Mast cell binding of neurotensin. II. Molecular conformation of neurotensin involved in the stereospecific binding to mast cell receptor sites.

Systematic substitution of the natural L-amino acids in neurotensin by their D isomers reveals that the COOH-terminal portion of this tridecapeptide is required for binding to mast cell receptors: D-amino acid replacements from Pro10 through Leu13 substantially decrease that binding. Either blockage of the COOH-terminal carboxyl group as with N-methylamidation, or formation of a cyclic structure by the inclusion of a disulfide bond, a Cys2,13 substitution, markedly reduces the specific binding to mast cell receptor sites. Modifications in the NH2-terminal portion of neurotensin do not affect the binding to mast cells. However, D-Arg8 and D-Arg9 substitutions increase binding by factors of 5- to 6-fold. The hydroxyl group at position 3 or 11 is not essential for binding since Phe3 or Phe11 is equivalent to Tyr3 or Tyr11. The COOH-terminal penta- and hexapeptides are able to displace approximately 70% 125I-neurotensin relative to the intact peptide. Of 18 other biologically active peptides tested, only xenopsin, a naturally occurring COOH-terminal analog of neurotensin, and bradykinin effectively compete in the binding assay to an extent of 60 and 100%, respectively. Histamine, diphenhydramine, and noradrenaline are ineffective in this regard.     

Rapid degradation of neurotensin by stimulated rat mast cells

A RIA towards neurotensin (NT) using C-terminal- and N-terminal-specific antisera was used to study degradation of this tridecapeptide by isolated rat mast cells. Incubation of NT (10 μM) with peritoneal or pleural mast cells resulted in a rapid loss of NT immunoreactivity (iNT), as measured by C-terminal-directed antiserum, with little effect on N-terminal iNT. The rate of the reaction was faster with pleural cells (T_1/2, 30 s) than with peritoneal cells (T_1/2, 180 s) and was > 10-fold slower in the presence of metabolic poisons. The enzyme(s) involved is most likely released from the cells during secretion, as NT was degraded by media conditioned by compound 48/80-stimulated mast cells 40–60 times faster than by media from unstimulated cells. This degradation by conditioned media was concentration dependent, pH dependent, and temperature sensitive. HPLC analyses indicated a near stoichiometric conversion of NT to NT(1–12) (66%) and NT(1–11) (34%) after incubation for 10–30 s with conditioned media. By 30 min only NT(1–11) and NT(1–10) were present. Phenanthroline (1 mM), an inhibitor of carboxypeptidase, prevented the loss of C-terminal iNT and the generation of NT(1–12) and NT(1–11). While NT(1–12) was effective in releasing histamine from mast cells in vitro and increasing vascular permeability in vivo, NT(1–11) was not. These results suggest that carboxypeptidase-like enzyme(s) could modulate the level and form of NT-related peptides in various states involving activation of mast cells.     

Mast cell binding of neurotensin. I. Iodination of neurotensin and characterization of the interaction of neurotensin with mast cell receptor sites.

Neurotensin was iodinated at equimolar concentrations of peptide, iodide, and chloramine-T, producing a labeled peptide with a specific activity of 1000 to 2000 Ci/mmol. Rat mast cells specifically and reversibly bound 1.27 pmol of neurotensin/10(6) cells with a reversible affinity, KD, of 154 nM. Optimum specific binding occurred betwen pH 6.8 and 7.2 under hypotonic conditions and dropped sharply as buffer concentration increased beyond 10 mM. The divalent cations Ca2+ and Mg2+ prevented binding with 50% inhibition at 1.5 and 4 mM, respectively. Binding was strongly and equally inhibited by the sodium and potassium salts of chloride, bromide, and iodide, and to a lesser degree by LiCl. Maximum binding of 125I-neurotensin occurred within 10 min at 0 degrees, and within 1.5 to 2 min binding was reduced to half-maximum in the presence of excess unlabeled neurotensin or upon 20-fold dilution in buffer. Both CaCl2 and NaCl were able to dissociate 60% of the total bound neurotensin: half the label bound was removed in 4 to 6 min. EDTA inhibited the binding only at high concentrations and no requirement was found for sulfhydryl groups, ATP, or a glycoprotein in the binding of neurotensin.     

A theoretical study of the sequence specificity in binding of lexitropsins to B-DNA

A theoretical study is presented on the binding to B-DNA of a series of lexitropsins, these ligands being netropsin derivatives in which one or both of the pyrrole rings have been replaced by imidazoles. The best complexes have been located by energy minimisation taking into account nucleic acid flexibility, ligand flexibility, explicit, mobile counterions and solvent dielectric effects. Calculations have been performed for two homopolymeric DNA receptor sequences, AT base sequence, which only decreases in the imidazole derivatives. These results emphasize the decisive role of the molecular electrostatic potential of the nucleic acid in determining the sequence selectivity of these ligands, as opposed to the postulated role of adenine C2 - pyrrole beta hydrogen contacts.     

Using RNase sequence specificity to refine the identification of RNA-protein binding regions

Massively parallel pyrosequencing is a high-throughput technology that can sequence hundreds of thousands of DNA/RNA fragments in a single experiment. Combining it with immunoprecipitation-based biochemical assays, such as cross-linking immunoprecipitation (CLIP), provides a genome-wide method to detect the sites at which proteins bind DNA or RNA. In a CLIP-pyrosequencing experiment, the resolutions of the detected protein binding regions are partially determined by the length of the detected RNA fragments (CLIP amplicons) after trimming by RNase digestion. The lengths of these fragments usually range from 50-70 nucleotides. Many genomic regions are marked by multiple RNA fragments. In this paper, we report an empirical approach to refine the localization of protein binding regions by using the distribution pattern of the detected RNA fragments and the sequence specificity of RNase digestion. We present two regions to which multiple amplicons map as examples to demonstrate this approach.     

Identification of residues involved in neurotensin binding and modeling of the agonist binding site in neurotensin receptor 1

The neurotensin receptor 1 (NTR1) subtype belongs to the family of G protein-coupled receptors and mediates most of the known effects of the neuropeptide including modulation of central dopaminergic transmission. This suggested that nonpeptide agonist mimetics acting at the NTR1 might be helpful in the treatment of Parkinson's disease and schizophrenia. Here, we attempted to define the molecular interactions between neurotensin-(8-13), the pharmacophore of neurotensin, and the rat NTR1. Mutagenesis of the NTR1 identified residues that interact with neurotensin. Structure-activity studies with neurotensin-(8-13) analogs identified the peptide residues that interact with the mutated amino acids in the receptor. By taking these data into account, computer-assisted modeling techniques were used to build a tridimensional model of the neurotensin-(8-13)-binding site in which the N-terminal tetrapeptide of neurotensin-(8-13) fits in the third extracellular loop and the C-terminal dipeptide binds to residues at the junction between the extracellular and transmembrane domains of the receptor. Interestingly, the agonist binding site lies on top of the previously described NTR1-binding site for the nonpeptide neurotensin antagonist SR 48692. Our data provide a basis for understanding at the molecular level the agonist and antagonist binding modes and may help design nonpeptide agonist mimetics of the NTR1.     

Sequence specificity of the human autonomously replicating sequence binding protein

We have previously found that the protein which bound to the essential region of human autonomously replicating sequences (ARS) could be the c-myc proto-oncogene product. Here we examined the binding specificity of human ARS binding protein purified from Burkitt's lymphoma Raji cells. Among several oligodeoxynucleotides, this protein bound to the fragments containing the sequence of 5'-CAPyCTCTNA-3'. Competition analysis revealed that the ARS binding protein could recognize not only the nucleotide sequences but also the high ordered structure.     

Neurotensin increases mortality and mast cells reduce neurotensin levels in a mouse model of sepsis

Sepsis is a complex, incompletely understood and often fatal disorder, typically accompanied by hypotension, that is considered to represent a dysregulated host response to infection. Neurotensin (NT) is a 13-amino-acid peptide that, among its multiple effects, induces hypotension. We find that intraperitoneal and plasma concentrations of NT are increased in mice after severe cecal ligation and puncture (CLP), a model of sepsis, and that mice treated with a pharmacological antagonist of NT, or NT-deficient mice, show reduced mortality during severe CLP. In mice, mast cells can degrade NT and reduce NT-induced hypotension and CLP-associated mortality, and optimal expression of these effects requires mast cell expression of neurotensin receptor 1 and neurolysin. These findings show that NT contributes to sepsis-related mortality in mice during severe CLP and that mast cells can lower NT concentrations, and suggest that mast cell-dependent reduction in NT levels contributes to the ability of mast cells to enhance survival after CLP.     

Drug binding in human P-glycoprotein causes conformational changes in both nucleotide-binding domains

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) uses ATP to transport many structurally diverse compounds out of the cell. It is an ABC transporter with two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Recently, we showed that the "LSGGQ" motif in one NBD ((531)LSGGQ(535) in NBD1; (1176)LSGGQ(1180) in NBD2) is adjacent to the "Walker A" sequence ((1070)GSSGCGKS(1077) in NBD2; (427)GNSGCGKS(434) in NBD1) in the other NBD (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2002) J. Biol. Chem. 277, 41303-41306). Drug substrates can stimulate or inhibit the ATPase activity of P-gp. Here, we report the effect of drug binding on cross-linking between the LSGGQ signature and Walker A sites (Cys(431)(NBD1)/C1176C(NBD2) and Cys(1074)(NBD2)/L531C(NBD1), respectively). Seven drug substrates (calcein-AM, demecolcine, cis(Z)-flupentixol, verapamil, cyclosporin A, Hoechst 33342, and trans(E)-flupentixol) were tested for their effect on oxidative cross-linking. Substrates that stimulated the ATPase activity of P-gp (calcein-AM, demecolcine, cis(Z)-flupentixol, and verapamil) increased the rate of cross-linking between Cys(431)(NBD1-Walker A)/C1176C(NBD2-LSGGQ) and between Cys(1074)(NBD2-Walker A)/L531C(NBD1-LSGGQ) when compared with cross-linking in the absence of drug substrate. By contrast, substrates that inhibited ATPase activity (cyclosporin A, Hoechst 33342, and trans(E)-flupentixol) decreased the rate of cross-linking. These results indicate that interaction between the LSGGQ motifs and Walker A sites must be essential for coupling drug binding to ATP hydrolysis. Drug binding in the transmembrane domains can induce long range conformational changes in the NBDs, such that compounds that stimulate or inhibit ATPase activity must decrease and increase, respectively, the distance between the Walker A and LSGGQ sequences.     

Role of sequence evolution and conformational dynamics in the substrate specificity and oligomerization mode of thymidylate kinases

Thymidylate kinase (TMK) is a key enzyme for the synthesis of DNA, making it an important target for the development of anticancer, antibacterial, and antiparasitic drugs. TMK homologs exhibit significant variations in sequence, residue conformation, substrate specificity, and oligomerization mode. However, the influence of sequence evolution and conformational dynamics on its quaternary structure and function has not been studied before. Based on extensive sequence and structure analyses, our study detected several non-conserved residues which are linked by co-evolution and are implicated in the observed variations in flexibility, oligomeric assembly, and substrate specificity among the homologs. These lead to differences in the pattern of interactions at the active site in TMKs of different specificity. The method was further tested on TMK from Sulfolobus tokodaii (StTMK) which has substantial differences in sequence and structure compared to other TMKs. Our analyses pointed to a more flexible dTMP-binding site in StTMK compared to the other homologs. Binding assays proved that the protein can accommodate both purine and pyrimidine nucleotides at the dTMP binding site with comparable affinity. Additionally, the residues responsible for the narrow specificity of Brugia malayi TMK, whose three-dimensional structure is unavailable, were detected. Our study provides a residue-level understanding of the differences observed among TMK homologs in previous experiments. It also illustrates the correlation among sequence evolution, conformational dynamics, oligomerization mode, and substrate recognition in TMKs and detects co-evolving residues that affect binding, which should be taken into account while designing novel inhibitors.     

Heavy and light chain variable single domains of an anti-DNA binding antibody hydrolyze both double- and single-stranded DNAs without sequence specificity

Anti-DNA antibodies (Abs) are of biomedical interest because they are associated with autoimmune diseases in human and mice. Previously we isolated an anti-DNA monoclonal Ab 3D8 from an autoimmune-prone MRL-lpr/lpr mouse. Here we have characterized DNA binding kinetics and hydrolyzing activities of the recombinant single chain variable fragment (scFv) and the single variable domains of heavy chain (VH) and light chain (VL) using various single-stranded (ss) and double-stranded (ds) DNA substrates. All the Abs bound to both ds- and ssDNAs without significant preferential sequence specificity showing scFv higher affinities (KD = approximately 17-74 nm) than VH (KD = approximately 2.4-8.4 microm) and VL (KD = approximately 3.2-72 microm), and efficiently hydrolyzed both ds- and ssDNAs without sequence specificity in a Mg2+-dependent manner, except for the poor activity of 3D8 scFv for ss-(dT)40. Elucidated crystal structure-based His to Ala mutations on the complementarity determining regions of VH (His-H35 --> Ala) and/or VL (His-L94 --> Ala) of 3D8 scFv significantly inhibited the catalytic activities, indicating that the His residues are involved in the catalytic mechanism of 3D8 scFv. However, the DNA hydrolyzing activities of single domain VH and VL were not affected by the mutations, indicative of their different catalytic mechanisms from that of 3D8 scFv. Our results demonstrate single domain Abs with DNase activities for the first time, which might provide new insights into substrate recognition and catalytic mechanisms of anti-DNA Abs.     

Functional expression in mast cells of chimeric receptors with antibody specificity

Cell Biochemistry and Biophysics - Chimeric genes composed of a single-chain Fv domain (scFv) of an antibody linked with receptor chains normally present in cells of hematopoietic origin were...     

Influence of stereochemistry of the sequence Arg-Gly-Asp-Xaa on binding specificity in cell adhesion

Peptides containing the tripeptide sequence Arg-Gly-Asp can duplicate or inhibit the cell attachment-promoting effects of fibronectin and vitronectin. Peptides analogous to a prototype peptide, Gly-Arg-Gly-Asp-Ser-Pro-Cys, the sequence of which was taken from the cell attachment site of fibronectin, were assayed for their relative abilities to inhibit the attachment of cells to a fibronectin or vitronectin substrate. A peptide having the L-Arg residue replaced with D-Arg showed no difference in this capacity, whereas substituting Gly with D-Ala or L-Asp with D-Asp resulted in completely inactive peptides. Replacement of L-Ser with D-Ser drastically reduced the influence that the resulting peptide had on the vitronectin interaction, but this peptide showed little difference in its effect on the binding of cells to fibronectin when compared with the prototype peptide. Furthermore, substitution of the Ser with L-Asn resulted in a peptide that had an apparent increased preference for the fibronectin receptor and decreased preference for the vitronectin receptor. Conversely, threonine in this position gave a peptide with increased preference for the vitronectin receptor, whereas L-Pro in this position gave a completely inactive peptide. Finally, by cyclicizing the prototype peptide to restrict its conformational flexibility, a peptide was obtained that was a much improved inhibitor of attachment of cells to vitronectin and yet nearly inactive with respect to the interactions of cells with fibronectin substrates. These studies lend support to the hypothesis that different Arg-Gly-Asp-directed adhesion receptors can recognize differences in the conformation and environment of the Arg-Gly-Asp tripeptide, and they establish the feasibility of obtaining synthetic probes that are more selective for individual receptors than are the peptides modeled after the natural sequences of adhesive extracellular matrix molecules.     

A role for both conformational selection and induced fit in ligand binding by the LAO protein

Molecular recognition is determined by the structure and dynamics of both a protein and its ligand, but it is difficult to directly assess the role of each of these players. In this study, we use Markov State Models (MSMs) built from atomistic simulations to elucidate the mechanism by which the Lysine-, Arginine-, Ornithine-binding (LAO) protein binds to its ligand. We show that our model can predict the bound state, binding free energy, and association rate with reasonable accuracy and then use the model to dissect the binding mechanism. In the past, this binding event has often been assumed to occur via an induced fit mechanism because the protein's binding site is completely closed in the bound state, making it impossible for the ligand to enter the binding site after the protein has adopted the closed conformation. More complex mechanisms have also been hypothesized, but these have remained controversial. Here, we are able to directly observe roles for both the conformational selection and induced fit mechanisms in LAO binding. First, the LAO protein tends to form a partially closed encounter complex via conformational selection (that is, the apo protein can sample this state), though the induced fit mechanism can also play a role here. Then, interactions with the ligand can induce a transition to the bound state. Based on these results, we propose that MSMs built from atomistic simulations may be a powerful way of dissecting ligand-binding mechanisms and may eventually facilitate a deeper understanding of allostery as well as the prediction of new protein-ligand interactions, an important step in drug discovery.     

Theoretical study of the sequence specificity in the covalent binding of the antitumor drug CC-1065 to DNA

A theoretical modelling is presented of the covalent adducts of the antitumor agent CC-1065 with B-DNA. The optimal complexes are obtained by energy minimisation, taking into account full structure flexibility, including the flexible rings of the ligand and DNA. The binding preference of CC-1065 with respect to base sequence is studied. The results obtained elucidate the origin of the preference for two AT base pairs on the 5'side of the modified adenine. The modifications of the DNA structure upon ligand covalent binding are discussed.     

Assessing the sequence specificity in the binding of Co(III) to DNA via a thermodynamic approach

The interaction specificities of Co(III) with DNA were investigated via consideration of thermodynamic characteristics of the duplex to single strand transition for DNA oligomers incubated in the presence of [Co(NH₃)₅(OH₂)] (ClO₄)₃. It has previously been demonstrated that incubation of the DNA oligomer [(5medC-dG)₄]₂ with this cobalt complex leads to coordination of the cobalt center to the DNA, presumably at N7 of guanine bases [D. C. Calderone, E. J. Mantilla, M. Hicks, D. H. Huchital, W. R. Murphy, Jr. and R. D. Sheardy, (1995) Biochemistry 34, 13841]. In this report, DNA oligomers of different sequence were incubated with [Co(NH₃)₅(OH₂)] (ClO₄)₃ via protocols developed previously and the treated oligomers were subjected to thermal denaturation for comparison to the untreated oligomers. The DNA oligomers were designed in order to investigate the sequence specificity, if any, in the reaction of the cobalt complex with DNA. The values of T_m, ΔH_uH, and Δn (the differential ion binding term) obtained from the thermal denaturations were used to assess the sequence specificity of the interaction. For all oligomers, treated or untreated, T_m and Δ_uH vary linearly with log [Na⁺] and hence the value of Δn is a function of the Na⁺ concentration. The results indicate no significant reaction between the cobalt complex and oligomers possessing isolated -GA- or -CG- sites; however, the thermodynamic characteristics of DNA oligomers possessing either an isolated -GG- site or an isolated -GC- site were altered by the treatment. Atomic absorption studies of the treated oligomers demonstrate that only the DNA oligomers possessing isolated -GG- or -GC- sites bind cobalt. Hence, the changes in the thermodynamic properties of these oligomers are a result of cobalt binding with a remarkable sequence specificity. © 1997 John Wiley & Sons, Inc. Biopoly 42: 549–599, 1997     

Dimerization of protein tyrosine phosphatase sigma governs both ligand binding and isoform specificity

Signaling through receptor protein tyrosine phosphatases (RPTPs) can influence diverse processes, including axon development, lymphocyte activation, and cell motility. The molecular regulation of these enzymes, however, is still poorly understood. In particular, it is not known if, or how, the dimerization state of RPTPs is related to the binding of extracellular ligands. Protein tyrosine phosphatase sigma (PTPsigma) is an RPTP with major isoforms that differ in their complements of fibronectin type III domains and in their ligand-binding specificities. In this study, we show that PTPsigma forms homodimers in the cell, interacting at least in part through the transmembrane region. Using this knowledge, we provide the first evidence that PTPsigma ectodomains must be presented as dimers in order to bind heterophilic ligands. We also provide evidence of how alternative use of fibronectin type III domain complements in two major isoforms of PTPsigma can alter the ligand binding specificities of PTPsigma ectodomains. The data suggest that the alternative domains function largely to change the rotational conformations of the amino-terminal ligand binding sites of the ectodomain dimers, thus imparting novel ligand binding properties. These findings have important implications for our understanding of how heterophilic ligands interact with, and potentially regulate, RPTPs.     

Properties of the naturally occurring soluble surface glycoprotein of ecotropic murine leukemia virus: binding specificity and possible conformational change after binding to receptor

Ecotropic murine leukemia virus (MuLV) infection is initiated by the interaction between the surface glycoprotein (SU) of the virus and its cell-surface receptor mCAT-1. We investigated the SU-receptor interaction by using a naturally occurring soluble SU which was encoded by the envelope (env) gene of a defective endogenous MuLV, Fv-4(r). Binding of the SU to mCAT-1-positive mouse cells was completed by 1 min at 37 degrees C. The SU could not bind to mouse cells that were persistently infected by ecotropic MuLVs (but not amphotropic or dualtropic MuLVs) or transfected with wild-type ecotropic env genes or a mutant env gene which can express only precursor Env protein that is restricted to retention in the endoplasmic reticulum. These cells were also resistant to superinfection by ecotropic MuLVs. Thus, superinfection resistance correlated with the lack of SU-binding capacity. After binding to the cells, the SU appeared to undergo some conformational changes within 1 min in a temperature-dependent manner. This was suggested by the different properties of two monoclonal antibodies (MAbs) reactive with the same C-terminal half of the Fv-4(r) SU domain, including a proline-rich motif which was shown to be important for conformation of the SU and interaction between the SU and the transmembrane protein. One MAb reacting with the soluble SU bound to cells was dissociated by a temperature shift from 4 to 37 degrees C. Such dissociation was not observed in cells synthesizing the SU or when another MAb was used, indicating that the dissociation was not due to a temperature-dependent release of the MAb but to possible conformational changes in the SU.     

A short sequence responsible for both phosphoinositide binding and actin binding activities of cofilin.

Cofilin is a widely distributed actin-modulating protein that has abilities to bind along the side of F-actin and to depolymerize F-actin. Both abilities of cofilin can be inhibited by phosphoinositides such as phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate (PIP2). We have previously shown that the synthetic dodecapeptide corresponding to Trp104-Met115 of cofilin is a potent inhibitor of actin polymerization (Yonezawa, N., Nishida, E., Iida, K., Kumagai, H., Yahara, I., and Sakai, H. (1991) J. Biol. Chem. 266, 10485-10489). In this study, we have found that the inhibitory effect of the synthetic dodecapeptide on actin polymerization is canceled specifically by phosphatidylinositol, phosphatidylinositol 4-monophosphate and PIP2. We further show that the dodecapeptide as well as cofilin binds to PIP2 molecules and inhibits PIP2 hydrolysis by phospholipase C. Thus, the actin-binding dodecapeptide sequence of cofilin may constitute a multifunctional domain in cofilin.