A combinatorial approach to protein docking with flexible side chains.

Rigid-body docking approaches are not sufficient to predict the structure of a protein complex from the unbound (native) structures of the two proteins. Accounting for side chain flexibility is an important step towards fully flexible protein docking. This work describes an approach that allows conformational flexibility for the side chains while keeping the protein backbone rigid. Starting from candidates created by a rigid-docking algorithm, we demangle the side chains of the docking site, thus creating reasonable approximations of the true complex structure. These structures are ranked with respect to the binding free energy. We present two new techniques for side chain demangling. Both approaches are based on a discrete representation of the side chain conformational space by the use of a rotamer library. This leads to a combinatorial optimization problem. For the solution of this problem, we propose a fast heuristic approach and an exact, albeit slower, method that uses branch-and-cut techniques. As a test set, we use the unbound structures of three proteases and the corresponding protein inhibitors. For each of the examples, the highest-ranking conformation produced was a good approximation of the true complex structure.     

Linckosides C-E, three new neuritogenic steroid glycosides from the Okinawan starfish Linckia laevigata

Three new steroid glycosides, linckosides C-E, were isolated from the Okinawan starfish Linckia laevigata. Their structures and partial stereochemistry were elucidated by spectroscopic methods and chemical derivatization. These metabolites are additional members of the linckosides that were previously discovered as a novel class of neuritogenic compounds. Each of them possesses two monosaccharide units at C-3 of a polyhydroxylated steroidal aglycon and at the side chain (C-28 or C-29). Linckosides C and D are the first steroids that possess a hydroxyisopropyl substituent at C-24 of the side chain. These compounds are not only potent inducers of neurite outgrowth on PC12 cells but also significant enhancers of nerve growth factor (NGF) to induce the neurite outgrowth. The structure-activity relationships within the linckosides revealed that the presence of xylopyranose at the side chain was important rather than arabinofuranose, but that the diversity of the side chain carbon skeleton was not.     

Structure-toxicity relationships in the amatoxin series. Synthesis of S-deoxy[gamma(R)-hydroxy-Ile3]-amaninamide, its crystal and molecular structure and inhibitory efficiency

The amatoxins, highly toxic components of Amanita mushrooms, strongly inhibit the DNA-dependent RNA polymerase II (or B) in eukaryotic cell nuclei. For optimal binding to the enzyme a gamma-hydroxyisoleucine side chain in the 3-position is important as in gamma-amanitin (compound 1), where the OH-group is bound in the [S]-configuration. Amanullin, a non-toxic component, having an oxygen-free isoleucine side chain no. 3, exhibits an inhibitory effect on RNA polymerase II about two orders of magnitude smaller than that of gamma-amanitin. An equal, relatively weak, inhibitory effect has previously been found with the synthetically obtained Ile3-analog 7. In the present paper the synthesis of an analog (2) bearing a gamma-hydroxyl group in the isoleucine side chain is described. The compound was found to have about the same inhibitory effect on RNA polymerase II from Drosophila embryos as amanullin and the Ile3-analog 7. Structure analysis by X-ray diffraction revealed that the hydroxyl group at the -carbon atom of side chain-3 has the [R]-configuration, the new analog thus being -deoxo[( )-hydroxy-[Ile3]-amaninamide. It follows that the [S]-configuration of this chiral center is a prerequisite to maximal toxicity. Crystallographic data demonstrating great similarity between the peptide backbones of the new analog and those of natural amatoxins are given.     

Synthesis and biological activities of thioether derivatives related to the antiestrogens tamoxifen and ICI 164384

The catalyzed coupling reaction of activated alcohol and mercaptan was used for the short and efficient synthesis of 14 thioether compounds. Two types of side chains, the methyl butyl alkylamide related to the pure steroidal antiestrogen ICI 164384 and the dimethylamino ethyloxy phenyl related to the clinically used nonsteroidal antiestrogen tamoxifen, were introduced by a thioether link on two types of nuclei (triphenylethane or estradiol). The new thioether derivatives were tested to assess their relative binding affinity for the estrogen receptor and their estrogenic or antiestrogenic activity in the ZR-75-1 (ER⁺) cell line. The results indicate that of the three types of compounds studied, only the nonsteroidal derivatives with an alkylamide side chain possess antiestrogenic activity. In the steroidal series, displacement of the alkylamide side chain from the 7 to the 6 position produced compounds with chemical characteristics similar to ICI 164384 or EM-139 but without antiestrogenic activity. In the nonsteroidal series of compounds with an aryl side chain, compounds with estrogenic activity were obtained. One compound, a nonsteroidal derivative with a methyl butyl alkylamide side chain 20, possesses a relative binding affinity for the estrogen receptor identical to EM-139 (1.1 and 1.2%, respectively) and a relatively good antiestrogenic activity that is 10-fold lower than EM-139 (IC₅₀ values of 250 and 25 nM, respectively). This nonsteroidal thioether with an alkylamide side chain is free of estrogenic activity.     

Optimum interaction of sterol side chains with phosphatidylcholine

The specificity of the interaction between the cholesterol side chain and egg phosphatidylcholine was precisely defined by examining the effect of three new analogues of cholesterol with modified side chains on the ordering of two steroid spin labels in liposomes. The complete side chain of cholesterol was shown to be required for maximum ordering. Sterols with side chains shorter or longer than cholesterol caused significantly less ordering.     

Optimum interaction of sterol side chains with phosphatidylcholine.

The specificity of the interaction between the cholesterol side chain and egg phosphatidylcholine was precisely defined by examining the effect of three new analogues of cholesterol with modified side chains on the ordering of two steroid spin labels in liposomes. The complete side chain of cholesterol was shown to be required for maximum ordering. Sterols with side chains shorter or longer than cholesterol caused significantly less ordering.     

A new amphipathy scale I. Determination of the scale from molecular dynamics data

Two new amphipathy scales elaborated from molecular dynamics data are presented. Their applications contribute for the identification of the hydrophobic or hydrophilic regions in proteins solely from the primary structure. The new amphipathy coefficients (AC) reflect the side chain/solvent molecules configurational energies. A polar (water) and an apolar solvent, CCl4, were used resulting in the two ACwater and ACCCl4 scales. These solvents were chosen to simulate the aqueous phases and the transmembrane ambients of cellular membranes where the membrane proteins act. The new amphipathy scales were compared with some previous scales determined by different methods, which were also compared between them, indicating more than 90% of the correlation coefficients are less than 0.9: the scales are strictly dependent on the methodologies used in their determination. The ACCCl4 scale is related with the size of side chain amino acids while ACwater is related with the hydrophobicity of side chain amino acids. The quality of the scales was confirmed by an example of application where ACwater was able to identify correctly the transmembrane, hydrophobic regions of a membrane protein. These results also indicate that water is an important factor responsible for the tertiary structure of membrane proteins.     

Effect of alkyl side chain variation on the electron-transfer activity of ubiquinone derivatives

The effect of the alkyl side chain of the ubiquinone molecule on the electron-transfer activity of ubiquinone in mitochondrial succinate-cytochrome c reductase is studied by using synthetic ubiquinone derivatives that possess the basic ubiquinone structure of 2,3-dimethoxy-5-methyl-1,4-benzoquinone with different alkyl side chains at the 6-position. The alkyl side chains vary in chain length, degree of saturation, and location of double bonds. When a ubiquinone derivative is used as an electron acceptor for succinate-ubiquinone reductase, an alkyl side chain of six carbons is needed to obtain the maximum activity. However, when it serves as an electron donor for ubiquinol-cytochrome c reductase or as a mediator in succinate-cytochrome c reductase, an alkyl side chain of 10 carbons gives maximal efficiency. Introduction of one or two isolated double bonds into the alkyl side chain of the ubiquinone molecule has little effect on electron-transfer activity. However, a conjugated double bond system in the alkyl side chain drastically reduces electron-transfer efficiency. The effect of the conjugated double bond system on the electron-transferring efficiency of ubiquinone depends on its location in the alkyl side chain. When location is far from the benzoquinone ring, the effect is minimal. These observations together with the results obtained from photoaffinity-labeling studies lead us to conclude that flexibility in the portion of the alkyl side chain immediately adjacent to the benzoquinone ring is required for the electron-transfer activity of ubiquinone.     

Protein side‐chain modeling with a protein‐dependent optimized rotamer library

Despite years of effort, the problem of predicting the conformations of protein side chains remains a subject of inquiry. This problem has three major issues, namely defining the conformations that a side chain may adopt within a protein, developing a sampling procedure for generating possible side‐chain packings, and defining a scoring function that can rank these possible packings. To solve the former of these issues, most procedures rely on a rotamer library derived from databases of known protein structures. We introduce an alternative method that is free of statistics. We begin with a rotamer library that is based only on stereochemical considerations; this rotamer library is then optimized independently for each protein under study. We show that this optimization step restores the diversity of conformations observed in native proteins. We combine this protein‐dependent rotamer library (PDRL) method with the self‐consistent mean field (SCMF) sampling approach and a physics‐based scoring function into a new side‐chain prediction method, SCMF–PDRL. Using two large test sets of 831 and 378 proteins, respectively, we show that this new method compares favorably with competing methods such as SCAP, OPUS‐Rota, and SCWRL4 for energy‐minimized structures. Proteins 2014; 82:2000–2017. © 2014 Wiley Periodicals, Inc.     

Crystal structure of extended-spectrum beta-lactamase Toho-1: insights into the molecular mechanism for catalytic reaction and substrate specificity expansion

The crystallographic structure of the class A beta-lactamase Toho-1, an extended-spectrum beta-lactamase with potent activity against expanded-spectrum cephems, has been determined at 1.65 A resolution. The result reveals that the Lys73 side chain can adopt two alternative conformations. The predominant conformation of Lys73 is different from that observed in the E166A mutant, indicating that removal of the Glu166 side chain changes the conformation of the Lys73 side chain and thus the interaction between Lys73 and Glu166. The Lys73 side chain would play an important role in proton relay, switching its conformation from one to the other depending on the circumstances. The electron density map also implies possible rotation of Ser237. Comparison of the Toho-1 structure with the structure of other class A beta-lactamases shows that the hydroxyl group of Ser237 is likely to rotate through interaction with the carboxyl group of the substrate. Another peculiarity is the existence of three sulfate ions positioned in or near the substrate-binding cavity. One of these sulfate ions is tightly bound to the active center, while the other two are held by a region of positive charge formed by two arginine residues, Arg274 and Arg276. This positively charged region is speculated to represent a pseudo-binding site of the beta-lactam antibiotics, presumably catching the methoxyimino group of the third-generation cephems prior to proper binding in the substrate-binding cleft for hydrolysis. This high-resolution structure, together with detailed kinetic analysis of Toho-1, provides a new hypothesis for the catalytic mechanism and substrate specificity of Toho-1.     

Benzothiophene and naphthalene derived constrained SERMs

For selective estrogen receptor modulators (SERMs), the orientation of the basic side chain relative to the SERM core has a significant impact on function. The synthesis and biological evaluation of two series of SERMs are disclosed, where the ligand side chain is constrained to adopt a defined orientation. Compounds where the side chain is forced into the plane of the SERM core have a different profile compared to those compounds where the side chain is pseudo-orthogonal, particularly with regard to antagonism of estradiol action on an Ishikawa uterine cell line.     

A new amphipathy scale: I. Determination of the scale from molecular dynamics data

Two new amphipathy scales elaborated from molecular dynamics data are presented. Their applications contribute for the identification of the hydrophobic or hydrophilic regions in proteins solely from the primary structure. The new amphipathy coefficients (AC) reflect the side chain/solvent molecules configurational energies. A polar (water) and an apolar solvent, CCl₄, were used resulting in the two AC_water and AC_CCl4 scales. These solvents were chosen to simulate the aqueous phases and the transmembrane ambients of cellular membranes where the membrane proteins act. The new amphipathy scales were compared with some previous scales determined by different methods, which were also compared between them, indicating more than 90% of the correlation coefficients are less than 0.9: the scales are strictly dependent on the methodologies used in their determination. The AC_CCl4 scale is related with the size of side chain amino acids while AC_water is related with the hydrophobicity of side chain amino acids. The quality of the scales was confirmed by an example of application where AC_water was able to identify correctly the transmembrane, hydrophobic regions of a membrane protein. These results also indicate that water is an important factor responsible for the tertiary structure of membrane proteins.     

Cutting edge: N-hydroxy peptides: a new class of TCR antagonists.

TCR antagonists are altered T cell epitopes that specifically inactivate T cells. Commonly, they are derived from agonists by amino acid side chain replacement at positions accessible to the TCR. In this paper we report for the first time that a main chain N-hydroxylation, which is not exposed at the surface of the MHC peptide complex, renders an agonist into an antagonist. These mimotopes are a new, yet undescribed class of TCR antagonists. The antagonists are about 100 times more potent than an unrelated peptide that competes for binding to the MHC molecule. The novel main chain modification enhances biostability and maintains side chain constitution and thus opens new prospects for the use of TCR antagonists in the treatment of pathological immune reactions.     

Synthesis and evaluation of some steroidal oximes as cytotoxic agents: structure/activity studies (I)

The side chain of a compound plays an important role in its biological function. In our studies, we have found that hydroximinosteroid derivatives with different side chains and position of hydroximino on ring A and B displayed remarkable distinct cytotoxicities against a diversity of cancer cell types. Presence of an oxime group on ring B and a hydroxy on ring A or B resulted in a higher cytotoxicity than other structural motifs. In addition, a cholesterol-type side chain at position 17 was required for the biological activity. Our findings provide new evidence showing the relationship between the chemical structure and biological function. The information obtained from the studies may be useful for the design of novel chemotherapeutic drugs.     

Metabolism of a novel side chain modified Delta8(14)-15-ketosterol, a potential cholesterol lowering drug: 28-hydroxylation by CYP27A1

The synthetic inhibitors of sterol biosynthesis, 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one and 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one, are of interest as potential cholesterol lowering drugs. Rapid metabolism of synthetic 15-ketosterols may lead to a decrease, or loss, of their potency to affect lipid metabolism. 3beta-Hydroxy-5alpha-cholest-8(14)-en-15-one is reported to be rapidly side chain oxygenated by rat liver mitochondria. In an attempt to reduce this metabolism, the novel side chain modified 15-ketosterol 3beta-Hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one was synthesized. We have examined the metabolism by recombinant human CYP27A1 of this novel side chain modified 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one and compared the rate of metabolism with that of the previously described 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. Both sterols were found to be efficiently metabolized by recombinant human CYP27A1. None of the two 15-ketosterols was significantly metabolized by microsomal 7alpha-hydroxylation. Interestingly, CYP27A1-mediated product formation was much lower with the side chain modified 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one than with the previously described 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. A surprising finding was that this novel side chain modified sterol was metabolized mainly in the C-28 position by CYP27A1. The data on 28-hydroxylation by human CYP27A1 provide new insights on the catalytic properties and substrate specificity of this enzyme. The finding that 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one with a modified side chain is metabolized at a dramatically slower rate than the previously described 15-ketosterol with unmodified side chain may be important for future development of synthetic cholesterol lowering sterols.     

Cholesterol side chain cleavage in rat adrenal supported by outer mitochondrial membrane NADH-semidehydroascorbate reductase

Rat adrenal mitochondria have an active rotenone-insensitive outer mitochondrial membrane NADH-semidehydroascorbate (NADH-SDA) reductase which supports cholesterol side chain cleavage at a rate equal to that supported by malate. Side chain cleavage activity supported by both of these electron donor systems is equally inhibited by cycloheximide. Catalase or butylated hydroxyanisole are required for the NADH-SDA reductase-supported cholesterol side chain cleavage. This requirement can be removed by briefly subjecting the mitochondrial preparations to -20 degrees C. Ascorbic acid alone or with malate is either inhibitory or has no effect on side chain cleavage activity. These observations demonstrate that outer mitochondrial membrane NADH-SDA reductase in rat adrenal functions to provide cytoplasmic reducing equivalents to intramitochondrial cytochrome P-450scc and provides a new explanation for the function of ascorbic acid in corticosteroidogenesis.     

Structural basis of neurophysin hormone specificity: Geometry, polarity, and polarizability in aromatic ring interactions

The structural origins of the specificity of the neurophysin hormone-binding site for an aromatic residue in peptide position 2 were explored by analyzing the binding of a series of peptides in the context of the crystal structure of liganded neurophysin. A new modeling method for describing the van der Waals surface of binding sites assisted in the analysis. Particular attention was paid to the unusually large (5 kcal/mol) difference in binding free energy between Phe and Leu in position 2, a value representing more than three times the maximum expected based on hydrophobicity alone, and additionally remarkable since modeling indicated that the Leu side chain was readily accommodated by the binding pocket. Although evidence was obtained of a weak thermodynamic linkage between the binding interactions of the residue 2 side chain and of the peptide alpha-amino group, two factors are considered central. (1) The bound Leu side chain can establish only one-third of the van der Waals contacts available to a Phe side chain. (2) The bound Phe side chain appears to be additionally stabilized relative to Leu by more favorable dipole and induced dipole interactions with nonaromatic polar and sulfur ligands in the binding pocket, as evidenced by examination of its interactions in the pocket, analysis of the detailed energetics of transfer of Phe and Leu side chains from water to other phases, and comparison with thermodynamic and structural data for the binding of residue 1 side chains in this system. While such polar interactions of aromatic rings have been previously observed, the present results suggest their potential for significant thermodynamic contributions to protein structure and ligand recognition.     

Mechanism of post-translational quinone formation in copper amine oxidases and its relationship to the catalytic turnover

Copper amine oxidases (CAOs) post-translationally construct a redox-active quinone from an amino acid side chain in their polypeptide chain. As such, these enzymes illustrate how nature is able to expand upon naturally-occurring side chains to create new, catalytically powerful functionalities. The active sites of the CAOs are highly unusual in their ability to catalyze two very different reactions: single-turnover, oxygen-dependent quinone formation, followed by catalytic oxidation (formally dehydrogenation) of amines. This review summarizes our current understanding of the pathway whereby the 2,4,5-trihydroxyphenylalanyl quinone (TPQ) cofactor is generated from the phenolic side chain of tyrosine. This reaction occurs spontaneously intermediates in the presence of O(2) and active site bound Cu(II), without the assistance of other proteins or cofactors. Ongoing work has focused on uncovering the details of the TPQ formation mechanism. A larger goal is to understand how a single active site is capable of supporting both quinone formation and subsequent catalytic turnover.     

Identification of secretin,vasoactive intestinal peptide and glucagon binding sites: from chimaeric receptors to point mutations

We have identified two basic residues that are important for the recognition of secretin and vasoactive intestinal peptide (VIP) by their respective receptors. These two peptides containing an Asp residue at position 3 interacted with an arginine residue in transmembrane helix 2 (TM2) of the receptor, and the lysine residue in extracellular loop 1 (ECL1) stabilized the active receptor conformation induced by the ligand. The glucagon receptor possesses a Lys instead of an Arg in TM2, and an Ile instead of Lys in ECL1; it markedly prefers a Gln side chain in position 3 of the ligand. Our results suggested that, in the wild-type receptor, the Ile side chain prevented access to the TM2 Lys side chain, but oriented the glucagon Gln(3) side chain to its proper binding site. In the double mutant, the ECL1 Lys allowed an interaction between negatively charged residues in position 3 of glucagon and the TM2 Arg, resulting in efficient receptor activation by [Asp(3)]glucagon as well as by glucagon.