Calculation of affinities of peptides for proteins

Several methodologies were employed to calculate the Gibbs standard free energy of binding for a collection of protein-ligand complexes, where the ligand is a peptide and the protein is representative for various protein families. Almost 40 protein-ligand complexes were employed for a continuum approach, which considers the protein and the peptide at the atomic level, but includes solvent as a polarizable continuum. Five protein-ligand complexes were employed for an all-atom approach that relies on a combination of the double decoupling method with thermodynamic integration and molecular dynamics. These affinities were also computed by means of the linear interaction energy method. Although it generally proved rather difficult to predict the absolute free energies correctly, for some protein families the experimental ranking order was correctly reproduced by the continuum and all-atom approach. Considerable attention has also been given to correctly analyze the affinities of charged peptides, where it is required to judge the effect of one or more ions that are being decoupled in an all-atom approach to preserve electroneutrality. The various methods are further judged upon their merits.     

Intermolecular Interactions and Protein Dynamics by Solid‐State NMR Spectroscopy

Understanding the dynamics of interacting proteins is a crucial step toward describing many biophysical processes. Here we investigate the backbone dynamics for protein GB1 in two different assemblies: crystalline GB1 and the precipitated GB1–antibody complex with a molecular weight of more than 300 kDa. We perform these measurements on samples containing as little as eight nanomoles of GB1. From measurements of site‐specific ¹⁵N relaxation rates including relaxation dispersion we obtain snapshots of dynamics spanning nine orders of magnitude in terms of the time scale. A comparison of measurements for GB1 in either environment reveals that while many of the dynamic features of the protein are conserved between them (in particular for the fast picosecond–nanosecond motions), much greater differences occur for slow motions with motions in the >500 ns range being more prevalent in the complex. The data suggest that GB1 can potentially undergo a small‐amplitude overall anisotropic motion sampling the interaction interface in the complex.     

Hydration in drug design. 1. Multiple hydrogen-bonding features of water molecules in mediating protein-ligand interactions

Summary Water is known to play an important rôle in the recognition and stabilization of the interaction between a ligand and its site. This has important implications for drug design. Analyses of 19 high-resolution crystal structures of protein-ligand complexes reveal the multiple hydrogen-bonding feature of water molecules mediating protein-ligand interactions. Most of the water molecules (nearly 80%) involved in bridging the protein and the ligand can make three or more hydrogen bonds when distance and bond angles are used as criteria to define hydrogen-bonding interactions. Isotropic B-factors have been used to take into account the mobility of water molecules. The water molecules at binding sites bridge the protein and ligand, and interact with other water molecules to form a complex network of interconnecting hydrogen bonds. Some water molecules at the site do not directly bridge between the protein and the ligand, but may contribute indirectly to the stability of the complex by holding bridging water molecules in the right position through a network of hydrogen bonds. These water networks are probably crucial for the stability of the protein-ligand complex and are important for any site-directed drug design strategies.     

Top-down ESI-ECD-FT-ICR mass spectrometry localizes noncovalent protein-ligand binding sites

Mass spectrometry (MS) with electrospray ionization (ESI) has the capability to measure and detect noncovalent protein-ligand and protein-protein complexes. However, information on the sites of ligand binding is not easily obtained by the ESI-MS methodology. Electron capture dissociation (ECD) favors cleavage of covalent backbone bonds of protein molecules. We show that this characteristic of ECD translates to noncovalent protein-ligand complexes, as covalent backbone bonds of protein complexes are dissociated, but the noncovalent ligand interaction is retained. For the complex formed from 140-residue, 14.5 kDa alpha-synuclein protein, and one molecule of polycationic spermine (202 Da), ECD generates product ions that retain the protein-spermine noncovalent interaction. Spermine binding is localized to residues 106-138; the ECD data are consistent with previous solution NMR studies. Our studies suggest that ECD mass spectrometry can be used to determine directly the sites of ligand binding to protein targets.     

A comprehensive characterization of the T-cell antigen receptor complex composition by microcapillary liquid chromatography-tandem mass spectrometry

It has become apparent that many intracellular signaling processes involve the dynamic reorganization of cellular proteins into complex signaling assemblies that have a specific subunit composition, function, and subcellular location. Since the elements of such assemblies interact physically, multiprotein signaling complexes can be isolated and analyzed. Recent technical advances in highly sensitive protein identification by electrospray-tandem mass spectrometry have dramatically increased the sensitivity with which such analyses can be performed. The T-cell antigen receptor (TCR) is an oligomeric transmembrane protein complex that is essential to T-cell recognition and function. The extracellular protein domains are responsible for ligand binding while intracellular domains generate and transduce signals in response to specific receptor-ligand interactions. We used microbore capillary chromatography-tandem mass spectrometry to investigate the composition of the TCR protein complex isolated from resting and activated cells of the murine T-cell line CD11.3. We identified all the previously known subunits of the TCR/CD3 complex as well as proteins previously not known to associate with the TCR. The catalytic activities of some of these proteins could potentially be used to interfere pharmacologically with TCR signaling.     

Statistical potential for modeling and ranking of protein-ligand interactions

Applications in structural biology and medicinal chemistry require protein-ligand scoring functions for two distinct tasks: (i) ranking different poses of a small molecule in a protein binding site and (ii) ranking different small molecules by their complementarity to a protein site. Using probability theory, we developed two atomic distance-dependent statistical scoring functions: PoseScore was optimized for recognizing native binding geometries of ligands from other poses and RankScore was optimized for distinguishing ligands from nonbinding molecules. Both scores are based on a set of 8,885 crystallographic structures of protein-ligand complexes but differ in the values of three key parameters. Factors influencing the accuracy of scoring were investigated, including the maximal atomic distance and non-native ligand geometries used for scoring, as well as the use of protein models instead of crystallographic structures for training and testing the scoring function. For the test set of 19 targets, RankScore improved the ligand enrichment (logAUC) and early enrichment (EF(1)) scores computed by DOCK 3.6 for 13 and 14 targets, respectively. In addition, RankScore performed better at rescoring than each of seven other scoring functions tested. Accepting both the crystal structure and decoy geometries with all-atom root-mean-square errors of up to 2 ? from the crystal structure as correct binding poses, PoseScore gave the best score to a correct binding pose among 100 decoys for 88% of all cases in a benchmark set containing 100 protein-ligand complexes. PoseScore accuracy is comparable to that of DrugScore(CSD) and ITScore/SE and superior to 12 other tested scoring functions. Therefore, RankScore can facilitate ligand discovery, by ranking complexes of the target with different small molecules; PoseScore can be used for protein-ligand complex structure prediction, by ranking different conformations of a given protein-ligand pair. The statistical potentials are available through the Integrative Modeling Platform (IMP) software package (http://salilab.org/imp) and the LigScore Web server (http://salilab.org/ligscore/).     

Impact of Stable Protein-protein Interaction on Protein Conformational Space

Backbone dihedral angle based clustering approach was applied to investigate the effect of protein complexation on backbone conformational space and the effect on protein dynamics. Three representative enzyme-inhibitor complexes and their comprised proteins were used as models for small-and moderate-sized globular proteins to compare available backbone conformational space before and after complexation. Microsecond time scale molecular dynamic simulations were generated to ensure sufficient statistics. The result suggests that stable protein-protein interactions lead to redistribution of protein backbone mobility and restriction of the protein backbone conformational space, especially for short time scale motions. Surprisingly, these effects are found to be uncorrelated with protein-protein interaction surface. Consistent with many experimental and computational observations, our results indicate that both induced-fit and conformational selection models play roles in stable protein complexation process, with the dominant role being different for different protein complexes.     

Selective detection of specific protein-ligand complexes by electrosonic spray-precursor ion scan tandem mass spectrometry

A novel mass spectrometric method for the selective detection of specific protein-ligand complexes is presented. The new method is based on electrosonic spray ionization of samples containing protein and ligand molecules, and mass spectrometric detection using the precursor ion scanning function on a triple quadrupole instrument. Mass-selected intact protein-ligand complex ions are subjected to fragmentation by means of collision-induced dissociation in the collision cell of the instrument, while the second mass analyzer is set to the m/z of protonated ligand ions or their alkali metal adducts. The method allows for the detection of only those ions which yield ions characteristic of the ligand molecules upon fragmentation. Since the scan range of first analyzer is set well above the m/z of the ligand ion, and the CID conditions are established to permit fragmentation of only loosely bound, noncovalent complexes, the method is specific to the detection of protein-ligand complexes under described conditions. Behavior of biologically specific and nonspecific complexes was compared under various instrumental settings. Parameters were optimized to obtain maximal selectivity for specific complexes. Specific and nonspecific complexes were found to show markedly different fragmentation characteristics, which can be a basis for selective detection of complexes with biological relevance. Preparation of specific and nonspecific complexes containing identical building blocks was attempted. Complex ions with identical stoichiometry but different origin showed the expected difference in fragmentation characteristics, which gives direct evidence for the different mechanism of specific versus nonspecific complex ion formation.     

SOS-NMR: a saturation transfer NMR-based method for determining the structures of protein-ligand complexes

An NMR-based alternative to traditional X-ray crystallography and NMR methods for structure-based drug design is described that enables the structure determination of ligands complexed to virtually any biomolecular target regardless of size, composition, or oligomeric state. The method utilizes saturation transfer difference (STD) NMR spectroscopy performed on a ligand complexed to a series of target samples that have been deuterated everywhere except for specific amino acid types. In this way, the amino acid composition of the ligand-binding site can be defined, and, given the three-dimensional structure of the protein target, the three-dimensional structure of the protein-ligand complex can be determined. Unlike earlier NMR methods for solving the structures of protein-ligand complexes, no protein resonance assignments are necessary. Thus, the approach has broad potential applications--especially in cases where X-ray crystallography and traditional NMR methods have failed to produce structural data. The method is called SOS-NMR for structural information using Overhauser effects and selective labeling and is validated on two protein-ligand complexes: FKBP complexed to 2-(3'-pyridyl)-benzimidazole and MurA complexed to uridine diphosphate N-acetylglucosamine.     

毒蕈碱型M1受体的同源模建和分子对接

采用同源模建方法对M1受体的三维结构进行了模拟, 将得到的模型分别与M受体完全激动剂乙酰胆碱和M1受体选择性激动剂占诺美林进行分子对接, 形成非特异性激动和特异性激动的受体-配体复合物. 用分子动力学模拟方法分别将未与小分子对接的M1受体、M1受体-乙酰胆碱复合物、M1受体-占诺美林复合物置于磷脂双膜中模拟10 ns. 将模拟后的蛋白质结构与包含活性分子的测试库对接并将结果打分, 以top5%富集因子(EF)作为评价依据, 用占诺美林优化后的M1受体模型的EF为8.0, 用乙酰胆碱优化后M1受体模型的EF为6.5, 非复合物的EF为1.5. 说明M1受体选择性激动剂复合物进行分子动力学模拟后得到的三维结构模型比较合理, 可以作为化合物虚拟筛选的模型对新化合物进行虚拟筛选, 为找到新的选择性M1受体激动剂奠定了基础.     

dbHDPLS: A database of human disease-related protein-ligand structures

Protein-ligand complexes perform specific functions, most of which are related to human diseases. The database, called as human disease-related protein-ligand structures (dbHDPLS), collected 8833 structures which were extracted from protein data bank (PDB) and other related databases. The database is annotated with comprehensive information involving ligands and drugs, related human diseases and protein-ligand interaction information, with the information of protein structures. The database may be a reliable resource for structure-based drug target discoveries and druggability predictions of protein-ligand binding sites, drug-disease relationships based on protein-ligand complex structures. It can be publicly accessed at the website: http://DeepLearner.ahu.edu.cn/web/dbDPLS/.     

Combining weak affinity chromatography, NMR spectroscopy and molecular simulations in carbohydrate-lysozyme interaction studies

By examining the interactions between the protein hen egg-white lysozyme (HEWL) and commercially available and chemically synthesized carbohydrate ligands using a combination of weak affinity chromatography (WAC), NMR spectroscopy and molecular simulations, we report on new affinity data as well as a detailed binding model for the HEWL protein. The equilibrium dissociation constants of the ligands were obtained by WAC but also by NMR spectroscopy, which agreed well. The structures of two HEWL-disaccharide complexes in solution were deduced by NMR spectroscopy using (1)H saturation transfer difference (STD) effects and transferred (1)H,(1)H-NOESY experiments, relaxation-matrix calculations, molecular docking and molecular dynamics simulations. In solution the two disaccharides β-d-Galp-(1→4)-β-D-GlcpNAc-OMe and β-D-GlcpNAc-(1→4)-β-D-GlcpNAc-OMe bind to the B and C sites of HEWL in a syn-conformation at the glycosidic linkage between the two sugar residues. Intermolecular hydrogen bonding and CH/π-interactions form the basis of the protein-ligand complexes in a way characteristic of carbohydrate-protein interactions. Molecular dynamics simulations with explicit water molecules of both the apo-form of the protein and a ligand-protein complex showed structural change compared to a crystal structure of the protein. The flexibility of HEWL as indicated by a residue-based root-mean-square deviation analysis indicated similarities overall, with some residue specific differences, inter alia, for Arg61 that is situated prior to a flexible loop. The Arg61 flexibility was notably larger in the ligand-complexed form of HEWL. N,N'-Diacetylchitobiose has previously been observed to bind to HEWL at the B and C sites in water solution based on (1)H NMR chemical shift changes in the protein whereas the disaccharide binds at either the B and C sites or the C and D sites in different crystal complexes. The present study thus highlights that protein-ligand complexes may vary notably between the solution and solid states, underscoring the importance of targeting the pertinent binding site(s) for inhibition of protein activity and the advantages of combining different techniques in a screening process.     

Protein-ligand binding free energy estimation using molecular mechanics and continuum electrostatics. Application to HIV-1 protease inhibitors

A method is proposed for the estimation of absolute binding free energy of interaction between proteins and ligands. Conformational sampling of the protein-ligand complex is performed by molecular dynamics (MD) in vacuo and the solvent effect is calculated a posteriori by solving the Poisson or the Poisson-Boltzmann equation for selected frames of the trajectory. The binding free energy is written as a linear combination of the buried surface upon complexation, SASbur, the electrostatic interaction energy between the ligand and the protein, Eelec, and the difference of the solvation free energies of the complex and the isolated ligand and protein, deltaGsolv. The method uses the buried surface upon complexation to account for the non-polar contribution to the binding free energy because it is less sensitive to the details of the structure than the van der Waals interaction energy. The parameters of the method are developed for a training set of 16 HIV-1 protease-inhibitor complexes of known 3D structure. A correlation coefficient of 0.91 was obtained with an unsigned mean error of 0.8 kcal/mol. When applied to a set of 25 HIV-1 protease-inhibitor complexes of unknown 3D structures, the method provides a satisfactory correlation between the calculated binding free energy and the experimental pIC5o without reparametrization.     

Flexible docking of ligands into synthetic receptors using a two-sided incremental construction algorithm

We present a new algorithm for the fast and reliable structure prediction of synthetic receptor-ligand complexes. Our method is based on the protein-ligand docking program FlexX and extends our recently introduced docking technique for synthetic receptors, which has been implemented in the program FlexR. To handle the flexibility of the relevant molecules, we apply a novel docking strategy that uses an adaptive two-sided incremental construction algorithm which incorporates the structural flexibility of both the ligand and synthetic receptor. We follow an adaptive strategy, in which one molecule is expanded by attaching its next fragment in all possible torsion angles, whereas the other (partially assembled) molecule serves as a rigid binding partner. Then the roles of the molecules are exchanged. Geometric filters are used to discard partial conformations that cannot realize a targeted interaction pattern derived in a graph-based precomputation phase. The process is repeated until the entire complex is built up. Our algorithm produces promising results on a test data set comprising 10 complexes of synthetic receptors and ligands. The method generated near-native solutions compared to crystal structures in all but one case. It is able to generate solutions within a couple of minutes and has the potential of being used as a virtual screening tool for searching for suitable guest molecules for a given synthetic receptor in large databases of guests and vice versa.     

Functional concerted motions in the bovine serum retinol-binding protein

The large concerted motions in the apo/holo bovine serum retinol- binding protein were studied using molecular dynamics simulation and 'essential dynamics' analysis. Initially, concerted motions were calculated from conformational differences between various crystal structures. The dynamic behaviour of the protein in the configurational space directions, described by these concerted motions, is analysed. This reveals that the large backbone dynamics of the protein is not influenced by the presence of retinol. Study of free retinol dynamics and retinol in the retinol binding site reveals that the protein binds retinol in a favourable conformation, as opposed to what has been previously described for the bovine cellular retinol-binding protein.     

Critical evaluation of mass spectrometric measurement of dissociation constants: accuracy and cross-validation against surface plasmon resonance and circular dichroism for the calmodulin-melittin system

We present a comprehensive study for determining the binding affinity of a protein-ligand complex, using mass spectrometric methods. Mass spectrometry has been used to study noncovalent interactions for a number of years. However, the use of soft ionization mass spectrometry for quantitative analysis of noncovalently bound complexes is not widely accepted. This paper reports a comparison of MS methods against established methods such as surface plasmon resonance (SPR) and circular dichroism (CD) whose suitability for the quantitative assessment of noncovalent interactions is well known. ESI titration and MALDI-SUPREX were used as representative mass spectrometric methods for this work. We chose to study the calmodulin-melittin complex that presents three challenges: (i) it exhibits a high affinity (low nanomolar KD); (ii) complexes are formed only in the presence of a coactivator, calcium ions in this case; and (iii) the protein and the complex show a different ionization efficiency. Dissociation constants were obtained from each method for the selected system and compared thoroughly to elucidate pros and cons of the selected methodologies in terms of their ability for the determination of binding constants of protein-ligand complexes. ESI titration, SPR, CD and MALDI-SUPREX yielded KD values in the low nanomolar range that are in general agreement with an older value reported in the literature. We also critically evaluated the limitations in particular of the MS methods and the associated data evaluation procedures. We present an improved evaluation of SUPREX data, as well as a detailed error analysis for all methods used.     

Protein ligand docking based on empirical method for binding affinity estimation

An empirical protein-ligand binding affinity estimation method, SCORE, was incorporated into a popular docking program, DOCK4. The combined program, ScoreDock, was used to reconstruct the 200 protein-ligand complex structures and found to give good results for the complexes with high binding affinities. A quality assessment method for docking results from ScoreDock was developed based on the whole test set and tested by additionally selected complexes. The method significantly improves the docking accuracy and was shown to be reliable in docking quality assessment. As a docking tool in structural based drug design, ScoreDock can screen out final hits directly based on the predicted negative logarithms of dissociation equilibrium constants of protein-ligand complexes, and can explicitly deal with structure water molecules, as well as metal atoms.     

Conserved core substructures in the overlay of protein-ligand complexes

The method of conserved core substructure matching (CSM) for the overlay of protein-ligand complexes is described. The method relies upon distance geometry to align structurally similar substructures without regard to sequence similarity onto substructures from a reference protein empirically selected to include key determinants of binding site location and geometry. The error in ligand position is reduced in reoriented ensembles generated with CSM when compared to other overlay methods. Since CSM can only succeed when the selected core substructure is geometrically conserved, misalignments only rarely occur. The method may be applied to reliably overlay large numbers of protein-ligand complexes in a way that optimizes ligand position at a specific binding site or subsite or to align structures from large and diverse protein families where the conserved binding site is localized to only a small portion of either protein. Core substructures may be complex and must be chosen with care. We have created a database of empirically selected core substructures to demonstrate the utility of CSM alignment of ligand binding sites in important drug targets. A Web-based interface can be used to apply CSM to align large collections of protein-ligand complexes for use in drug design using these substructures or to evaluate the use of alternative core substructures that may then be shared with the larger user community. Examples show the benefit of CSM in the practice of structure-based drug design.     

Combining Neutron Scattering,Deuteration Technique,and Molecular Dynamics Simulations to Study Dynamics of Protein and Its Surface Water Molecules

Protein internal dynamics is essential for its function.Exploring the internal dynamics of protein molecules as well as its connection to protein structure and function is a central topic in biophysics.However,the atomic motions in protein molecules exhibit a great degree of complexities.These complexities arise from the complex chemical composition and superposition of different types of atomic motions on the similar time scales,and render it challenging to explicitly understand the microscopic mechanism governing protein motions,functions,and their connections.Here,we demonstrate that,by using neutron scattering,molecular dynamics simulation,and deuteration technique,one can address this challenge to a large extent.     

PROLIX: rapid mining of protein-ligand interactions in large crystal structure databases

A central problem in structure-based drug design is understanding protein-ligand interactions quantitatively and qualitatively. Several recent studies have highlighted from a qualitative perspective the nature of these interactions and their utility in drug discovery. However, a common limitation is a lack of adequate tools to mine these interactions comprehensively, since exhaustive searches of the protein data bank are time-consuming and difficult to perform. Consequently, fundamental questions remain unanswered: How unique or how common are the protein-ligand interactions observed in a given drug design project when compared to all complexed structures in the protein data bank? Which interaction patterns might explain the affinity of a tool compound toward unwanted targets? To answer these questions and to enable the systematic and comprehensive study of protein-ligand interactions, we introduce PROLIX (Protein Ligand Interaction Explorer), a tool that uses sophisticated fingerprint representations of protein-ligand interaction patterns for rapid data mining in large crystal structure databases. Our implementation strategy pursues a branch-and-bound technique that enables mining against thousands of complexes within a few seconds. Key elements of PROLIX include (i) an intuitive interface that enables users to formulate complex queries easily, (ii) exceptional speed for results retrieval, and (iii) a sophisticated results summarization. Herein we describe the algorithms developed to enable complex queries and fast retrieval of search results, as well as the intuitive aspects of the user interface and summarization viewer.