Interactions In Ternary Complexes Research Articles

RNA:RNA interaction in ternary complexes resolved by chemical probing.

RNA regulation can be performed by a second targeting RNA molecule, such as in the microRNA regulation mechanism. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) probes the structure of RNA molecules and can resolve RNA:protein interactions, but RNA:RNA interactions have not yet been addressed with this technique. Here, we apply SHAPE to investigate RNA-mediated binding processes in RNA:RNA and RNA:RNA-RBP complexes. We use RNA:RNA binding by SHAPE (RABS) to investigate microRNA-34a (miR-34a) binding its mRNA target, the silent information regulator 1 (mSIRT1), both with and without the Argonaute protein, constituting the RNA-induced silencing complex (RISC). We show that the seed of the mRNA target must be bound to the microRNA loaded into RISC to enable further binding of the compensatory region by RISC, while the naked miR-34a is able to bind the compensatory region without seed interaction. The method presented here provides complementary structural evidence for the commonly performed luciferase-assay-based evaluation of microRNA binding-site efficiency and specificity on the mRNA target site and could therefore be used in conjunction with it. The method can be applied to any nucleic acid-mediated RNA- or RBP-binding process, such as splicing, antisense RNA binding, or regulation by RISC, providing important insight into the targeted RNA structure.

Protein Ligand Interactions Using Surface Plasmon Resonance.

Surface Plasmon Resonance (SPR) is a powerful biophysical method for characterizing small molecule binding to proteins. Owing to its ability to characterize binary inteactions between warheads and E3 ligases or substrates, SPR is a useful tool for the development of targeted protein degraders. SPR is also an effective method for optimizing linkers and characterizing ternary complex interactions that are mediated by heterobifunctional ligands (Roy et al. ACS Chem Biol 14:361-368, 2019). Recent advances in the throughput of modern instruments have improved the ability of SPR to rapidly triage ligands based on binding kinetics and affinity, making this technique invaluable for driving degraderoptimization. This chapter describes the characterization of ligands binding to the Thalidomide Binding Domain of mouse Cereblon (mCRBN-TBD) using the Biacore 8K+.

Ternary Complexes Stabilized by Chalcogen and Alkaline-Earth Bonds: Crucial Role of Cooperativity and Secondary Noncovalent Interactions.

High-level G4 calculations show that the strength of chalcogen interactions is enhanced dramatically if chalcogen compounds simultaneously form alkaline-earth bonds. This phenomenon is studied by exploring binary YX2 ⋅⋅⋅N-Base complexes and two types of ternary MCl2 ⋅⋅⋅YX2 ⋅⋅⋅N-Base, YX2 ⋅⋅⋅N-Base⋅⋅⋅MCl2 complexes, in which YX2 is a chalcogen compound (Y=S, Se; X=F, Cl), the N-Bases are sp, sp2 , and sp3 bases (NCH, HN=CH2 , NH3 ), and MCl2 are alkaline-earth BeCl2 or MgCl2 derivatives. Starting from the chalcogen-bonded complexes YX2 ⋅⋅⋅NH3 and YX2 ⋅⋅⋅HN=CH2 , the binding site of a new incoming alkaline-earth bond is found, surprisingly, to depend on the nature of the halogen atom attached to the chalcogen. For the YF2 binary complexes the association site is the F atom of the YF2 subunit, whereas for YCl2 it is the N atom of the nitrogen base. Regarding YX2 ⋅⋅⋅NCH complexes, N is the most favorable site for an alkaline-earth interaction in ternary complexes, regardless of which YX2 derivative is used. The explanation relies on the interplay of all the noncovalent interactions involved: the strong cooperativity between chalcogen and alkaline-earth bonds, and the appearance of secondary noncovalent interactions in the form of hydrogen bonds.

Insights into the cooperativity between multiple interactions of dimethyl sulfoxide with carbon dioxide and water.

Interactions of dimethyl sulfoxide with carbon dioxide and water molecules which induce 18 significantly stable complexes are thoroughly investigated. An addition of CO2 or H2 O molecules into the DMSO⋯1CO2 and DMSO⋯1H2 O systems leads to an increase in the stability of the resulting complexes, in which it is larger for a H2 O addition than a CO2 . The overall stabilization energy of the DMSO⋯1,2CO2 is mainly contributed by the S=O⋯C Lewis acid-base interaction, whereas the O - H⋯O hydrogen bond plays a significant role in stabilizing complexes of DMSO⋯1,2H2 O and DMSO⋯1CO2 ⋯1H2 O. Remarkably, the complexes of DMSO⋯2H2 O are found to be more stable than DMSO⋯1CO2 ⋯1H2 O and DMSO⋯2CO2 . The level of the cooperativity of multiple interactions in ternary complexes tends to decrease in going from DMSO⋯2H2 O to DMSO⋯1CO2 ⋯1H2 O and finally to DMSO⋯2CO2 . It is generally found that the red shift of the O - H bond involved in an O - H⋯O hydrogen bond increases while the blue shift of a C - H bond in a C - H⋯O hydrogen bond decreases when a cooperative effect occurs in ternary complexes as compared to those of the corresponding binary complexes. © 2018 Wiley Periodicals, Inc.

Co-operativity in non-covalent interactions in ternary complexes: a comprehensive electronic structure theory based investigation

The structure and stability of various ternary complexes in which an extended aromatic system such as coronene interacts with ions/atoms/molecules on opposite faces of the π-electron cloud were investigated using ab initio calculations. By characterizing the nature of the intermolecular interactions using an energy decomposition analysis, it was shown that there is an interplay between various types of interactions and that there are co-operativity effects, particularly when different types of interactions coexist in the same system. Graphical abstract Weak OH-π, π-π and van der Waals-π ternary systems are stabilized through dispersion interactions. Cation-π ternary systems are stabilized by through-space electrostatic interactions.

Carbene–aerogen bonds: an ab initio study

ABSTRACTThrough the use of ab initio calculations, the possibility of formation of σ-hole interaction between ZO3 (Z = Ar, Kr and Xe) and carbene species is investigated. Since singlet carbenes show a negative electrostatic potential on their divalent carbon atom, they can favourably interact with the positive electrostatic potential generated by the σ-hole of Z atom of ZO3. The characteristic of this interaction, termed as ‘carbene–aerogen’ bond, is analysed in terms of geometric, interaction energies and electronic features. The energy decomposition analysis indicates that for all complexes analysed here, the electrostatic energy is more negative than the polarisation or dispersion energy term. According to the electron density analysis, some partial covalent character can be ascribed to Xe⋅⋅⋅C interactions. In addition, the carbene–aerogen bond exhibits cooperative effects with the H⋅⋅⋅O hydrogen-bonding interaction in ternary complexes where both interactions coexist. For a given carbene, the amount of these cooperative effects increases with the size of the Z atom. The results obtained in this work may be helpful for the extension and future application of σ-hole intermolecular interactions as well as coordination chemistry.

Tuning of chalcogen bonds by cation-π interactions: cooperative and diminutive effects.

The tunability of Y···N chalcogen bond via formation of a cation-π interaction in ternary complexes M(+)-PhYH-NH3, M(+)-PhYH-NCH, and M(+)-PhCCCN-YHF (M = Li, Na; Y = Se, Te) is investigated at MP2/aug-cc-pVDZ computational level. Our results indicate that the strength of Y···N and cation-π interactions in the ternary complexes depends on the role of the aromatic molecule. That is, a cooperative effect is evident if aromatic molecule acts as the Lewis acid and Lewis base, simultaneously, while a diminutive effect is observed when the aromatic molecule acts only as the Lewis base in both Y···N and cation-π interactions. For a given aromatic system, the shortening or lengthening of Y···N distances is more important for Li(+) complexes than Na(+) counterparts. The mechanism of cooperative/diminutive effects in the ternary complexes is studied by molecular electrostatic potential (MEP) and topological analysis of the electron density.

Formation of Cucurbit[8]uril-Based Supramolecular Hydrogel Beads Using Droplet-Based Microfluidics.

Herein we describe the use of microdroplets as templates for the fabrication of uniform-sized supramolecular hydrogel beads, assembled by supramolecular cross-linking of functional biopolymers with the macrocyclic host molecule, cucurbit[8]uril (CB[8]). The microdroplets were formed containing diluted hydrogel precursors in solution, including the functional polymers and CB[8], in a microfluidic device. Subsequent evaporation of water from collected microdroplets concentrated the contents, driving the formation of the CB[8]-mediated host-guest ternary complex interactions and leading to the assembly of condensed three-dimensional polymeric scaffolds. Rehydration of the dried particles gave monodisperse hydrogel beads. Their equilibrium size was shown to be dependent on both the quantity of material loaded and the dimensions of the microfluidic flow focus. Fluorescein-labeled dextran was used to evaluate the efficacy of the hydrogel beads as a vector for controlled cargo release. Both passive, sustained release (hours) and triggered, fast release (minutes) of the FITC-dextran was observed, with the rate of sustained release dependent on the formulation. The kinetics of release was fitted to the Ritger-Peppas controlled release equation and shown to follow an anomalous (non-Fickian) transport mechanism.

The influence of substituent effect on noncovalent interactions in ternary complexes stabilized by hydrogen-bonding and halogen-bonding

The ternary complexes stabilized by three types of noncovalent interactions, that is, H-bonding, X-bonding and N…C interaction, were investigated. In all investigated systems the bromine atom plays a dual Lewis acid–Lewis base role. The acidity/basicity of the Br-moiety is modulated by the substituent effect. Substituents attached to the benzene ring in para-position with respect to Br atom change the ability of that atom to interact through H- and X-bonding. The structural, energetic and electron-density based properties of the systems were investigated. The three-body contributions to interaction energies were estimated in order to assess the cooperative or anticooperative character of interrelation between three types of interactions present in the investigated systems. It was found that rather anticooperative effect is present for all variously substituted systems.

Structural model of haptoglobin and its complex with the anticoagulant ecotin variants: structure–activity relationship study and analysis of interactions

Recently the literature described the binding of Haptoglobin (HP) with ecotin, a fold-specific serine-proteases inhibitor with an anticoagulant profile and produced by Escherichia coli. In this work, we used some in silico and in vitro techniques to evaluate HP 3D-fold and its interaction with wild-type ecotin and two variants. Our data showed HP models conserved trypsin fold, in agreement to the in vitro immunological recognition of HP by trypsin antibodies. The analysis of the three ecotin–HP complexes using the mutants RR and TSRR/R besides the wild type revealed several hydrogen bonds between HP and ecotin secondary site. These data are in agreement with the in vitro PAGE assays that showed the HP-RR complex in native gel conditions. Interestingly, the ternary complex interactions varied depending on the inhibitor structure and site-directed mutation. The interaction of HP with TSRR/R involved new residues compared to wild type, which infers a binding energy increase caused by the mutation.

An ab initio study of cooperative effects in ternary complexes X:CNH:Z with X, Z=CNH, FH, ClH, FCl, and HLi: structures, binding energies, and spin–spin coupling constants across intermolecular bonds

A systematic ab initio investigation has been carried out to determine the structures, binding energies, and spin-spin coupling constants of ternary complexes X:CNH:Z and corresponding binary complexes X:CNH and CNH:Z, for X, Z = CNH, FH, ClH, FCl, and HLi. The enhanced binding energies of ternary complexes X:CNH:Z for fixed X as a function of Z decrease in the same order as the binding energies of the binary complexes CNH:Z. In contrast, the enhanced binding energies of the ternary complexes for fixed Z as a function of X do not decrease in the same order as the binding energies of the binary complexes X:CNH, a consequence of the increased stabilities of ternary complexes FCl:CNH:Z due to very strong chlorine-shared halogen bonds. For complexes in which the X···CNH interaction is a D-H···C hydrogen bond for D-H the proton-donor group (N-H, F-H, or Cl-H), spin-spin coupling constants (1)J(D-H) and (2h)J(D-C) in ternary complexes X:CNH:Z decrease in absolute value as the binding energies of binary complexes CNH:Z and the enhanced binding energies of the ternary complexes for fixed X as a function of Z also decrease. However, (2X)J(F-C) increases as the enhanced binding energies of the ternary complexes FCl:CNH:Z decrease, a consequence of the nature of the chlorine-shared halogen bond. The one-bond coupling constants (1)J(N-H) for the CNH···Z interaction in ternary complexes vary significantly, depending on the nature of the X···CNH interaction. The largest values of (1)J(N-H) are found for ternary complexes with FCl as X. Two-bond coupling constants (2h)J(N-A) for A the proton-acceptor atom of Z, and (2d)J(N-H) decrease in absolute value in the order of decreasing enhancement energies of ternary complexes X:CNH:Z for fixed Z as a function of X.

Ternary inclusion complexes of cyclodextrins with spin-labeled indoles and hydrocarbons. The formation of 2: 1: 2 complexes and partial dissociation of spin-labeled guests in the presence of a hydrocarbon liquid phase

The interaction of ternary complexes γ-or β-cyclodextrin (CD)-spin-labeled indole (pyrrolidine (1) or piperidine (2) derivative)-hydrocarbon (benzene or cyclohexane) with the liquid phase of these hydrocarbons gives rise to a new type of complexes (s) the ESR spectra of which are indicative of a much lower rotational mobility and its weaker temperature dependence for the spin-labeled guests compared with the initial ternary complexes (w). The formation of s-complexes is accompanied by a decrease in the proportion of the initial w-complexes. This gives rise to several isosbestic points in the ESR spectra, which indicates the formation of s-complexes from w-complexes. The rotational diffusion coefficient of 2 in s-complexes decreases 6–10-fold with respect to its value for w-complexes and the libration amplitude of 1 in the s-complexes decreases to 5–6° at 295 K. Transition between the two types of complexes occurs with characteristic times of 103–105 s and is reversible: upon the removal of the hydrocarbon, the proportion of the strongly immobilized s-signal decreases, while upon its addition, it increases again. These results indicate that the strongly immobilized ESR signals belong to 2: 1: 2 complexes, which are formed upon the insertion of the second hydrocarbon molecule into the initial ternary 2: 1: 1 w-complexes. According to PM3 quantum-chemical calculations, 2: 1: 2 complexes are stable in energy and the geometry of hydrocarbon arrangement depends on the structure of the spin-labeled guest.

Platelet Factor 4 (PF4) Modulation of Endothelial Protein C Receptor and Thrombomodulin Enhancements of Protein C Activation and TAFI Activation by Thrombin.

Protein C (PC) activation by thrombin on endothelial cells normally involves thrombomodulin (TM) and the endothelial cell protein C receptor (EPCR), and dysfunction of this mechanism is implicated in a variety of pathologies. Platelet factor 4 (PF4), a releasable platelet alpha-granule protein, stimulates PC activation in vitro and in vivo; PF4 stimulates PC activation in the presence but not in the absence of TM and this requires the PC Gla-domain (Slungaard et al). In contrast to PC activation, we found that thrombin/TM-mediated activation of thrombin-activatable fibrinolysis inhibitor (TAFI) is inhibited by PF4. In assays where TAFI-dependent inhibition of clot lysis was promoted by thrombin/TM-mediated TAFI activation, PF4 dose-dependently inhibited TAFI's anti-fibrinolytic effects. In the absence of TM, PF4 did not affect TAFI activation by thrombin alone and did not alter the clot-lysis times in normal plasma. Thus, the effects of PF4 on thrombin/TM actions are enhancing for PC activation and inhibitory for TAFI activation such that as PF4 concentration increases, the ratio for relative rates of PC and TAFI activation changes more than 40-fold to favor selective PC activation. The current model to explain PF4 stimulation of PC activation involves electrostatic bridging of the negatively charged Gla-domain of PC and the negatively charged glycosaminoglycan moiety of TM by the highly positively charged PF4. In assays using purified soluble reactants components, EPCR abrogated PF4 stimulation of PC activation by thrombin/TM. On 293 cells expressing TM but not EPCR, PF4 enhanced PC activation by thrombin, and this effect was inhibited by soluble EPCR (IC50 ~ 1.0 μM). Based on these and previous observations, we hypothesized that the binding sites for EPCR and PF4 on the Gla-domain of PC may overlap and, if so, that PF4 could interfere with EPCR-dependent APC cytoprotective activities involving protease activated recpotor-1 activation. Indeed, studies showed that PC and APC binding to 293 cells expressing EPCR was inhibited by PF4 (IC50 ~ 1.0 μM) and that PF4 concentrations which inhibited APC binding to EPCR significantly impaired EPCR-dependent anti-apoptotic activity of APC in assays of staurosporine-induced endothelial cell apoptosis. PF4 enhanced EPCR-dependent PC activation on endothelial cells at PF4 concentrations which did not significant inhibit PC binding to EPCR. On K293 cells transfected with both TM and EPCR, PF4 at low concentrations enhanced EPCR-dependent PC activation. Anti-EPCR antibodies abrogated both EPCR-dependent and PF4-dependent stimulation of PC activation on cells containing TM and EPCR, further suggesting a direct interaction between PF4 and EPCR. Proof for this interaction came from experiments showing that PF4 bound to immobilized soluble EPCR. Thus, these results suggest that on endothelial cells, PF4 enhances APC generation by enhancement of the ability of both TM and EPCR to promote PC activation while inhibiting TAFI activation. Therefore, the current model for stimulation of PC activation by PF4 involving thrombin/TM-PF4-PC quaternary ternary complex interactions does not adequately predict the effects of PF4 on PC activation on cells in the presence of EPCR. We propose a new model to explain the effects of PF4 on the generation of APC on endothelial cells in which the tetrameric character of PF4 promotes multiple simultaneous interactions of PF4 in a macromolecular complex with thrombin, TM, PC and EPCR.

Single Molecule Mechanical Probing of the SNARE Protein Interactions

Exocytotic release of neurotransmitters is mediated by the ternary soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors (SNAREs) complex, comprised of syntaxin (Sx), synaptosome-associated protein of 25 kDa (SNAP25), and synaptobrevin 2 (Sb2). Since exocytosis involves the nonequilibrium process of association and dissociation of bonds between molecules of the SNARE complex, dynamic measurements at the single molecule level are necessary for a detailed understanding of these interactions. To address this issue, we used the atomic force microscope in force spectroscopy mode to show from single molecule investigations of the SNARE complex, that Sx1A and Sb2 are zippered throughout their entire SNARE domains without the involvement of SNAP25. When SNAP25B is present in the complex, it creates a local interaction at the 0 (ionic) layer by cuffing Sx1A and Sb2. Force loading rate studies indicate that the ternary complex interaction is more stable than the Sx1A-Sb2 interaction.

Mouse p56 Blocks a Distinct Function of Eukaryotic Initiation Factor 3 in Translation Initiation

Members of the p56 family of mammalian proteins are strongly induced in virus-infected cells and in cells treated with interferons or double-stranded RNA. Previously, we have reported that human p56 inhibits initiation of translation by binding to the "e" subunit of eukaryotic initiation factor 3 (eIF3) and subsequently interfering with the eIF3/eIF2.GTP.Met-tRNAi (ternary complex) interaction. Here we report that mouse p56 also interferes with eIF3 functions and inhibits translation. However, the murine protein binds to the "c" subunit, not the "e" subunit, of eIF3. Consequently, it has only a marginal effect on eIF3.ternary complex interaction. Instead, the major inhibitory effect of mouse p56 is manifested at a different step of translation initiation, namely the binding of eIF4F to the 40 S ribosomal subunit.eIF3.ternary complex. Thus, mouse and human p56 proteins block different functions of eIF3 by binding to its different subunits.

Thioflavin T Is a Fluorescent Probe of the Acetylcholinesterase Peripheral Site That Reveals Conformational Interactions between the Peripheral and Acylation Sites

Three-dimensional structures of acetylcholinesterase (AChE) reveal a narrow and deep active site gorge with two sites of ligand binding, an acylation site at the base of the gorge, and a peripheral site near the gorge entrance. Recent studies have shown that the peripheral site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, but the question of whether the peripheral site makes other contributions to the catalytic process remains open. A possible role for ligand binding to the peripheral site that has long been considered is the initiation of a conformational change that is transmitted allosterically to the acylation site to alter catalysis. However, evidence for conformational interactions between these sites has been difficult to obtain. Here we report that thioflavin T, a fluorophore widely used to detect amyloid structure in proteins, binds selectively to the AChE peripheral site with an equilibrium dissociation constant of 1.0 microm. The fluorescence of the bound thioflavin T is increased more than 1000-fold over that of unbound thioflavin T, the greatest enhancement of fluorescence for the binding of a fluorophore to AChE yet observed. Furthermore, when the acylation site ligands edrophonium or m-(N, N,N-trimethylammonio)trifluoroacetophenone form ternary complexes with AChE and thioflavin T, the fluorescence is quenched by factors of 2.7-4.2. The observation of this partial quenching of thioflavin T fluorescence is a major advance in the study of AChE for two reasons. First, it allows thioflavin T to be used as a reporter for ligand reactions at the acylation site. Second, it indicates that ligand binding to the acylation site initiates a change in the local AChE conformation at the peripheral site that quenches the fluorescence of bound thioflavin T. The data provide strong evidence in support of a conformational interaction between the two AChE sites.

Substrate dependence of amiloride- and soman-induced conformation changes of butyrylcholinesterase as evidenced by high-pressure perturbation

Previous results on butyrylcholinesterase-catalyzed hydrolysis of o-nitrophenylbutyrate in the presence of soman, an irreversible inhibitor of cholinesterases, suggested that reversible binding of soman preceding enzyme phophonylation induced a new enzyme conformational state (E′). The purpose of the present study was to determine whether this effect depends on soman itself or is dependent on the presence and nature of substrate or ligand. First, we examined the effect of amiloride, a reversible cholinesterase effector, upon the butyrylcholinesterase-catalyzed hydrolysis of nitrophenyl esters. The effect of amiloride was found to be dependent on the position ortho or para of the substrate nitro group: amiloride acts as a non-linear reversible activator of p-nitrophenyl ester hydrolysis and as a non-linear reversible inhibitor of o-nitrophenyl ester hydrolysis. Second, the effect of amiloride upon hydrolysis of o/ p-nitrophenylbutyrate was also studied under perturbing conditions, i.e., as a function of pressure (1–1600 bar) in the presence and absence of soman. Results show that the effect of reversible soman binding on butyrylcholinesterase activity in the presence of amiloride depends on the position of the substrate nitro group and amiloride concentration. Molecular modelling suggests that the presence of amiloride determines the orientation of ortho- and para-nitrophenyl esters in the active-site gorge. The nitro group of o-nitrophenylbutyrate interacts with the oxyanion hole via hydrogen bonds and its phenyl ring interacts with amiloride whose heterocycle faces Trp-82. The nitro group of p-nitrophenylbutyrate does not interact with the oxyanion hole but points towards Tyr-332; the phenyl ring of p-nitrophenylbutyrate interacts with amiloride but there is no steric constraint on the acyl chain. Thus, the network of interactions in ternary complexes is tighter with o-nitrophenylbutyrate as the substrate. There is no evidence for the existence of amiloride and/or soman-induced E′ state when p-nitrophenylbutyrate is the substrate. On the other hand, reversible binding of amiloride and/or soman induces new active conformational states that may be either binary (or ternary) enzyme-ligand complex or new free enzyme conformation resulting from long-lived ligand-induced enzyme conformational change when o-nitrophenylbutyrate is the substrate. These ligand-induced states are stabilized by high pressure.

Interligand interaction in ternary complexes of Zn(II) and Cd(II) with dipeptides and aminoacids

The stability constants of binary and ternary complexes [MA], [Ma2], and [MAL] (where M = Zn(II) or Cd(II); A = glycylglycine, glycyl L-alanine, glycyl L-leucine; L = α-alanine phenylalanine, tyrosine, tryptophan, or L-histidine) in aqueous medium have been determined potentometrically at 25 °C and an ionic strength of 0.2 M NaClO4 (0.2 mol dm−3) using a computer system. It is observed that Δ log K of MAL complexes has low negative or positive values. Probable reasons have been discussed.

T cell receptor antagonism mediated by interaction between T cell receptor junctional residues and peptide antigen analogues.

A major objective of new rational immunosuppressive therapies is to be able to inhibit deleterious T cell responses in an Ag-specific manner. Recently, a novel approach to inducing Ag-specific nonresponsiveness in T cells, termed receptor or TCR antagonism, has been described. Several analogues of the influenza hemagglutinin (H3) peptide HA307-319 were shown to block recognition of the native HA307-319 peptide by a DR1-restricted T cell clone at concentrations 1,000- to 10,000-fold less than required to competitively inhibit HA307-319 binding to DR1. These Ag analogues that inhibited T cell recognition differed from the stimulatory antigenic peptide only at single positions. How such limited changes affect the ternary complex interactions of TCR, peptide, and DR remains unclear. In the present study, we have utilized two different DR5-restricted T cell clones that exhibit mutually exclusive specificities for HA peptides with or without substitutions at position 313 to investigate the molecular requirements for receptor antagonism. In this reciprocal model system, we show that a single peptide/DR complex can antagonize one T cell clone while stimulating another. A comparison of nucleotide sequences derived from the TCR of the T cell clones that were either antagonized or stimulated by the peptide analogue/DR5 complex indicated that the different responses of these T cells result from minor differences in the amino acid residues in junctional regions that most likely interact directly with position 313 of the peptide analogues. Our results suggest that TCR antagonism is an Ag-specific phenomenon in which T cells are inhibited by interactions involving TCR residues required for the recognition of conventional Ag and the altered residues in peptide analogues.

Suppression of rpsL phenotypes by tuf mutations reveals a unique relationship between translation elongation and growth rate

We have found a simple relationship between bacterial growth rate and the translation elongation rate. Thus, for a set of defined ribosomal protein S12 mutations which reduce the efficiency of the ternary complex ribosome interaction (and restrict the frequency of translational errors) there is a linear relationship between growth rate and translation elongation rate. When these mutants are combined with defined EF-Tu mutants (which increase the probability of translational errors) both the elongation rate and growth rate reductions are reversed. The reductions and reversals are described by a unique linear relationship. We interpret this to mean that these two types of mutation exert opposing effects on the same molecular interaction. We suggest that this interaction is in the initial selection of the aminoacyl-tRNA on the ribosome. The slope of the relationship between translation elongation rate and growth rate, defined in per cent of the wild-type rates, is close to 1. Interestingly, the reversal of the elongation and growth phenotypes is incomplete, suggesting that the ribosomal mutants have an additional defect which is not compensated for by the ternary complex interaction. Our results show that the efficiency of the ternary complex ribosome interaction limits the translation elongation rate, which in turn correlates with changes in exponential growth rate.