Protein-RNA Docking Benchmark v3.0 Integrated With Binding Affinity.

The motor enzymes that belong to the family of RNA helicases catalyze the strand separation of duplex RNA via ATP hydrolysis. Among these enzymes, Escherichia coli DbpA is a unique RNA helicase because it possesses ATPase-specific activity toward the peptidyl transferase center in 23 S ribosomal RNA. For this reason, it has been the subject of numerous biochemical and structure-function studies. The ATP-stimulated unwinding activity of DbpA toward specific and nonspecific RNA duplexes has been demonstrated. However, the underlying molecular and structural basis, which facilitates its helicase activities, is presently not known. We combined time-dependent limited proteolysis digestion, fluorescence spectroscopy, and three-dimensional structural homology modeling techniques to study the structural conformations of DbpA with respect to its binding to stoichiometric ratios of RNA and cofactors. We show that the conformational state of DbpA is markedly different in the ADP-bound state than in any other state (ATP- or RNA-bound). These results, together with structural homology studies, suggest that a hinge region located in the core domain of DbpA mediates such conformational changes.

As a part of studies on the mechanism of action of antibiotics polyoxins, effects of various N-aminoacyl derivatives of polyoxin C and other polyoxins on chitin-UDP acetylglucosaminyl-transferase (EC 2.4.1.16, chitin synthetase) prepared from phytopathogenic fungus Piricularia oryzae were investigated. It was found that they inhibited the enzyme in competition with the substrate UDP-N-acetylglucosamine, Inhibitor constants, Ki, for these polyoxins were determined and the values of binding affinity, −ΔGbind of the inhibitors to the enzyme were calculated from the Ki values. In addition, by using these −ΔGbind values the values of partial binding affinity, −Δg for the several atoms and atomic groups or the several moieties contained in the polyoxin J molecule were estimated. From the results obtained, it was concluded that the carbamoylpolyoxamic acid moiety of polyoxins helps to stabilize the polyoxin-enzyme complex through the contributions of its oxygen atom at C−1″, amino group at C−2″, hydroxyl groups at C−3″ and C−4″, aliphatic carbon chain and terminal carbamoyloxy group. The results obtained by the kinetic investigation using various nucleotides and nucleotide sugars suggested that there was a specific binding site on the enzyme corresponding to the uridine moiety of UDP-N-acetylglucosamine, and that the pyrimidine nucleoside moiety of polyoxins was also bound to this site.

This paper describes novel adaptations of optically sectioned planar format assays to screen compounds for their affinities to materials surfaces. The novel platform, which we name optically sectioned indicator displacement assays (O-IDA), makes use of displaceable dyes in a format adaptable to high-throughput multiwell plate technologies. We describe two approaches: the first being where the dye exhibits fluorescence in both the surface bound and unbound state and the second, where fluorescence is lost upon displacement of the dye from the surface. Half maximal inhibitory concentration (IC50), binding affinity (Ki), and binding free energy (ΔGads) values can be extracted from the raw data. Representative biomolecules were tested for interactions with silica in an aqueous environment and ZnO(0001)-Zn and (10-10) facets in a nonaqueous environment. We provide the first experimental values for both the binding of small molecules to silica and the facet-dependent ZnO binding affinity of key amino acids associated with ZnO-specific oligopeptides. The specific data will be invaluable to those studying interactions at interfaces both experimentally and computationally. O-IDA provides a general framework for the high-throughput screening of molecule binding to materials surfaces, which has important applications in drug delivery, (bio-) catalysis, biosensing, and biomaterial engineering.

RNA-binding proteins play many essential roles in the regulation of gene expression. In the cell, mRNA molecules and their precursors are always bound by proteins. RNA binding protein that are involved in RNA processing, cellular localization, gene expression, regulation, transcription and translation have been identified, and structural domains involved in RNA recognition have been described (Siomi et al., 1997; Cusack et al. 1999; Stefl et al. 2005). RNA-binding proteins are an extremely diverse group of proteins, reflecting the different functional requirements of different types of RNA molecules (Andreev et al. 2004). However, despite their obvious functional importance, the specific mechanisms of protein RNA interactions are still poorly understand. Identification of the most putative RNA-binding residues in these proteins is an important and challenging problem of molecular recognition. Despite the significant increase in the number of structures for RNA-protein complexes in the last few years, the molecular basis of specificity remains unclear even for the best studied protein families. Very few studies (Jeong et al. 2004) have been addressed so far to the important problem of predicting RNA-interacting sites in the protein as a critical goal in the field of molecular recognition. We have developed a method for identification of RNA-binding residues using machine learning approaches. Several machine learning techniques and feature selection methods based on protein composition, sequence, charge and structural information have been investigated for their classification accuracy. In detailed cross validation analysis using non redundant RNA-protein complexes deposited in the protein data bank, our method shows satisfactory performance for identification of RNA-binding residues.

The calcium- and integrin-binding protein 1 (CIB1) is a ubiquitous Ca(2+)-binding protein and a specific binding partner for the platelet integrin αIIb cytoplasmic domain, which confers the key role of CIB1 in hemostasis. CIB1 is also known to be involved in apoptosis, embryogenesis, and the DNA damage response. In this study, the solution structures of both Ca(2+)-CIB1 and Mg(2+)-CIB1 were determined using solution-state NMR spectroscopy. The methyl groups of Ile, Leu, and Val were selectively protonated to compensate for the loss of protons due to deuteration. The solution structure of Ca(2+)-CIB1 possesses smaller opened EF-hands in its C-domain compared with available crystal structures. Ca(2+)-CIB1 and Mg(2+)-CIB1 have similar structures, but the N-lobe of Mg(2+)-CIB1 is slightly more opened than that of Ca(2+)-CIB1. Additional NMR experiments, such as chemical shift perturbation and methyl group solvent accessibility as measured by a nitroxide surface probe, were carried out to further characterize the structures of Ca(2+)-CIB1 and Mg(2+)-CIB1 as well as their interactions with the integrin αIIb cytoplasmic domain. NMR measurements of backbone amide proton slow motion (microsecond to millisecond) dynamics confirmed that the C-terminal helix of Ca(2+)-CIB1 is displaced upon αIIb binding. The EF-hand III of both Ca(2+)-CIB1 and Mg(2+)-CIB1 was identified to be directly involved in the interaction of CIB1 with αIIb. Together, these data illustrate that CIB1 behaves quite differently from related EF-hand regulatory calcium-binding proteins, such as calmodulin or neuronal calcium sensor proteins.

SelB is an elongation factor needed for the co-translational incorporation of selenocysteine. Selenocysteine is coded by a UGA stop codon in combination with a specific downstream mRNA hairpin. In bacteria, the C-terminal part of SelB recognizes this hairpin, while the N-terminal part binds GTP and tRNA in analogy with elongation factor Tu (EF-Tu). We present the crystal structure of a C-terminal fragment of SelB (SelB-C) from Moorella thermoacetica at 2.12 A resolution, solved by a combination of selenium and yttrium multiwavelength anomalous dispersion. This 264 amino acid fragment contains the entire C-terminal extension beginning after the EF-Tu-homologous domains. SelB-C consists of four similar winged-helix domains arranged into the shape of an L. This is the first example of winged-helix domains involved in RNA binding. The location of conserved basic amino acids, together with data from the literature, define the position of the mRNA-binding site. Steric requirements indicate that a conformational change may occur upon ribosome interaction. Structural observations and data in the literature suggest that this change happens upon mRNA binding.

Protein–RNA recognition often induces conformational changes in binding partners. Consequently, the solvent accessible surface area (SASA) buried in contact estimated from the co-crystal structures may differ from that calculated using their unbound forms. To evaluate the change in accessibility upon binding, we compare SASA of 126 protein-RNA complexes between bound and unbound forms. We observe, in majority of cases the interface of both the binding partners gain accessibility upon binding, which is often associated with either large domain movements or secondary structural transitions in RNA-binding proteins (RBPs), and binding-induced conformational changes in RNAs. At the non-interface region, majority of RNAs lose accessibility upon binding, however, no such preference is observed for RBPs. Side chains of RBPs have major contribution in change in accessibility. In case of flexible binding, we find a moderate correlation between the binding free energy and change in accessibility at the interface. Finally, we introduce a parameter, the ratio of gain to loss of accessibility upon binding, which can be used to identify the native solution among the flexible docking models. Our findings provide fundamental insights into the relationship between flexibility and solvent accessibility, and advance our understanding on binding induced folding in protein-RNA recognition.

Lantana camara mengandung beberapa jenis flavonoid seperti Umuhengerin, Lantadene A, Lantadene B, Ictero -genin. Salah satu mekanisme kerja flavonoid sebagai antiinflammatoryis dengan menghambat ceptor siklooksigenase (COX). Penelitian ini bertujuan untuk mengetahui afinitas senyawa flavonoid yang terkandung dalam L. camara dengan reseptor COX-1 sebagai antiinflamasi dan untuk mengetahui ikatan hidrogen yang dapat dibentuk dengan metode molecular docking. Pengujian in silico dilakukan berdasarkan metode desain obat yang dibantu komputer. Alat-alat yang digunakan dalam penelitian ini, perangkat lunak PyRx, perangkat lunak PyMol, Pubchem (senyawa database), PDB (Protein Data Bank), dan Po-seView. Berdasarkan hasil docking dan pembahasan di atas dapat disimpulkan bahwa Icterogenin com-pound dan senyawa Umuhengerin adalah senyawa yang paling efektif dalam proses antiinflamasi, Icterogenin com-pound memiliki nilai RMSD 41,1 Å dan nilai afinity mengikat -8,8 dan senyawa Umuhengerin memiliki val-ue RMSD 1, 61 Å dan nilai afinitas pengikatan -8.0. Ini menunjukkan bahwa senyawa tersebut memiliki nilai RMSD dari senyawa Icterogenin. Namun, senyawa Icterogenin memiliki ikatan hidrogen yang lebih kuat dan lebih efisien daripada senyawa Umuhengerin karena nilai afinitas pengikatan terendah.

In silico docking studies of 50 selected compounds from Millettia dielsiana Harms ex Diels (family Leguminosae) were docked into the binding pocket of the PI3K/mTOR protein. In there, compounds trans−3−O-p-hydroxycinnamoyl ursolic acid (1) and 5,7,4′−trihydroxyisoflavone 7−O−β−D−apiofuranosyl−(1→6)−β−D−glucopyranoside (2) are predicted to be very promising inhibitors against PI3K/mTOR. They direct their cytotoxic activity against Hepatocellular carcinoma with binding affinity (BA) values, the pulling work spent to the co-crystallized ligand from the binding site of PI3K/mTOR (W and Fmax), and the non-equilibrium binding free energy (∆GneqJar) as BA values = −9.237 and −9.083 kcal/mol, W = 83.5 ± 10.6 kcal/mol with Fmax = 336.2 ± 45.3 pN and 126.6 ± 21.7 kcal/mol with Fmax = 430.3 ± 84.0 pN, and ∆GneqJar = −69.86074 and −101.2317 kcal/mol, respectively. In molecular dynamic simulation, the RMSD value of the PI3K/mTOR complex with compounds (1 and 2) was in the range of 0.3 nm to the end of the simulation. Therefore, the compounds (1 and 2) are predicted to be very promising inhibitors against PI3K/mTOR. The crude extract, ethyl acetate fraction and compounds (1 and 2) from Millettia dielsiana exhibited moderate to potent in vitro cytotoxicity on Hepatocellular carcinoma cell line with IC50 values of 81.2 µg/mL, 60.4 µg/mL, 23.1 μM, and 16.3 μM, respectively, and showed relatively potent to potent in vitro antioxidant activity on mouse hepatocytes with ED50 values of 24.4 µg/mL, 19.3 µg/mL, 30.7 μM, and 20.5 μM, respectively. In conclusion, Millettia dielsiana and compounds (1 and 2) are predicted to have very promising cytotoxic activity against Hepatocellular carcinoma and have a hepatoprotective effect.

Melanoma represents the most malignant type of skin cancer. It is estimated that approximately 100,000 new cases of melanoma were diagnosed in 2022, resulting in over 7600 deaths in the United States alone. Recently, anticancer peptides (ACPs) have emerged as novel therapeutic agents for cancer, offering higher potency, biocompatibility, and fewer adverse reactions in host cells. One of the druggable targets of melanoma is the melanoma inhibitor of apoptosis (ML-IAP), conventionally inhibited by the nonapeptide AVPIAQKSE. The current study is aimed at enhancing both the binding affinity and safety profile of this peptide through in silico peptide engineering. Initially, the 3D structure of the protein was downloaded from the Protein Data Bank (PDB) (ID: 1OXQ) and prepared. The hotspot residues at the interface were detected using Discovery Studio Client 2021. Afterwards, saturation mutagenesis was conducted to discover the best potential amino acid substitutions with a positive impact on the binding affinity. The lead candidates were docked to the receptor via HPEPDOCK 2. Additionally, the safety profile was assessed using the ToxIBTL and AllerCatPro 2 servers. Finally, molecular dynamics simulations and principal component analysis were performed to check the stability of the best complexes. HVPIAQKSE, WVPWAQKSE, and HVPWAQKSE were the best mutants that could be superior to the original peptide in terms of binding affinity as well as safety profile. MD results confirmed the stability, flexibility, reduced local motions, conformational changes, and more compact structure upon binding the receptor for 200 ns, which deserve in vitro validation as a better melanoma ACP therapeutic option. These variants displayed increased flexibility, reduced conformational alterations and local motions, and a more compact configuration, suggesting greater stability compared with the reference peptide.

Ligand conformational strain energy (LCSE) plays an important role in virtual screening and lead optimization. While various studies have provided insights into LCSE for small-molecule ligands in the Protein Data Bank (PDB), conclusions are inconsistent mainly due to small datasets, poor quality control of crystal structures, and molecular mechanics (MM) or low-level quantum mechanics (QM) calculations. Here, we built a high-quality dataset (LigBoundConf) of 8145 ligand-bound conformations from PDB crystal structures and calculated LCSE at the M062X-D3/ma-TZVPP (SMD)//M062X-D3/def2-SVP(SMD) level for each case in the dataset. The mean/median LCSE is 4.6/3.7 kcal/mol for 6672 successfully calculated cases, which is significantly lower than the estimates based on molecular mechanics in many previous analyses. Especially, when removing ligands with nonaromatic ring(s) that are prone to have large LCSEs due to electron density overfitting, the mean/median LCSE was reduced to 3.3/2.5 kcal/mol. We further reveal that LCSE is correlated with several ligand properties, including formal atomic charge, molecular weight, number of rotatable bonds, and number of hydrogen-bond donors and acceptors. In addition, our results show that although summation of torsion strains is a good approximation of LCSE for most cases, for a small fraction (about 6%) of our dataset, it underestimates LCSEs if ligands could form nonlocal intramolecular interactions in the unbound state. Taken together, our work provides a comprehensive profile of LCSE for ligands in PDB, which could help ligand conformation generation, ligand docking pose evaluation, and lead optimization.

Investigating the binding between proteins and aptamers, such as peptides or RNA molecules, is of crucial importance both for understanding the molecular mechanisms that regulate cellular activities and for therapeutic applications in several pathologies. Here, a new computational procedure, employing mainly docking, clustering analysis, and molecular dynamics simulations, was designed to estimate the binding affinities between a protein and some RNA aptamers, through the investigation of the dynamical behavior of the predicted molecular complex. Using the state-of-the-art software catRAPID, we computationally designed a set of RNA aptamers interacting with the TAR DNA-binding protein 43 (TDP-43), a protein associated with several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). We thus devised a computational protocol to predict the RNA-protein molecular complex, so that the structural and dynamical behavior of such a complex can be investigated through extensive molecular dynamics simulation. We hypothesized that the coordinated and synchronized motion of the protein-binding residues, when in contact with RNA molecule, is a critical requisite in order to have a stable binding. Indeed, we calculated the motion covariance exhibited by the interface residues during molecular dynamics simulation: we tested the results against experimental measurements of binding affinity (in this case, the dissociation constant) for six RNA molecules, resulting in a linear correlation of about 0.9. Our findings suggest that the synchronized movement of interface residues plays a pivotal role in ensuring the stability within RNA-protein complexes, moreover providing insights into the contribution of each interface residue. This promising pipeline could thus contribute to the design of RNA aptamers interacting with proteins.

Tubulointerstitial nephritis is a cardinal renal manifestation of leptospirosis. LipL32, a major lipoprotein and a virulence factor, locates on the outer membrane of the pathogen Leptospira. It evades immune response by recognizing and adhering to extracellular matrix components of the host cell. The crystal structure of Ca(2+)-bound LipL32 was determined at 2.3 A resolution. LipL32 has a novel polyD sequence of seven aspartates that forms a continuous acidic surface patch for Ca(2+) binding. A significant conformational change was observed for the Ca(2+)-bound form of LipL32. Calcium binding to LipL32 was determined by isothermal titration calorimetry. The binding of fibronectin to LipL32 was observed by Stains-all CD and enzyme-linked immunosorbent assay experiments. The interaction between LipL32 and fibronectin might be associated with Ca(2+) binding. Based on the crystal structure of Ca(2+)-bound LipL32 and the Stains-all results, fibronectin probably binds near the polyD region on LipL32. Ca(2+) binding to LipL32 might be important for Leptospira to interact with the extracellular matrix of the host cell.

Protein-RNA complexes are increasingly important in our understanding of cell signaling, metabolism, and transcription. Electrostatic interactions play dominant role in stabilizing such complexes. Using conventional computational approaches, very long simulations of both bound and unbound states are required to obtain accurate estimates of complex dissociation constants (Kd). Here, we derive a simple formula that offers an alternative approach based on the theory of fluctuations. Our method extracts a strong correlate to experimental Kd values using short molecular dynamics simulations of the bound complex only. To test our method, we compared the computed relative Kd values to our experimentally measured values for the U1A-Stem Loop 2 (SL2) RNA complex, which is one of the most-studied protein-RNA complexes. Additionally we also included several experimental values from the literature, to enlarge the data set. We obtain a correlation of r = 0.93 between theoretical and measured estimates of Kd values of the mutated U1A protein-RNA complexes relative to the wild type dissociation constant. The proposed method increases the efficiency of relative Kd values estimation for multiple protein mutants, allowing its applicability to protein engineering projects.

The cryo-EM structure of ETB-Gi complex, obtained using the Fusion-G system, provides insights into the distinct activation mechanism and G-protein promiscuity of ETB, aiding in the design of ETB agonists for therapeutic applications.