Interaction of PLD1b with actin in antigen-stimulated mast cells.

Phosphatidic acid, the product of phospholipase D catalysed phosphatidylcholine hydrolysis is an important signalling molecule that has been implicated in regulation of actin cytoskeleton remodelling and secretion from mast cells. We show that human PLD1b (hPLD1b) is an actin-binding protein and the N-terminus is predominantly involved in this interaction. Protein kinase C (PKC) is a major upstream regulator of PLD activity and PKC phosphorylation sites have been identified within the N-terminus of PLD1b at serine 2 and threonine 147. Over-expression of wild type hPLD1b in mast cells showed that antigen stimulation significantly enhanced co-localisation of PLD1b with actin structures. Mutation of serine 2 to alanine abolished antigen-induced co-localisation whereas mutation of threonine 147 had less dramatic effects on co-localisation. The absence of co-localisation of PLD1b (S2A) with actin coincides with a significant decrease in PLD activity in cells expressing the PLD1b (S2A) mutant. In resting RBL-2H3 cells, mutation of serine 2 to aspartate resulted in constitutive co-localisation of PLD with the actin cytoskeleton, coincident with restored PLD activity. These results reveal that serine 2 is an important regulatory site involved in controlling PLD enzyme activity and the interaction between PLD and actin.

Anti-B4-blocked ricin immunotoxin shows therapeutic efficacy in four different SCID mouse tumor models.

Anti-CD19 monoclonal antibody anti-B4 (IgG1) conjugated to the novel toxin-blocked ricin forms a potent immunotoxin, anti-B4-blocked ricin, that kills greater than 4.5 logs of CD19-positive cells in vitro after a 24-h exposure to a conjugate concentration of 5 x 10(-9) M (1.11 micrograms/ml). The efficacy of anti-B4-blocked ricin in vivo was assessed in survival models of SCID mice bearing either a human B-cell lymphoma (Namalwa), a human non-T and non-B acute lymphoblastic leukemia (Nalm-6), or a murine B-cell lymphoma transfected with the human CD19 gene (300B4). In one model, 5 x 10(7) tumor cells were injected i.p., and 1 h later the mice were treated with i.v. bolus injections of anti-B4-blocked ricin at 100 micrograms/kg/day for 5 days. Controls included similar treatment with anti-B4 antibody (72 micrograms/kg/day or 2 mg/kg/day for 5 days) alone or with the isotype-matched nonspecific immunotoxin, N901-blocked ricin (100 micrograms/kg/day). In a second model, 4 x 10(6) tumor cells were injected i.v., and 7 days later mice were treated i.v. as above. Anti-B4-blocked ricin showed efficacy by killing in vivo up to 3 logs of tumor cells, which was manifested in significant prolongation of the life of the treated animals. Only very limited or no effects were observed in animals treated with either anti-B4 antibody alone or N901-blocked ricin control conjugate. The concentration of anti-B4-blocked ricin in the blood of animals was 150 ng/ml after the first i.v. injection and about 800 ng/ml following the fifth injection of conjugate. This increase may be due to damage to the reticuloendothelial system by anti-B4-blocked ricin, since the rate of clearance of carbon from blood also decreased 5-fold after five injections as compared to the rate after only one injection. These studies indicate that anti-B4-blocked ricin has the potential to increase survival times of hosts with malignant disease.

The binding of guanine nucleotide to N-ras p21--a phosphorous and proton magnetic resonance study.

One dimensional [1H] and [31P] nuclear magnetic resonance (NMR) studies have been carried out on purified wild type and mutant (Gly-12----Asp) N-ras protein expressed at high level in E. coli. Both proteins were isolated as stable 1:1 molar complexes with GDP with the upper limit for the first order rate constant for nucleotide dissociation 3 x 10(-4)s-1. From observation of the [31P] NMR spectrum after the addition of GTP it was concluded that the rate of nucleotide hydrolysis is appreciably greater than that of nucleotide exchange. Differences in the [31P] spectra of mutant and wild type proteins suggest that the mutation has a direct influence on the catalytic step. [1H] NMR spectra obtained for both mutant and wild type p21 were consistent with proteins of considerable stability and the addition of urea to concentrations of 4M appeared to cause little disruption in secondary structure. Additionally, the protein environment of the bound nucleotide remained well defined in the presence of a number of added reagents and over the pH range 5.8-9.5. The data are discussed in the light of the known crystal structure for H-ras p21 and indicate that the transforming mutation of aspartate for glycine-12 results in structural perturbations near the nucleotide binding site.

The binding of organic ions to phospholipid bilayers.

The binding of organic anions and cations, mainly tetraphenylboride and tetraphenylarsonium, to phospholipid membranes has been studied using an NMR method. Binding is appreciable and is affected by cholesterol in the membrane and counterions in solution. The passage of the organic anions through the membrane has also been followed. These measurements indicate that it is naive to use organic anions to measure membrane potentials in a simple manner.

Identification of a surface actin-binding site on myosin.

The surface accessibility of mobile domains of rabbit fast muscle myosin subfragment-1 isoenzymes (subfragment-1(A1), (A2)) influenced by interaction with actin has been investigated by proton magnetic resonance spectroscopy using the soluble paramagnetic reagents Cr(CN)6(3-), Fe(CN)6(3-), Mn2+ and the Gd3+ salt of 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid as probes. Anionic probes interact principally with lysine residues disposed close to other non-charged sidechains in both isoenzymes. Additional resonances in subfragment-1(A1) not present in subfragment-1(A2) are also observed to be affected, notably the sharp signal at 3.23 ppm which derives from a -N+ (CH3)3 group found in the N-terminal segment of the A1 light chain, showing that this domain of interaction with actin (Prince et al. (1981) Eur. J. Biochem. 121, 213-219) is situated at a surface location. Different probes identify a heterogeneity in the location and function of mobile sidechains. These results suggest a configurational lability in the various parts of the myosin head, differentially constrained upon interaction with actin and consistent with a structure composed of relatively rigid domains linked by more flexible regions.

Proton magnetic resonance studies on [methionine]-enkephalin and beta-endorphin in aqueous solution.

Proton magnetic resonance studies of [Met5]-enkephalin (lipotropin 61-65) in aqueous solution indicate a conformational preference for the pentapeptide backbone. The structural differences between [Met5]-enkephalin and other, more flexible peptides have been investigated using paramagnetic probe techniques. An outline structure for beta-endorphin (lipotropin 61-91) in aqueous solution is obtained from binding studies using Gd(III) as a relaxation probe.

Proton magnetic resonance studies on proteolytic fragments of troponin-C. Structural homology with the native molecule.

Comparison of proton magnetic resonance spectra of a tryptic and a thrombin fragment of troponin-C with that of the native protein has identified the domain of the molecule influenced by Ca2+ binding to the lower affinity regions I and II of troponin-C. The binding of Ca2+ to these sites results in a subtle alteration of the tertiary fold of the N-terminal half of troponin-C involving weakened contacts between several hydrophobic groups. The role and kinetics of the movements within the troponin-C molecule associated with binding at the regulatory sites are discussed.

Interaction of calmodulin with phospholamban and caldesmon: comparative studies by 1H-NMR spectroscopy.

In order to identify comparative aspects of the interaction of calmodulin with its target proteins, proton magnetic-resonance studies of complex formation between calmodulin and defined segments of phospholamban and caldesmon have been undertaken. Residues 3-15 in the cytoplasmic region of phospholamban, an integral membrane protein of cardiac sarcoplasmic reticulum believed to regulate the calcium pumping ATPase, are shown to contribute to interaction with calmodulin. Using wheat germ calmodulin specifically modified with a spin-label to provide the spectral means for spatial localisation, these residues of phospholamban were correlated with binding in the vicinity of the probe attached to Cys-27 in the N-terminal domain of calmodulin. This interaction, relevant to the mechanism of calmodulin-dependent phosphorylation of phospholamban that relieves its inhibitory influence on the calcium pump, provides a useful model system for comparative study of the properties of calmodulin-binding domains. We contrast here a calmodulin-binding segment in the C-terminal region of caldesmon localised by 1H-NMR study of the interface(s) between the two proteins. These observations are discussed in the context of other calmodulin-binding sequences.

Comparison of the calcium- and magnesium-induced structural changes of troponin--C. A proton magnetic resonance study.

Previous proton magnetic resonance studies of the effects of Ca(II) on the structure of rabbit skeletal muscle troponin--C have shown that Ca(II) binding to the two high affinity sites of troponin--C both directs and stabilizes the folding of much of the structure. Ca(II) binding by the two low affinity sites of troponin--C causes changes in the environment of largely hydrophobic residues. We have now examined the structural changes caused by Mg(II) and by Ca(II) in the presence of excess Mg(II). Successive addition of Mg(II) to metal-free troponin--C leads to broadly similar, but not identical, structural changes to those previously assigned to Ca(II) binding at the high affinity sites. None of the changes previously assigned to Ca(II) binding to the low affinity sites was observed. Since Mg(II) does not bind to the low affinity sites, these results confirm the previous assignments. The spectral differences between Mg(II) and Ca(II) show that the degree of backbone folding and interactions between a group of hydrophobic residues (one or more Val, Leu, Ile; two or more Phe) are different for the two cations. In the presence of excess Mg(II), at a molar ratio that may exist in vivo (approx. 40 : 1 mol ratio Mg(II): Ca(II), titration with Ca(II) leads to a displacement of Mg(II) and to all the structural changes previously observed for Ca(II) alone. However, in the presence of Mg(II) the distinction between high and low affinity sites is blurred as judged by the overlap of the spectral changes associated with each of the binding sites. This result, together with the observation that Mg(II) promotes structural changes different from Ca(II), suggests a structural basis for the observation that the Ca(II) threshold for the activation of tension in some myofibrils is increased in the presence of high Mg(II) concentrations.

The nature of the trifluoperazine binding sites on calmodulin and troponin-C.

We have employed 1H-nuclear magnetic resonance spectroscopy to study the interaction of the drug trifluoperazine with calmodulin and troponin-C. Distinct trifluoperazine-binding sites exist in the N- and C-terminal halves of both proteins. Each site consists of a group of hydrophobic side-chains brought into proximity by the Ca2+-dependent juxtaposition of two alpha-helical segments of the protein, each, in turn, belonging to a different Ca2+-binding site in the protein half. The trifluoperazine-induced inhibition of the biological activating ability of calmodulin appears to result from conformational restrictions conferred upon the protein by the bound drug.

Characterization of the actin-binding site on the alkali light chain of myosin.

1H-NMR experiments on myosin subfragment-1 (S1) isoenzymes, containing either the A1 or the A2 alkali light chains (S1(A1) or S1(A2)), have previously suggested the 41-residue proline, alanine and lysine-rich N-terminal extension of A1 to constitute a mobile 'domain' in solution. This segment of the molecule is immobilised in the presence of actin (Prince et al. (1981) Eur. J. Biochem. 121, 213-219). We now establish that the A1 light chain interacts with actin directly, and furthermore, that the binding site appears to be restricted to the terminal 41 residues. This observation carries important consequences for both the structure of the actomyosin complex and the role of myosin isoenzymes. Using the proteinase, thrombin, a technique has been developed in which the A1 light chain is cleaved, releasing the N-terminal 'tail' from an A2-like fragment. The method is shown to be widely applicable to light chains from a variety of sources. The isolated N-terminal fragments from rabbit skeletal and bovine cardiac muscle have been shown to interact directly with actin by a combination of affinity chromatography and 1H-NMR experiments. The 1H-NMR results are similar to those obtained earlier with S1 (ibid) and suggest the terminal alpha-N-trimethylalanine residue (Henry et al. (1982) FEBS Lett. 144, 11-15) to participate in the interaction.

Structure and function of X-Pro dipeptide repeats in the TonB proteins of Salmonella typhimurium and Escherichia coli.

The TonB protein is required for several outer membrane transport processes in bacteria. A short 33-residue peptide segment of TonB has been studied by 1H and 13C nuclear magnetic resonance spectroscopy. The sequence of this peptide segment contains multiple Glu-Pro and Lys-Pro dipeptide repeats that maintain rigid, elongated structures and flank a short connecting segment that adopts a beta-strand configuration. This TonB peptide is shown to interact specifically with the FhuA protein, the outer membrane receptor for ferrichrome-iron, providing the first direct evidence that the TonB protein interacts with outer membrane receptors. Interaction with the FhuA protein involves the extended structural element containing positively charged Lys-Pro repeats, and suggests a functional role for this segment of the TonB protein. As TonB is anchored in the cytoplasmic membrane the protein must, uniquely, span the periplasm. These data, together with studies described in the accompanying paper, suggest a model by which TonB serves to transduce conformational information over extended distances, from the cytoplasmic membrane to the outer membrane.

TonB protein of Salmonella typhimurium. A model for signal transduction between membranes.

The tonB gene product is required for several outer membrane transport processes in bacteria. The tonB gene from Salmonella typhimurium was sequenced and found to be similar to that of Escherichia coli. The TonB protein is highly proline-rich and includes an unusual segment consisting of multiple X-Pro dipeptide repeats. A synthetic peptide corresponding to this segment has been used to raise anti-TonB antibodies. TonB was shown to be associated with the cytoplasmic membrane, apparently anchored via a single hydrophobic N-terminal segment. Protease accessibility studies, and the use of a series of TonB-beta-lactamase fusions, showed that the rest of the TonB protein is periplasmic. Unusually, export of TonB is not accompanied by cleavage of the N-terminal signal peptide. In the accompanying paper, we show that TonB interacts directly with the outer membrane FhuA (TonA) receptor. Thus, TonB must span the periplasm, providing a link between the cytoplasmic membrane and receptors in the outer membrane. On the basis of these data, and those published by other laboratories, we propose a model whereby TonB serves as a "mechanical" linkage that, by transmitting protein conformational changes from the cytoplasmic membrane across the periplasm, acts as a means of coupling energy to outer membrane transport processes. Such a mechanism has general implications for signal transduction within and between proteins.

Multiple binding sites on the CH2 domain of IgG for mouse Fc gamma R11.

Important mammalian defensive functions such as phagocytosis are triggered in leukocytes by the interaction of the Fc region of IgG with cell surface receptors (Fc gamma R). The CH2 domain of IgG has been implicated previously as the site of interaction with human and mouse Fc gamma R. This domain was mapped for interaction with mouse Fc gamma R11 expressed by the macrophage-like cell line P388D1, using two panels of a total of 32 site-directed mutants of mouse IgG2b and chimeric human IgG3 monoclonal antibodies. Two potential binding sites have been identified: one in or within the vicinity of the lower hinge site on IgG for human Fc gamma R1, and one within the binding site on IgG for Clq. The three mutant IgGs (Gly 237----Ala, Asn 297----Ala, and Glu 318----Ala) which do not interact in complexed form also fail to bind as monomers. A 1H NMR study of the three non-binding monomeric mutants suggests that the mutations are largely site-specific, indicating that IgG interacts with mouse Fc gamma R11 at two regions within the CH2 domain. This interaction dictates phagocytosis mediated by Fc gamma R11 of the P388D1 cell line.

The interaction of actin with dystrophin.

Proton NMR spectroscopy of synthetic peptides corresponding to defined regions of human dystrophin has been employed to study the interaction with F-actin. No evidence of interaction with a C-terminal region corresponding to amino acid residues 3429-3440 was obtained. F-actin restricted the mobility of residues 19-27 in a synthetic peptide corresponding to residues 10-32. This suggests that this is a site of F-actin interaction in the intact dystrophin molecule. Identical sequences to that of residues 19-22 in dystrophin, namely Lys-Thr-Phe-Thr are also present in the N-terminal regions of the alpha-actinins implying this is also a site of F-actin interaction with alpha-actinin.

Binding sites involved in the interaction of actin with the N-terminal region of dystrophin.

Two actin-binding sites have been identified on human dystrophin by proton NMR spectroscopy of synthetic peptides corresponding to defined regions of the polypeptide sequence. These are Actin-Binding Site 1 (ABS1) located at residues 17-26 and Actin-Binding Site 2 (ABS2) in the region of residues 128-156. Using defined fragments of the actin amino acid sequence, ABS1 has been shown to bind to actin in the region represented by residues 83-117 and ABS2 to the C-terminal region represented by residues 350-375. These dystrophin-binding sites lie on the exposed domain in the actin filament.

Sequence-imposed structural constraints in the TonB protein of E. coli.

The solution conformation of a 33-residue peptide segment, derived from the TonB protein which is implicated in bacterial membrane transport processes, has been investigated using high-resolution proton magnetic resonance techniques. This proline-rich peptide possesses sequence-imposed sections of elongated secondary structure that must be retained in the native protein configuration. These structural constraints provide elements of stiffness that imply a purely structural role for TonB and are relevant to the subcellular location and biological role of the protein. On the basis of these data we suggest that this protein spans the periplasmic space, linking the inner and outer membrane components of TonB-dependent transport systems.

Sequential phosphorylation of adjacent serine residues on the N-terminal region of cardiac troponin-I: structure-activity implications of ordered phosphorylation.

We have used NMR spectroscopy to monitor the phosphorylation of a peptide corresponding to the N-terminal region of human cardiac troponin-I (residues 17-30), encompassing the two adjacent serine residues of the dual phosphorylation site. An ordered incorporation of phosphate catalysed by PKA was observed, with phosphorylation of Ser-24 preceding that of Ser-23. Diphosphorylation induced a conformational transition in this region, involving the specific association of the Arg-22 and Ser-24P side-chains, and maximally stabilised when both phosphoserines were in the di-anionic form. The results suggest that the second phosphorylation at Ser-23 of cardiac troponin-I is of particular significance in the mechanism by which adrenaline regulates the calcium sensitivity of the myofibrillar actomyosin Mg-ATPase.

Characterisation of the effects of mutation of the caldesmon sequence 691glu-trp-leu-thr-lys-thr696 to pro-gly-his-tyr-asn-asn on caldesmon-calmodulin interaction.

We have investigated the functional properties of a mutant (Cg1) derived from the C-terminal 99 amino acids of chicken caldesmon, 658-756 (658C) where the sequence 691glu-trp-leu-thr-lys-thr696 is changed to pro-gly-his-tyr-asn-asn. Cg1 bound Ca2+-calmodulin with (1/7)th of the affinity as compared to 658C or whole caldesmon. NMR titrations indicate that the contacts of Ca2+-calmodulin with the Trp-722 region of the peptide are retained but that those at the mutated site are lost. Most importantly Ca2+-calmodulin is not able to reverse the Cg1-induced inhibition. We conclude that the interaction of calmodulin with this caldesmon sequence is crucial for the reversal of caldesmon inhibition of actin-tropomyosin activation of myosin ATPase. The results are interpreted in terms of multisite attachment of actin and Ca2+-calmodulin to overlapping sequences in caldesmon domain 4b.

Calcium and magnesium binding by parvalbumin. A proton magnetic resonance spectral study.

Proton magnetic resonance spectroscopy has been used to monitor the kinetics and nature of the conformational transitions induced by binding of calcium and magnesium to carp parvalbumin. Rate constants have been determined for the exchange between the cation dependent conformational states of the protein in solution. These data enable a description of the kinetics and mechanism controlling the calcium flux in vivo during contraction.