NMR-assisted computational studies of peptidomimetic inhibitors bound in the hydrophobic pocket of HIV-1 glycoprotein 41

Due to the inherently flexible nature of a protein–protein interaction surface, it is difficult both to inhibit the association with a small molecule, and to predict how it might bind to the surface. In this study, we have examined small molecules that mediate the interaction between a WWI motif on the C-helix of HIV-1 glycoprotein-41 (gp41) and a deep hydrophobic pocket contained in the interior N-helical trimer. Association between these two components of gp41 leads to virus–cell and cell–cell fusion, which could be abrogated in the presence of an inhibitor that binds tightly in the pocket. We have studied a comprehensive combinatorial library of α-helical peptidomimetics, and found that compounds with strongly hydrophobic side chains had the highest affinity. Computational docking studies produced multiple possible binding modes due to the flexibility of both the binding site and the peptidomimetic compounds. We applied a transferred paramagnetic relaxation enhancement experiment to two selected members of the library, and showed that addition of a few experimental constraints enabled definitive identification of unique binding poses. Computational docking results were extremely sensitive to side chain conformations, and slight variations could preclude observation of the experimentally validated poses. Different receptor structures were required for docking simulations to sample the correct pose for the two compounds. The study demonstrated the sensitivity of predicted poses to receptor structure and indicated the importance of experimental verification when docking to a malleable protein–protein interaction surface.     

Computational Characterization of Binding of Small Molecule Inhibitors to HIV‐1 gp41

Developing orally available small molecule inhibitors of HIV‐1 fusion has attracted significant interest over many years. Frey had recently reported several synthetic compounds which are experimentally shown to inhibit cell‐cell fusion in the low micromolar range. We carried out computational study to help identify possible binding modes by docking these compounds onto the hydrophobic pocket on gp41 and to characterize structures of binding complexes. The detailed gp41‐molecule binding interactions and free energies of binding are obtained through molecular dynamics simulation and MM‐PBSA calculation. Specific molecular interactions in the gp41‐inhibitor complexes are identified. The present computational study complements the corresponding experimental investigation and helps establish a good starting point for further refinement of small molecular gp41 inhibitors.     

Estimation of important binding sites in compounds that interact with proteins

Proteins are one of the important substances in understanding biological activity, and many of them express the function by binding to other proteins or small molecules (ligands) on the molecular surface. This interaction often occurs in the hollows (pockets) on the molecular surface of the protein. It is known that when pockets are similar in structure and physical properties, they are likely to express similar functions and to bind similar ligands. Therefore, exploring the similarity of the structure and physical properties in pockets is very useful because it leads to the discovery of new ligands that are likely to bind. In addition, exploring the important structure when binding to the protein significant spot in the ligand will provide useful knowledge for the development of new ligands.In this study, we propose a method to search for proteins containing pockets that are structurally and physically similar to significant spot in the pocket of the analyzed protein, and to extract significant spots in the ligands that bind to them. We use feature points as data. Feature points are the 3-dimensional points that are extracted from 3D structure data of proteins with feature values quantifying hydrophobicity and electrostatic potential. The corresponding feature points are extracted by comparing structurally and physically the pockets of the search target proteins with the significant spot of the analyzed protein. By evaluating the similarity based on the comparison results of the feature values given to the extracted feature points, we search for proteins that are similar to the analyzed protein. From the ligands that bind to the searched proteins, atoms that are near the protein pocket and similar to the atoms in ligand binding to the analyzed protein are extracted. The site constituted by the extracted atoms is defined as a significant spot in the ligand.As a result of classifying ligands binding to the protein by using the extracted significant spot in the ligand, the effectiveness of the proposed method was confirmed.     

A Virtual Active Compound Produced from the Negative Image of a Ligand-binding Pocket, and its Application to in-silico Drug Screening

We developed a new structure-based in-silico screening method using a negative image of a ligand-binding pocket and a multi-protein–compound interaction matrix. Based on the structure of the ligand pocket of the target protein, we designed a negative image, which consists of virtual atoms whose radii are close to those of carbon atoms. The virtual atoms fit the pocket ideally and achieve an optimal Coulomb interaction. A protein–compound docking program calculates the protein–compound interaction matrix for many proteins and many compounds including the negative image, which can be treated as a virtual compound. With specific attention to a vector of docking scores for a single compound with many proteins, we selected a compound whose score vector was similar to that of the negative image as a candidate hit compound. This method was applied to representative target proteins and showed high database enrichment with a relatively quick procedure.     

NMR of membrane-associated peptides and proteins

In living cells, membrane proteins are essential to signal transduction, nutrient use, and energy exchange between the cell and environment. Due to challenges in protein expression, purification and crystallization, deposition of membrane protein structures in the Protein Data Bank lags far behind existing structures for soluble proteins. This review describes recent advances in solution NMR allowing the study of a select set of peripheral and integral membrane proteins. Surface-binding proteins discussed include amphitropic proteins, antimicrobial and anticancer peptides, the HIV-1 gp41 peptides, human alpha-synuclein and apolipoproteins. Also discussed are transmembrane proteins including bacterial outer membrane beta-barrel proteins and oligomeric alpha-helical proteins. These structural studies are possible due to solubilization of the proteins in membrane-mimetic constructs such as detergent micelles and bicelles. In addition to protein dynamics, protein-lipid interactions such as those between arginines and phosphatidylglycerols have been detected directly by NMR. These examples illustrate the unique role solution NMR spectroscopy plays in structural biology of membrane proteins.     

Nanoscale substrate recognition by porphyrin dendrimers with patched structures

Dendrimer technology has enabled us to build macromolecules with nanosized defined structures. By introducing unsymmetrical patched structures in dendrimers, sophisticated artificial receptors exhibiting nanoscale substrate recognition can be obtained. In this review article, our recent studies on molecular recognition by porphyrin dendrimers with patched structures are summarized. Three topics are presented: (1) oligopeptide-patched dendrimers as a nanoscale receptor of cytochrome c protein; (2) pocket dendrimers as a nanoscale receptor for bimolecular guest accommodation; and (3) energy transfer in unsymmetrical dendrimers. These dendrimers nicely mimic proteins and enzymes, and also act as photofunctional artificial receptors, in which porphyrin’s strong photoabsorption and intense fluorescence signals can respond sensitively to the substrate binding.     

Computational fragment-based drug design to explore the hydrophobic sub-pocket of the mitotic kinesin Eg5 allosteric binding site

Eg5, a mitotic kinesin exclusively involved in the formation and function of the mitotic spindle has attracted interest as an anticancer drug target. Eg5 is co-crystallized with several inhibitors bound to its allosteric binding pocket. Each of these occupies a pocket formed by loop 5/helix α2 (L5/α2). Recently designed inhibitors additionally occupy a hydrophobic pocket of this site. The goal of the present study was to explore this hydrophobic pocket with our MED-SuMo fragment-based protocol, and thus discover novel chemical structures that might bind as inhibitors. The MED-SuMo software is able to compare and superimpose similar interaction surfaces upon the whole protein data bank (PDB). In a fragment-based protocol, MED-SuMo retrieves MED-Portions that encode protein-fragment binding sites and are derived from cross-mining protein-ligand structures with libraries of small molecules. Furthermore we have excluded intra-family MED-Portions derived from Eg5 ligands that occupy the hydrophobic pocket and predicted new potential ligands by hybridization that would fill simultaneously both pockets. Some of the latter having original scaffolds and substituents in the hydrophobic pocket are identified in libraries of synthetically accessible molecules by the MED-Search software. Ksenia Oguievetskaia and Laetitia Martin-Chanas contributed equally to this work.     

Peptide affinity chromatography media that bind N fusion proteins under chaotropic conditions

To design a generic purification platform and to combine the advantages of fusion protein technology and matrix-assisted refolding, a peptide affinity medium was developed that binds inclusion body-derived N^pro fusion proteins under chaotropic conditions. Proteins were expressed in Escherichia coli using an expression system comprising the autoprotease N^pro from Pestivirus, or its engineered mutant called EDDIE, with C-terminally linked target proteins. Upon refolding, the autoprotease became active and cleaved off its fusion partner, forming an authentic N-terminus. Peptide ligands binding to the autoprotease at 4 M urea were screened from a combinatorial peptide library. A group of positive peptides were identified and further refined by mutational analysis. The best binders represent a common motif comprising positively charged and aromatic amino acids, which can be distributed in a random disposition. Mutational analysis showed that exchange of a single amino acid within the peptide ligand caused a total loss of binding activity. Functional affinity media comprising hexa- or octapeptides were synthesized using a 15-atom spacer with terminal sulfhydryl function and site-directed immobilization of peptides derivatized with iodoacetic anhydride. The peptide size was further reduced to dipeptides comprising only one positively charged and one aromatic amino acid. Based on this, affinity media were prepared by immobilization of a poly amino acid comprising lysine or arginine, and tryptophan, phenylalanine, or tyrosine, respectively, in certain ratios. Binding capacities were in the range of 7–15 mg protein mL⁻¹ of medium, as could be shown for several EDDIE fusion proteins. An efficient protocol for autoproteolytic cleavage using an on-column refolding method was implemented.     

Enhancement of α-helix mimicry by an α/β-peptide foldamer via incorporation of a dense ionic side-chain array

We report a new method for preorganization of α/β-peptide helices, based on the use of a dense array of acidic and basic side chains. Previously we have used cyclically constrained β residues to promote α/β-peptide helicity; here we show that an engineered ion pair array can be comparably effective, as indicated by mimicry of the CHR domain of HIV protein gp41. The new design is effective in biochemical and cell-based infectivity assays; however, the resulting α/β-peptide is susceptible to proteolysis. This susceptibility was addressed via introduction of a few cyclic β residues near the cleavage site, to produce the most stable, effective α/β-peptide gp41 CHR analogue identified. Crystal structures of an α- and α/β-peptide (each involved in a gp41-mimetic helix bundle) that contain the dense acid/base residue array manifest disorder in the ionic side chains, but there is little side-chain disorder in analogous α- and α/β-peptide structures with a sparser ionic side-chain array. These observations suggest that dense arrays of complementary acidic and basic residues can provide conformational stabilization via Coulombic attractions that do not require entropically costly ordering of side chains.     

Toward fully synthetic carbohydrate-based HIV antigen design: on the critical role of bivalency

Synthetic gp120331-335 glycopeptide fragments carrying hybrid and high-mannose type N-linked glycans were evaluated for binding to broadly neutralizing antibody 2G12 using surface plasmon resonance technology. None of the hybrid-type constructs demonstrated binding to 2G12. In the high-mannose series, the "Cys dimer" construct, presenting two undecasaccharide glycans, showed significantly higher binding than the Cys-protected monomer. The binding of the dimeric structure was further investigated in competition with recombinant gp120. The data suggest that gp120 and its designed synthetic epitope construct bind to the same site on 2G12.     

Protein engineering ofDe novo protein with predesigned structure and activity

Thede novo protein albebetin has been engineered (J. Mol. Biol. 1992,225, 927–931) to form a predesigned tertiary fold that has not yet been observed in natural proteins. Analysis of albebetin expressed in a cell-free system and inEscherichia coli revealed its compactness, relative stability, and the secondary structure close to the predesigned one. The blast-transforming biological activity of human interferon was grafted to albebetin by attachment of an eight amino acid interferon fragment to the N-terminus of albebetin next to its first methionine residue. The chimeric protein was expressed in a wheat germ cell-free translation system and tested for its structural properties, receptor binding, and biological activity. According to the tests, albebetin incorporating the active interferon fragment has a compact and relatively stable structure, and binds the murine thymocyte recep or effectively. It activates the blast transformation reaction of thymo yte cells even more efficiently than human interferon at low concentrations.     

Stabilization centers and protein stability

The well-balanced stability of protein structures allows large-scale fluctuations, which are indispensable in many biochemical functions, ensures the long-term persistence of the equilibrium structure and it regulates the degradation of proteins to provide amino acids for biosynthesis. This balance is studied in the present work with two sets of proteins by analyzing stabilization centers, defined as certain clusters of residues involved in cooperative long-range interactions. One data set contains 56 proteins, which belong to 16 families of homologous proteins, derived from organisms of various physiological temperatures. The other set is composed of 31 major histocompatibility complex (MHC)–peptide complexes, which represent peptide transporters complexed with peptide ligands that apparently contribute to the stabilization of the MHC proteins themselves. We show here that stabilization centers, which had been identified as special clusters of residues that protect the protein structure, evolved to serve also as regulators of function – related degradation of useless protein as part of protein housekeeping. Received: 25 August 2000 / Accepted: 6 September 2000 / Published online: 21 December 2000     

A study of the interactions between carboplatin and blood plasma proteins using size exclusion chromatography coupled to inductively coupled plasma mass spectrometry

To study the carboplatin–protein interaction, a sensitive method using size exclusion chromatography coupled to inductively coupled plasma mass spectrometry (SEC–ICP–MS) was developed. The complexes formed between plasma proteins and carboplatin were monitored and identified with this method. Composite blood plasma samples from patients who were undergoing chemotherapy were analyzed, and carboplatin was found to bind plasma proteins. In addition, blank plasma samples were spiked with carboplatin and were analyzed as a time course study, and the results confirmed that carboplatin formed complexes with plasma proteins, primarily albumin and γ-globulin. To further substantiate the study, these two proteins were incubated with carboplatin. The binding between carboplatin and these proteins was then characterized qualitatively and quantitatively. In addition to a one-to-one binding of Pt to protein, protein aggregation was observed. The kinetics of the binding process of carboplatin to albumin and γ-globulin was also studied. The initial reaction rate constant of carboplatin binding to albumin was determined to be 0.74 M⁻¹ min⁻¹, while that for γ-globulin was 1.01 M⁻¹ min⁻¹, which are both lower than the rate constant of the cisplatin–albumin reaction previously reported.     

Mutant tyrosine kinases with unnatural nucleotide specificity retain the structure and phospho-acceptor specificity of the wild-type enzyme

The direct substrates of one protein kinase in a cell can be identified by mutation of the ATP binding pocket to allow an unnatural ATP analog to be accepted exclusively by the engineered kinase. Here, we present structural and functional assessment of peptide specificity of mutant protein kinases with unnatural ATP analogs. The crystal structure (2.8 A resolution) of c-Src (T338G) with N(6)-(benzyl) ADP bound shows that the creation of a unique nucleotide binding pocket does not alter the phospho-acceptor binding site of the kinase. A panel of optimal peptide substrates of defined sequence, as well as a degenerate peptide library, was utilized to assess the phospho-acceptor specificity of the engineered "traceable" kinases. The specificity profiles for the mutant kinases were found to be identical to those of their wild-type counterparts.     

Construction of artificial signal transducers on a lectin surface by post-photoaffinity-labeling modification for fluorescent saccharide biosensors

A new general method, post-photoaffinity-labeling modification (PPALM), for constructing fluorescent saccharide biosensors based on naturally occurring saccharide-binding proteins, lectins, is described in detail. An active-site-directed incorporation of a masked reactive site into a lectin was conducted by using a photoaffinity labeling technique followed by demasking and then chemical modification to yield a fluorescent lectin. Two photoaffinity labeling reagents were designed and synthesized in this study. The labeling reagent with a photoreactive site appended through a disulfide link to a mannoside unit was bound to the saccharide-binding pocket of the lectin concanavalin A (Con A). After light irradiation, the mannoside unit was cleaved by reduction. The unique thiol group thus produced was site-specifically modified with various fluorescent groups (dansyl, coumarin, or dimethylaminobenzoate derivatives) to afford fluorescent Con As. The labeling site was characterized by protease-catalyzed digestion followed by HPLC, MALDI-TOF MS, and tandem mass-mass spectrometry; these methods indicated that the photolabeling step is remarkably site specific. Strong fluorescence was observed in the engineered Con A with a fluorophore, and the emission changed sensitively upon saccharide complexation. The binding constants for various saccharides were determined by fluorescence titration and demonstrated that the binding selectivity and affinity of the engineered Con As are comparable to those of native Con A. The red shift of the emission maximum, the decrease in the fluorescence anisotropy of the dansyl unit, and the increase in the twisted intramolecular charge transfer emission caused by sugar binding to the engineered Con A explicitly indicate that the microenvironment of the appended fluorophores changes from a restricted and relatively hydrophobic environment into a rather freely mobile and hydrophilic environment.     

Biomineralization of a Cadmium Chloride Nanocrystal by a Designed Symmetrical Protein

We have engineered a metal‐binding site into the novel artificial β‐propeller protein Pizza. This new Pizza variant carries two nearly identical domains per polypeptide chain, and forms a trimer with three‐fold symmetry. The designed single metal ion binding site lies on the symmetry axis, bonding the trimer together. Two copies of the trimer associate in the presence of cadmium chloride in solution, and very high‐resolution X‐ray crystallographic analysis reveals a nanocrystal of cadmium chloride, sandwiched between two trimers of the protein. This nanocrystal, containing seven cadmium ions lying in a plane and twelve interspersed chloride ions, is the smallest reported to date. Our results indicate the feasibility of using rationally designed symmetrical proteins to biomineralize nanocrystals with useful properties.     

Preparation of a novel composite particles and its application in the fluorescent detection of proteins

A new fluorescence method for the detection of proteins with novel composite nanoparticles (CdS/PPA) has been developed. The composite nanoparticles have been prepared through an in-situ polymerization method under ultrasonic irradiation. The surface of the composite nanoparticles was covered with functional groups (-COOH). These groups may play a major role in the improving the water solubility and biocompatibility of the nanoparticles. The composite particles is combined with proteins in NaAc-HCl buffer solution (pH=1.99), which can result in strong fluorescence, and the response is linearly proportional to the concentration of proteins. In λem/λex=650 nm/365 nm place (the stoke’ shift is 285 nm), its fluorescent strength reaches the maximum. Under the optimum conditions, the linear range is 0.10–20.0 μg·ml⁻¹ with the detection limit of 41 ng·ml⁻¹ for HSA, and 0.10–15.0 μg·ml⁻¹ with the detection limit of 35 ng·ml⁻¹ for Human γ-IgG . The method has been applied to the determination of the total protein in human serum samples collected from the hospital and the results are satisfactory.     

Study of the conformation of γ-zeins in purified maize protein bodies by FTIR and NMR spectroscopy

The γ-zeins are a mixture of 16, 27, and 50-kDa polypeptides which are important in the formation and stabilization of protein bodies (PB). These organelles are used for deposition of zeins, the water-insoluble storage proteins in maize. The nature of the physical interaction between proteins in the assembly and stabilization of PB are fairly well known. It is suggested the repeated hexapeptide sequence (PPPVHL)₈ in the N-terminus is responsible for aggregation of the γ-zeins on the PB surface. Despite this importance, there is little information about the native conformation of γ-zeins. In this work, we have analyzed the secondary structures of γ-zeins in purified protein bodies from two maize cultivars, in the solid state, by FTIR and NMR spectroscopy. The results revealed that γ-zeins in their physiological state are comprise similar proportions of α-helix and β-sheet, 33 and 31% as determined by FTIR. It was not possible to state if the polyproline II (PPII) conformation is present in the solid-state structure of γ-zeins, as has been demonstrated for the hexapeptide in solution. Because of the similarity of the solid-state NMR spectra of γ and α-zeins in the α carbon region we attributed their contributions to the β-sheet structures rather than to the PPII conformation or a mixture of these extended structures.     

全新设计蛋白质的折叠机制

蛋白质全新设计和折叠研究是从两个不同的方向来理解蛋白质序列-结构-功能关系这一结构生物学重要问题. 蛋白质全新设计取得的成功实例一定程度上检验了人们对蛋白质结构和相互作用理解的准确性, 但它们中多数所表现的不同于天然蛋白质的折叠动力学特征也表明, 要达到最终的功能化实现目标还面临着不少的挑战. 本文综述了蛋白质全新设计的发展过程及现状, 蛋白质折叠研究在实验、理论及模拟方面的研究进展, 以及全新设计蛋白质的折叠机制的研究现状. 阐述了深入了解全新设计蛋白质与天然蛋白质折叠机制的不同, 可以为进一步有效地合理化设计蛋白质提供有益的参考.