembrane domain. The results provide insight into the relative affinity of PLB for plausible sites of interaction on SERCA. Overall, the molecular dynamics simulations support a 2‐site model in which PLB interacts first with the high affinity conventional site and then relocates to a nearby secondary site when SERCA binds calcium.

Clickable Antifouling Polymer Brushes for Polymer Pen Lithography.

Protein-repellent reactive surfaces that promote localized specific binding are highly desirable for applications in the biomedical field. Nonspecific adhesion will compromise the function of bioactive surfaces, leading to ambiguous results of binding assays and negating the binding specificity of patterned cell-adhesive motives. Localized specific binding is often achieved by attaching a linker to the surface, and the other side of the linker is used to bind specifically to a desired functional agent, as e.g. proteins, antibodies, and fluorophores, depending on the function required by the application. We present a protein-repellent polymer brush enabling highly specific covalent surface immobilization of biorecognition elements by strain-promoted alkyne-azide cycloaddition click chemistry for selective protein adhesion. The protein-repellent polymer brush is functionalized by highly localized molecular binding sites in the low micrometer range using polymer pen lithography (PPL). Because of the massive parallelization of writing pens, the tunable PPL printed patterns can span over square centimeter areas. The selective binding of the protein streptavidin to these surface sites is demonstrated while the remaining polymer brush surface is resisting nonspecific adsorption without any prior blocking by bovine serum albumin (BSA). In contrast to the widely used BSA blocking, the reactive polymer brushes are able to significantly reduce nonspecific protein adsorption, which is the cause of biofouling. This was achieved for solutions of single proteins as well as complex biological fluids. The remarkable fouling resistance of the polymer brushes has the potential to improve the multiplexing capabilities of protein probes and therefore impact biomedical research and applications.

Molecular Dynamics Simulations Suggest Multiple Binding Sites for Phospholamban on SERCA

Molecular Dynamics Simulations Suggest Multiple Binding Sites for Phospholamban on SERCA

Abstract 3909: Interactions of glioma cells with chlorotoxin: an ATR-FTIR spectroscopy study

Abstract Chlorotoxin is a scorpion-derived peptide that preferentially binds to tumor cells of neuroectodermal origin, like glioma, but not to normal non-transformed cells. We have previously reported the development of a targeted nanodelivery system using chlorotoxin. Chlorotoxin has been studied as an imaging and targeting agent for drug and radioisotope delivery, but the mechanism of interaction of chlorotoxin with cancer cells has not been well understood and it needs to be further evaluated in order to optimize its use as a targeting compound for cancer cells. In this study, we used U87 human glioma cell line to evaluate the binding kinetics of chlorotoxin by Attenuated Total Reflection Fourier Transform Infra-Red (ATR-FTIR) spectroscopy. As a sensitive and label free technique, ATR-FTIR is a powerful diagnostic tool for the comparison of cancer cells with normal cells. First, we characterized the signature spectra of chlorotoxin and U87 cells by assigning and evaluating the infrared absorption bands and their second derivatives. Next, we studied the spectral differences between U87 cells incubated with and without chlorotoxin for incremental time periods ranging from 15 minutes to 24 hours. We also examined the metabolic changes in U87 cells induced with chlorotoxin at different incubation time points by measuring the ratios of integrated areas corresponding to different chemical conformations of proteins, lipids, carbohydrates, and nucleic acids. Our results revealed spectral changes in U87 cells at different stages of incubation with chlorotoxin. The most notable change occurred after 30 minutes of incubation, where the band assigned to CH3 bending of lipid membranes shifted from 1456 cm-1 to 1450 cm-1. Consequently, a new shoulder appeared at wavenumber of 1466 cm-1 assigned to CH2 bending of lipid membranes for U87 cells treated with chlorotoxin. These changes indicate a direct interaction due to the presence of a molecular binding site between chlorotoxin and the lipids of the cell membrane. Another significant alternation occurred after 1 hour of incubation of U87 cells with chlorotoxin in the spectral region assigned for nucleic acids. This interaction included a large upshift of the band at 1036 cm-1 to 1051 cm-1 assigned to C-O stretching vibrations of ribose ring in RNA due to the hydrogen bonding with chlorotoxin. These findings suggest at least two different interactions of chlorotoxin with U87 human glioma cells that can be further explored for improving chlorotoxin containing drug delivery systems. We are currently using the ATR-FTIR method to conduct control experiments by measuring possible interactions of chlorotoxin with normal human astrocytes. Citation Format: Rana Falahat, Marzenna Wiranowska, Ryan Toomey, Norma Alcantar. Interactions of glioma cells with chlorotoxin: an ATR-FTIR spectroscopy study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3909.

Stereochemical Recognition of Helicenes on Metal Surfaces.

The chiral recognition among biomolecules is fundamentally important for many processes of life, including the stereochemistry of evolution. Of special interest is chiral recognition during crystallization of racemates, when either homochiral recognition leads to a conglomerate of homochiral crystals or heterochiral recognition dominates resulting in a racemic compound. The complex nature of molecular recognition at the level of nucleation and crystal growth renders it difficult to understand and calls for manageable model systems. Notably, the approach of studying aggregation of molecules at surfaces under well-defined conditions includes the benefit of the availability of a multitude of highly sensitive investigation methods, of which scanning tunneling microscopy (STM) with its submolecular resolution is tremendously valuable. Heterogeneous nucleation at surfaces is strongly favored over homogeneous nucleation in solution; hence, surfaces are significantly involved in stereochemical recognition during crystallization. Helicenes are a fascinating class of chiral compounds with outstanding optical activity. These π-conjugated, ortho-fused, aromatic hydrocarbons are promising candidates for organic electronic devices such as sensors, circular dichroic photonics, liquid crystal displays or spin filters. But in particular the defined footprint of their terminal benzo rings on a surface makes them interesting for studying stereochemical recognition with different single crystalline surfaces and the impact this has, in turn, on intermolecular recognition. In this Account, we describe the self-assembly of helicenes on metal surfaces with the focus on stereochemical recognition in two-dimensional structures. Using the isomeric all-carbon helicenes, heptahelicene and dibenzohelicene as examples, different aggregation phenomena on different surfaces of single crystalline copper, silver, and gold are investigated. By means of STM different modes of transmission of molecular handedness from single molecules into extended two-dimensional supramolecular structures are identified. For the problem of racemate versus conglomerate crystallization, the impact of surface and molecular structure and their interplay are analyzed. This leads to detailed conclusions about the importance of the match of molecular and surface binding sites for long-range self-assembly. The absence of polar groups puts emphasis on van der Waals interaction and their maximization by steric overlap of molecular parts in enantiomeric and diastereomeric interactions. With STM as a manipulation tool, dimers are manually separated in order to analyze their chiral composition. And finally, new nonlinear cooperative effects induced by small enantiospecific bias are discovered that lead to single enantiomorphism in two-dimensional racemate crystals as well as in racemic multilayered films. By means of these model studies many details that govern chiral recognition at surfaces are rationalized.

Single Tripyridyl–Triazine Molecular Junction with Multiple Binding Sites

We present an electronic characterization of a single molecular junction of 2,4,6-tris(2′,2″,2‴-pyridyl)-1,3,5-triazine (TPTZ) with multiple metal–molecule binding sites using scanning tunneling microscopy-based break junction method under ambient conditions. The TPTZ molecule consists of a centered triazine moiety and surrounding three 2-pyridyl groups. The benzene rings containing a N atom in TPTZ act as molecular binding sites for bridging a gap between two Au electrodes to form a single molecular junction. Because the N atom at the ortho-position in the 2-pyridyl groups is spatially hidden from the electrode surfaces, the single molecular junction forms via direct metal−π couplings. We demonstrated that the single TPTZ molecular junctions exhibit highly conductive character up to 10–1 G0 (G0 = 2e2/h), which is due to the effect of the direct metal−π coupling. We found three preferential conductance states of ca. 10–1, 10–2, and 10–4 G0, which suggests that the single TPTZ molecular junctions have thre...

Computational allosteric ligand binding site identification on Ras proteins.

A number of computational techniques have been proposed to expedite the process of allosteric ligand binding site identification in inherently flexible and hence challenging drug targets. Some of these techniques have been instrumental in the discovery of allosteric ligand binding sites on Ras proteins, a group of elusive anticancer drug targets. This review provides an overview of these techniques and their application to Ras proteins. A summary of molecular docking and binding site identification is provided first, followed by a more detailed discussion of two specific techniques for binding site identification in ensembles of Ras conformations generated by molecular simulations.

Integral Molecular Sizes Up Ebola

Genetic Engineering & Biotechnology NewsVol. 36, No. 1 Corporate ProfileIntegral Molecular Sizes Up EbolaMembrane Protein Specialist Maps Ebola's Binding Sites to Advance Vaccine DiscoveryGail DuttonGail DuttonSearch for more papers by this authorPublished Online:26 Dec 2015https://doi.org/10.1089/gen.36.01.05AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byProduction, characterization, and epitope mapping of monoclonal antibodies of ribosomal protein S3 (rpS3)6 October 2021 | Animal Cells and Systems, Vol. 25, No. 5 Volume 36Issue 1Jan 2016 Information© 2016 by GEN PublishingTo cite this article:Gail Dutton.Integral Molecular Sizes Up Ebola.Genetic Engineering & Biotechnology News.Jan 2016.8-9.http://doi.org/10.1089/gen.36.01.05Published in Volume: 36 Issue 1: December 26, 2015PDF download

Characterization of the dextran-binding domain in the glucan-binding protein C of Streptococcus mutans.

Streptococcus mutans produces multiple glucan-binding proteins (Gbps), among which GbpC encoded by the gbpC gene is known to be a cell-surface-associated protein involved in dextran-induced aggregation. The purpose of the present study was to characterize the dextran-binding domain of GbpC using bioinformatics analysis and molecular techniques. Bioinformatics analysis specified five possible regions containing molecular binding sites termed GB1 through GB5. Next, truncated recombinant GbpC (rGbpC) encoding each region was produced using a protein expression vector and five deletion mutant strains were generated, termed CDGB1 through CDGB5 respectively. The dextran-binding rates of truncated rGbpC that included the GB1, GB3, GB4 and GB5 regions in the upstream sequences were higher than that of the construct containing GB2 in the downstream region. In addition, the rates of dextran-binding for strains CDGB4 and CD1, which was entire gbpC deletion mutant, were significantly lower than for the other strains, while those of all other deletion mutants were quite similar to that of the parental strain MT8148. Biofilm structures formed by CDGB4 and CD1 were not as pronounced as that of MT8148, while those formed by other strains had greater density as compared to that of CD1. Our results suggest that the dextran-binding domain may be located in the GB4 region in the interior of the gbpC gene. Bioinformatics analysis is useful for determination of functional domains in many bacterial species.

Covalent immobilization of molecularly imprinted polymer nanoparticles on a gold surface using carbodiimide coupling for chemical sensing

One challenging task in building (bio)chemical sensors is the efficient and stable immobilization of receptor on a suitable transducer. Herein, we report a method for covalent immobilization of molecularly imprinted core–shell nanoparticles for construction of robust chemical sensors. The imprinted nanoparticles with a core–shell structure have selective molecular binding sites in the core and multiple amino groups in the shell. The model Au transducer surface is first functionalized with a self-assembled monolayer of 11-mercaptoundecanoic acid. The 11-mercaptoundecanoic acid is activated by treatment with carbodiimide/N-hydroxysuccinimide and then reacted with the core–shell nanoparticles to form amide bonds. We have characterized the process by studying the treated surfaces after each preparation step using atomic force microscopy, scanning electron microscopy, fluorescence microscopy, contact angle measurements and X-ray photoelectron spectroscopy. The microscopy results show the successful immobilization of the imprinted nanoparticles on the surface. The photoelectron spectroscopy results further confirm the success of each functionalization step. Further, the amino groups on the MIP surface were activated by electrostatically adsorbing negatively charged Au colloids. The functionalized surface was shown to be active for surface enhanced Raman scattering detection of propranolol. The particle immobilization and surface enhanced Raman scattering approach described here has a general applicability for constructing chemical sensors in different formats.

EMMPRIN/CD147 is a novel coreceptor of VEGFR-2 mediating its activation by VEGF.

EMMPRIN/CD147 is mainly known for its protease inducing function but a role in promoting tumor angiogenesis has also been demonstrated. This study provides evidence that EMMPRIN is a new coreceptor for the VEGFR-2 tyrosine kinase receptor in both endothelial and tumor cells, as it directly interacts with it and regulates its activation by its VEGF ligand, signalling and functional consequences both in vitro and in vivo. Computational docking analyses and mutagenesis studies identified a molecular binding site in the extracellular domain of EMMPRIN located close to the cell membrane and containing the amino acids 195/199. EMMPRIN is overexpressed in cancer and hence is able to further potentiate VEGFR-2 activation, suggesting that a combinatory therapy of an antiangiogenic drug together with an inhibitor of EMMPRIN/VEGFR-2 interaction may have a greater impact on inhibiting angiogenesis and malignancy.

Covalent immobilization of molecularly imprinted polymer nanoparticles using an epoxy silane

Molecularly imprinted polymers (MIPs) can be used as antibody mimics to develop robust chemical sensors. One challenging problem in using MIPs for sensor development is the lack of reliable conjugation chemistry that allows MIPs to be fixed on transducer surface. In this work, we study the use of epoxy silane to immobilize MIP nanoparticles on model transducer surfaces without impairing the function of the immobilized nanoparticles. The MIP nanoparticles with a core–shell structure have selective molecular binding sites in the core and multiple amino groups in the shell. The model transducer surface is functionalized with a self-assembled monolayer of epoxy silane, which reacts with the core–shell MIP particles to enable straightforward immobilization. The whole process is characterized by studying the treated surfaces after each preparation step using atomic force microscopy, scanning electron microscopy, fluorescence microscopy, contact angle measurements and X-ray photoelectron spectroscopy. The microscopy results show that the MIP particles are immobilized uniformly on surface. The photoelectron spectroscopy results further confirm the action of each functionalization step. The molecular selectivity of the MIP-functionalized surface is verified by radioligand binding analysis. The particle immobilization approach described here has a general applicability for constructing selective chemical sensors in different formats.

Predicting Peptide Binding Sites on Protein Surfaces by Clustering Chemical Interactions

Short peptides play important roles in cellular processes including signal transduction, immune response and transcription regulation. Correct identification of the peptide binding site on a given protein surface is of great importance not only for mechanistic investigation of these biological processes but also for therapeutic development. In the present study, we developed a novel computational approach, referred to as ACCLUSTER, for predicting the peptide binding sites on protein surfaces. Specifically, we use the twenty standard amino acids as probes to globally scan the protein surface. The poses forming good chemical interactions with the protein are identified, followed by clustering with the Density-Based Spatial Clustering of Applications with Noise (DBSCAN) technique. Finally, these clusters are ranked based on their sizes. The cluster with the largest size is predicted as the putative binding site. Assessment of ACCLUSTER was performed on a diverse test set of 251 non-redundant protein-peptide complexes. The results were compared with the performance of POCASA, a pocket detection method for ligand binding site prediction. Peptidb, another protein-peptide database that contains both bound structures and unbound or homologous structures was used to test the robustness of ACCLUSTER. The performance of ACCLUSTER was also compared with PepSite2 and PeptiMap, two recently developed methods developed for identifying peptide binding sites. The results showed that ACCLUSTER is a promising method for peptide binding site prediction. Additionally, ACCLUSTER was also shown to be applicable to small molecular binding site prediction and protein-protein interface prediction. Notably, ACCLUSTER is based on physical interactions rather than informatics training, and can easily be extended to other macromolecular systems, including systems like peptide-RNA complexes which lack sufficient structural data and therefore pose challenges to machine-learning based methods.

Molecular binding sites are located near the interface of intrinsic dynamics domains (IDDs).

We provide evidence supporting that protein-protein and protein-ligand docking poses are functions of protein shape and intrinsic dynamics. Over sets of 68 protein-protein complexes and 240 nonhomologous enzymes, we recognize common predispositions for binding sites to have minimal vibrations and angular momenta, while two interacting proteins orient so as to maximize the angle between their rotation/bending axes (>65°). The findings are then used to define quantitative criteria to filter out docking decoys less likely to be the near-native poses; hence, the chances to find near-native hits can be doubled. With the novel approach to partition a protein into "domains" of robust but disparate intrinsic dynamics, 90% of catalytic residues in enzymes can be found within the first 50% of the residues closest to the interface of these dynamics domains. The results suggest an anisotropic rather than isotropic distribution of catalytic residues near the mass centers of enzymes.