Site-specific platinum(II) cross-linking in a ribozyme active site

The function of RNA depends on its ability to adopt complex and dynamic structures, and the incorporation of site-specific cross-linking probes is a powerful method for providing distance constraints that are valuable in RNA structural biology. Here we describe a new RNA-RNA cross-linking strategy based on Pt(II) targeting of specific phosphorothioate substitutions. In this strategy cis-diammine Pt(II) complexes are kinetically recruited and anchored to a phosphorothioate substitution embedded within a structured RNA. Substitution of the remaining exchangeable Pt(II) ligand with a nucleophile supplied by a nearby RNA nucleobase results in metal-mediated cross-links that are stable during isolation. This type of cross-linking strategy was explored within the catalytic core of the Hammerhead ribozyme (HHRz). When a phosphorothioate substitution is installed at the scissile bond normally cleaved by the HHRz, Pt(II) cross-linking takes place to nucleotides G8 and G10 in the ribozyme active site. Both of these positions are predicted to be within ~8 ? of a phosphorothioate-bound Pt(II) metal center. Cross-linking depends on Mg(2+) ion concentration, reaching yields as high as 30%, with rates that indicate cation competition within the RNA three-helix junction. Cross-linking efficiency depends on accurate formation of the HHRz tertiary structure, and cross-links are not observed for RNA helices. Combined, these results show promise for using kinetically inert Pt(II) complexes as new site-specific cross-linking tools for exploring RNA structure and dynamics.     

Analysis of receptor-ligand binding by photoaffinity cross-linking

Photoaffinity cross-linking is a fast developing technology for biomolecular interactions,including receptor-ligand binding.The chemical mechanisms of the most commonly used photoactivatable probes and their respective photochemistry are summarized.This review focuses on the expanding utilities of this technology as a result of recent advances in the(i)identification of receptor contact sites,(ii)monitoring ligand-induced receptor conformational changes,(iii)identification of global binding surfaces,(iv)binding mode analysis using bifunctional photo-probes,(v)application of biosynthetic photo-probes,and(vi)examples of novel target discovery using this technology.Limitations and future potential of this approach are also discussed.     

Tightening of active site interactions en route to the transition state revealed by single-atom substitution in the guanosine-binding site of the Tetrahymena group I ribozyme

Protein enzymes establish intricate networks of interactions to bind and position substrates and catalytic groups within active sites, enabling stabilization of the chemical transition state. Crystal structures of several RNA enzymes also suggest extensive interaction networks, despite RNA's structural limitations, but there is little information on the functional and the energetic properties of these inferred networks. We used double mutant cycles and presteady-state kinetic analyses to probe the putative interaction between the exocyclic amino group of the guanosine nucleophile and the N7 atom of residue G264 of the Tetrahymena group I ribozyme. As expected, the results supported the presence of this interaction, but remarkably, the energetic penalty for introducing a CH group at the 7-position of residue G264 accumulates as the reaction proceeds toward the chemical transition state to a total of 6.2 kcal/mol. Functional tests of neighboring interactions revealed that the presence of the CH group compromises multiple contacts within the interaction network that encompass the reactive elements, apparently forcing the nucleophile to bind and attack from an altered, suboptimal orientation. The energetic consequences of this indirect disruption of neighboring interactions as the reaction proceeds demonstrate that linkage between binding interactions and catalysis hinges critically on the precise structural integrity of a network of interacting groups.     

Probing general base catalysis in the hammerhead ribozyme

Recent structural and computational studies have shed new light on the catalytic mechanism and active site structure of the RNA cleaving hammerhead ribozyme. Consequently, specific ribozyme functional groups have been hypothesized to be directly involved in general/acid base catalysis. In order to test this hypothesis, we have developed an affinity label to identify the functional general base in the S. mansoni hammerhead ribozyme. The ribozyme was reacted with a substrate analogue bearing a 2'-bromoacetamide group in place of the nucleophilic 2'-hydroxyl group which would normally be deprotonated by a general base. The electrophilic 2'-bromoacetamide group is poised to alkylate the general base, which is subsequently identified by footprinting analysis. Herein, we demonstrate alkylation of N1 of G12 in the hammerhead ribozyme in a pH and [Mg(2+)] dependent manner that is consistent with the native cleavage reaction. These results provide substantial evidence that deprotonated N1 of G12 functions directly as a general base in the hammerhead ribozyme; moreover, our experiments provide evidence that the pKa of G12 is perturbed downward in the context of the active site structure. We also observed other pH-independent alkylations, which do not appear to reflect the catalytic mechanism, but offer further insight into ribozyme conformation and structure.     

Characterization of an RNA active site: interactions between a Diels-Alderase ribozyme and its substrates and products

Ribozymes have recently been shown to catalyze the stereoselective formation of carbon-carbon bonds between small organic molecules. The interactions of these Diels-Alderase ribozymes with their substrates and products have now been elucidated by chemical substitution analysis by using 44 different, systematically varied analogues. RNA-diene interaction is governed by stacking interactions, while hydrogen bonding and metal ion coordination appear to be less important. The diene has to be an anthracene derivative, and substituents at defined positions are permitted, thereby shedding light on the geometry of the binding site. The dienophile must be a five-membered maleimidyl ring with an unsubstituted reactive double bond, and a hydrophobic side chain makes a major contribution to RNA binding. The ribozyme distinguishes between different enantiomers of chiral substrates and accelerates cycloadditions with both enantio- and diastereoselectivity. The stereochemistry of the reaction is controlled by RNA-diene interactions. The RNA interacts strongly and stereoselectively with the cycloaddition products, requiring several structural features to be present. Taken together, the results highlight the intricacy of ribozyme active sites which can control chemical reaction pathways based on minute differences in substrate stereochemistry and substitution pattern.     

Probing Akt-inhibitor interaction by chemical cross-linking and mass spectrometry

The serine/threonine kinase Akt is a critical enzyme that regulates cell survival. As high Akt activity has been shown to contribute to the pathogenesis of various human malignancies, inhibition of Akt activation is a promising therapeutic strategy for cancers. We have previously demonstrated that changes in Akt interdomain arrangements from a closed to open conformation occur upon Akt-membrane interaction, which in turn allows Akt phosphorylation/activation. In the present study, we demonstrate a novel strategy to discern mechanisms for Akt inhibition based on Akt conformational changes using chemical cross-linking and ¹⁸O labeling mass spectrometry. By quantitative comparison of two interdomain cross-linked peptides, which represent the proximity of the domains involved, we found that the binding of Akt to an inhibitor (PI analog) caused the open interdomain conformation where the PH and regulatory domains moved away from the kinase domain, even before interacting with membranes, subsequently preventing translocation of Akt to the plasma membrane. In contrast, the interdomain conformation remained unchanged after incubating with another type of inhibitor (peptide TCL1). Subsequent interaction with unilamellar vesicles suggested that TCL1 impaired particularly the opening of the PH domain for exposing T308 for phosphorylation at the plasma membrane. This novel approach based on the conformation-based molecular interaction mechanism should be potentially useful for drug discovery efforts for specific Akt inhibitors or anti-tumor agents.     

Probing the stereochemistry of the active site of gamma-glutamyl transpeptidase using sulfur derivatives of l-glutamic acid

Gamma-glutamyl transpeptidase (GGT) catalyses the transfer of a gamma-glutamyl moiety from a donor substrate to different acceptors, such as amino acids and water. GGT is known to display relatively low stereospecificity with respect to the alpha-stereocentre of its donor substrates. In this study we have studied its stereospecificity with respect to the stereocentre at the delta-position of different analogues of L-glutamic acid. Notably, L-methionine sulfoxide is well-recognised whereas L-methionine sulfone and L-methionine sulfoximine are not. Furthermore, when the synthetic gamma-diastereoisomers of L-methionine sulfoxide were separated and tested, it was discovered that GGT shows remarkable stereospecificity at the gamma-position, binding the S(C)S(S) diastereoisomer with a K(i) of 3.5 mM, whereas the S(C)R(S) diastereoisomer is not recognised. Finally, using a sulfoxide as a new pharmacophore for GGT, we have synthesized and tested an analogue of glutathione to obtain a very promising competitive inhibitor with a K(i) of (53 +/- 3) microM.     

Reduction of an aldehyde by a NADH/Zn2+ -dependent redox active ribozyme

We report here the ability of an alcohol dehydrogenase (ADH) ribozyme to reduce a benzaldehyde. While the ribozyme was initially evolved in vitro based on the activity for the NAD+-dependent oxidation of the benzyl alcohol, we found that this ADH ribozyme is also capable of reducing the aldehyde in the presence of NADH and Zn2+. The rate acceleration gained by ribozyme catalysis was more than 6 orders of magnitude larger than the spontaneous reaction. Although the reversibility of phosphordiester and acyl transfer reactions catalyzed by ribozymes was known, that of other chemical reactions has not been well established. This study has demonstrated the reversibility of a hydride transfer chemistry catalyzed by the ADH ribozyme. Most interestingly, the ribozyme shares many features with the protein ADHs, e.g., reversibility and NADH/Zn2+ dependence.     

Probing the active site of trypsin with rose bengal: insights into the photodynamic inactivation of the enzyme

In this work the active site of trypsin has been probed with the dye rose bengal. The dye binds competitively to the enzyme, and it can be used as a probe of the active site of the enzyme. On the basis of the emission wavelength, the binding site of trypsin is relatively polar and is similar to that of acetone in its polarity. The triplet state of rose bengal is quenched by trypsin. This quenching may be caused by the tryptophan and tyrosine residues that are in the near vicinity of the trypsin active site. This quenching can compete with the formation of singlet oxygen from the excited triplet state of rose bengal. We demonstrate that the singlet oxygen involved in the photoinactivation of trypsin is produced by the free rose bengal in solution and the bound dye is incapable of producing singlet oxygen. This explains the lack of correlation between photoinactivation efficiency and sensitizer binding capability previously reported by Wade and Spikes.     

Probing three-dimensional structure of bovine serum albumin by chemical cross-linking and mass spectrometry

Serum albumin is the principal transporter of fatty acids that are otherwise insoluble in circulating plasma. While the crystal structure of human serum albumin (HSA) as well as its binding with fatty acids has been characterized, the three dimensional structure of bovine serum albumin (BSA) has not been determined although both albumins share 76% sequence homology. In this study we used mass spectrometry coupled with chemical cross-linking, to probe the tertiary structure of BSA. BSA was modified with lysine specific cross-linkers, bis(sulfosuccinimidyl) suberate (BS(3)), disuccinimidyl suberate (DSS) or disuccinimidyl glutarate (DSG), digested with trypsin and analyzed by tandem mass spectrometry. With O-18 labeling during the digestion, through-space cross-linked peptides were readily identified in mass spectra by a characteristic 8 Da shift. From the cross-linked peptides identified in this study, we found that 12 pairs of lysine residues were separated within 20 A, while 5 pairs were spaced between 20 and 24 A. The spatial distance constraints generated from five K-K pairs in BSA were consistent with the corresponding distance obtained from the crystal structure of HSA, although only six equivalent K-K pairs could be compared. According to our data, the distance between K235 of IIA and K374 of IIB domain in BSA was farther by 7-11 A than that expected from the crystal structure of HSA, suggesting structural differences between BSA and HSA in this region. The distance constraints obtained for lysine residues using various cross-linkers should be valuable in assisting the determination of the 3-D structure of BSA.     

Dihydrogen activation by a diruthenium analogue of the Fe-only hydrogenase active site

The photochemical reaction of Ru2(S2C3H6)(CO)4(PCy3)2 (1) and H2 gives the dihydride Ru2(S2C3H6)(mu-H)(H)(CO)3(PCy3)2 (2). NMR and crystallographic studies reveal mutually trans basal phosphine ligands and both bridging and terminal hydrides. Ru2(S2C2H4)(CO)4(PCy3)2 behaves similarly. Other HX substrates undergo photoaddition to 1, affording Ru2(S2C3H6)(mu-H)(X)(CO)3(PCy3)2 for X = OTs (3a), Cl (3b), and SPh (3c). Treatment of Ru2(S2C3H6)(mu-H)(H)(CO)3(PCy3)2 with [H(OEt2)]BArF4 (ArF = B(C6H3-3,5-(CF3)2) in CD2Cl2 gives [Ru2(S2C3H6)(mu-H)(CO)3(PCy3)2(H2)]+ (4), which catalyzes H2-D2 exchange. The reaction of 2 with [D(OEt2)]BArF4 gave [Ru2(S2C3H6)(mu-H)(CO)3(PCy3)2(HD)]+ (JH-D = 31 Hz). These studies provide the first models for the Fe-only hydrogenases that bear dihydrogen and terminal hydrido ligands.     

RNA targeting at any chosen site of interest by the novel RNA-protein hybrid ribozyme and the gene discovery based on the hybrid ribozyme libraries

RNA targeting by the RNA-protein hybrid ribozymes, whose protein part can specifically bind to the RNA helicase, is described.     

Site-specific cross-linking of nucleic acids using the abasic site

[formula: see text] An efficient site-specific cross-linking reaction between two carbohydrate residues present in two complementary DNA sequences is described. One oligodeoxynucleotide, 5'd(GGCTGA*CTGCG)3', carries an amino nucleophile tethered to the 2'-hydroxyl of an adenosine residue (A*). The target electrophile is an abasic site generated in the complementary sequence, 5'd(CGCAGDCAGCC)3' (D represents the deoxyribose). The cross-linking reaction was carried out by a reductive amination reaction in > 95% yield.     

Solid-phase synthesis of photoaffinity probes: highly efficient incorporation of biotin-tag and cross-linking groups

A benzophenone cross-linking group and a biotin-tag hybrid, resin 1a, attached to our novel resin 2 was readily converted to the photoaffnity probe 20 by condensation with the ligand carboxylic acid 19 and cleavage from the resin without purification.     

Aminoacyl-tRNA synthesis by a resin-immobilized ribozyme

We report herein a new method for the aminoacylation of tRNA, using a resin-immobilized ribozyme and the cyanomethyl ester (CME) of an amino acid substrate. The oxidized form of the ribozyme was immobilized on a hydrazine resin via covalent linkage. We performed aminoacylation of tRNAs using this ribozyme-resin to isolate aminoacyl-tRNAs. The column was recycled up to 5 times without significant activity loss. Thus, our ribozyme-based aminoacylation system has significant potential to be a powerful and practical technique for supplying various nonnatural aminoacyl-tRNAs for a highly efficient in vitro translation system.     

Analysing and understanding the active site by IR spectroscopy

IR spectroscopy is a technique particularly adapted for understanding the mechanism of catalytic reactions, being able to probe the surface mechanisms at the molecular level. In this critical review the main advances in the field are presented, both under the aspects of the in situ and operando approaches. A broad view of the most authoritative literature of the domain is given, based largely on the experience built up at the LCS laboratory in the last decades. After having presented the general methodology to observe a potential active site directly or by probe molecule adsorption, several examples illustrate the qualitative and quantitative analysis of the physical-chemical properties of the surface entities. The last part of the review is dedicated to the discrimination of the role of the active site and its links with the catalytic steps; the hot problem of the reaction intermediates and their visibility via spectroscopic techniques is critically addressed (138 references).     

Probing the conformational space available to inhibitors in the thermolysin active site using Monte Carlo/energy minimization techniques

The conformational space available to four inhibitors of the bacterial enzyme thermolysin has been searched in the enzyme binding site using a method that combines Monte Carlo type techniques with energy minimization for exploration of the conformational potential energy hypersurface. Molecular mechanics methodology using the AMBER force field was employed for computation of the molecular energetics. Solvation energies were also included in the calculations by employing a technique that estimates hydration energies based on the exposed solvent accessible surface area for each atom of the inhibitor and active site. It was found that in each case, the crystallographically observed conformation was among the low energy conformers discovered. In fact, in three of the calculations it was the lowest energy conformation. The methodology described in this article is expected to be quite useful for studies involving computer aided design and evaluation of enzyme inhibitors.     

Allosterically activated Diels-Alder catalysis by a ribozyme

We describe the allosteric control of Diels-Alder reactions by a small organic effector, theophylline. This is achieved by converting a Diels-Alder ribozyme into an allosterically regulated system. In contrast to other published systems, we have a bond-forming reaction with two small-molecule substrates and multiple turnover. This system could be very attractive for the development of assays for a variety of analytes and can be regarded as a prototype of fully synthetic signaling cascades.     

Photonics of the hemoglobin active site

The results of a pico- and nanosecond laser absorption spectroscopy study of the rebinding of the oxygen molecule by the triliganded hemoglobin tetramer in the R-state are presented.