Donald J. Winzor

Structural Basis for Assembly and Function of a Heterodimeric Plant Immune Receptor

Universal Immune Function Certain pathogen effectors are detected in plants by cytoplasmic receptors. First solving the crystal structures of Arabidopsis receptors, Williams et al. (p. 299; see the Perspective by Nishimura and Dangl) discovered that in the resting state, the structures form a heterodimer that readies the complex for effector binding and keeps the signaling domains from firing too early. Once the pathogen effector binds, the structure of the complex shifts such that the signaling domains can form a homodimer to initiate downstream signaling. Similarities between these plant-pathogen receptors and Toll-like receptors in animals suggest the molecular mechanisms may translate broadly. A heterodimer stands at the ready; a homodimer responds with action. [Also see Perspective by Nishimura and Dangl] Cytoplasmic plant immune receptors recognize specific pathogen effector proteins and initiate effector-triggered immunity. In Arabidopsis, the immune receptors RPS4 and RRS1 are both required to activate defense to three different pathogens. We show that RPS4 and RRS1 physically associate. Crystal structures of the N-terminal Toll–interleukin-1 receptor/resistance (TIR) domains of RPS4 and RRS1, individually and as a heterodimeric complex (respectively at 2.05, 1.75, and 2.65 angstrom resolution), reveal a conserved TIR/TIR interaction interface. We show that TIR domain heterodimerization is required to form a functional RRS1/RPS4 effector recognition complex. The RPS4 TIR domain activates effector-independent defense, which is inhibited by the RRS1 TIR domain through the heterodimerization interface. Thus, RPS4 and RRS1 function as a receptor complex in which the two components play distinct roles in recognition and signaling.

Evaluation of equilibrium constants for antigen-antibody interactions by solid-phase immunoassay: the binding of paraquat to its elicited mouse monoclonal antibody

A procedure is devised for simple graphical evaluation of the association constant for antibody-antigen interactions from data obtained by conventional solid-phase immunoassay for antigen. Application of the method to situations involving a univalent antigen is illustrated by means of ELISA data for the interactions of paraquat with its elicited murine monoclonal antibody, and the Fab fragment derived therefrom. Although the completely general situation with both antibody and antigen multivalent is not amenable to study by the present procedure, the quantitative expression is readily modified to accommodate antigen multivalency provided that the univalent Fab fragment may be substituted for the multivalent antibody (IgG or IgM) as partitioning species in the solid-phase immunoassay.

Rationalization of the effects of compatible solutes on protein stability in terms of thermodynamic nonideality

Inhibition by compatible solutes such as proline and glycine betaine of the rate of coagulation, at 60 degrees C, of bovine serum albumin in 0.1 M acetate buffer, pH 5, is used as a model system to substantiate the concept that the production of high concentrations of osmolytes by plants and other organisms in response to stress (e.g., drought) results in stabilization of native enzyme structures via nonspecific excluded volume effects. The paradoxical situation whereby this effect of compatible solutes counters to some extent the protein-precipitating effect of poly(ethylene glycol) is also seemingly resolved.

Textilinins from Pseudonaja textilis textilis. Characterization of two plasmin inhibitors that reduce bleeding in an animal model.

The incidence of vein-graft occlusion associated with myocardial infarction and thrombosis following the use of the plasmin inhibitor, aprotinin, to reduce blood loss during vascular surgery has prompted the isolation of an alternative kinetically distinct inhibitor of plasmin from the venom ofPseudonaja textilis. This inhibitor has been called textilinin (Txln) and two distinct forms have been isolated from the Brown-snake venom (molecular weight, 6688 and 6692). A comparison of plasmin inhibitor constants for aprotinin and the Txlns 1 and 2 indicated that the former bound very tightly (inhibitor constant,Ki ≈ 10−11 mol/l), while both of the latter bound less tightly (Ki ≈ 10−9 mol/l). Homogeneity of Txlns 1 and 2 was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry. A sequence difference of six amino acids was observed between the two forms of Txln. Txln 1 and 2 showed, respectively, 45 and 43% homology with aprotinin, while there was 58 and 55% homology, respectively, with a plasmin inhibitor from the venom of eastern Taipan,Oxyuranus scutellatus. Both Txlns have six cysteines, like other inhibitors of this group, and homology was determined by alignment of these cysteines. Both have been shown to reduce blood loss by about 60% in a murine tail vein bleeding model. It is proposed that the kinetic profiles of Txln 1 and 2 for plasmin allow the arrest of haemorrhage without the possible threat of thrombosis.

Direct analysis of solute self-association by sedimentation equilibrium

An improved procedure is described for the characterization of solute self-association by sedimentation equilibrium, Whereas previous statistical-mechanical approaches to allowance for the effects of thermodynamic nonideality have entailed tedious iteration because of their specification of activity coefficients in terms of the equilibrium concentrations of all species, such reliance upon knowledge of the solution composition is avoided by the adaptation of an alternative statistical-mechanical formulation [T. L. Hill and Y. D. Chen (1973) Biopolymers, Vol. 12, pp. 1285-1312 ] in which thermodynamic nonideality is expressed in terms of total solute concentration. The development of an analysis in terms of a relationship with total solute concentration as the experimental variable allows this attribute of the Adams-Fujita approach to be retained without sacrifice of statistical-mechanical rigor. Its use is illustrated by application to Rayleigh interferometric records of sedimentation equilibrium distributions reflecting α-chymotrypsin dimerization and lysozyme self-association.

THE INTERACTION OF CIMETIDINE WITH RAT LIVER MICROSOMES

The binding of cimetidine to rat liver microsomes in M/15 phosphate buffer, pH 7.9, has been investigated by difference spectroscopy and also by equilibrium partition studies, the latter method providing the more definitive characterization of the interaction in the pharmacologically relevant, low micromolar range of drug concn. In addition, the effect of cimetidine on the rate of dilution-induced displacement of [3H]cimetidine from rat liver microsomes has been used to justify consideration of the binding results in terms of two distinct and independent classes of microsomal site, governed by dissociation constants of 8.3 and 104 microM under the above conditions. By demonstrating unequivocally the existence of the stronger interaction, this investigation has provided an acceptable experimental basis for considering the undesired side effect of cimetidine in concomitant use with a number of other drugs to be the consequence of its inhibition of their monooxygenase-dependent metabolism.

Analytical exclusion chromatography

This review summarizes the development of exclusion chromatography, also termed gel filtration, molecular-sieve chromatography and gel permeation chromatography, for the quantitative characterization of solutes and solute interactions. As well as affording a means of determining molecular mass and molecular mass distribution, the technique offers a convenient way of characterizing solute self-association and solute-ligand interactions in terms of reaction stoichiometry and equilibrium constant. The availability of molecular-sieve media with different selective porosities ensures that very little restriction is imposed on the size of solute amenable to study. Furthermore, access to a diverse array of assay procedures for monitoring the column eluate endows analytical exclusion chromatography with far greater flexibility than other techniques from the viewpoint of solute concentration range that can be examined. In addition to its widely recognized prowess as a means of solute separation and purification, exclusion chromatography thus also possesses considerable potential for investigating the functional roles of the purified solutes.

Synthesis and evaluation of affinity adsorbents for glycoproteins: an artificial lectin.

A combination of rational design based on mimicking natural protein-carbohydrate interactions and solid-phase combinatorial chemistry has led to the identification of an affinity ligand which displays selectivity for the mannose moiety of glycoproteins. The ligand, denoted 18/18 and comprising a triazine scaffold bis-substituted with 5-aminoindan, has been synthesised in solution, characterised by TLC, 1H-NMR and MS. When immobilised to amine-derivatised agarose at concentrations >24 micromol/g moist weight gel, ligand 18/18 selectively binds glucose oxidase. The adsorbed enzyme was quantitatively eluted with 0.5 M alpha-D-methyl-mannoside and to a lesser extent with the equivalent glucoside. An investigation of the comparative retention times of saccharidic solutes showed that significant retardation was observed for alpha-D-mannose, mannobiose and mannan, with little or no evidence for selective retention of other saccharides, with the exception of alpha-L-fucose. Interestingly, alpha-L-fucose and alpha-D-mannose share an identical configuration of the hydroxyl groups on C-2, C-3 and C-4. Analysis of Scatchard plots from partition equilibrium studies on the interaction of glucose oxidase and the p-nitrophenyl-glycosides of D-mannose, D-glucose, L-fucose and D-galactose with immobilised 18/18 establish that the affinity constants (K(AX)) for the enzyme, the glycosides of mannose, glucose and fucose, and the p-nitrophenyl-galactoside are 4.3x10(5) M(-1), 1.9X10(4) M(-1) and 1.2x10(4) M(-1) respectively. 1H-NMR studies on the interaction of alpha-D-methyl-mannoside with ligand 18/18 in solution confirm the involvement of the hydroxyl group in the C-2 position. Molecular modelling suggests the formation of four hydrogen bonds between the hydroxyl groups at positions C-2, C-3 and C-4 of alpha-D-methyl-mannoside and the bridging and ring nitrogen atoms of the triazine scaffold, with aromatic stacking of a second ligand against the carbohydrate face. The greater specificity of ligand 18/18 for mannose and glucose than for galactose parallels that exhibited by concanavalin A.

Effect of sucrose on the dimerization of α-chymotrypsin allowance for thermodynamic nonideality arising from the presence of a small inert solute

The space-filling effects of sucrose on the dimerization of alpha-chymotrypsin have been investigated by sedimentation equilibrium studies on the enzyme in acetate-chloride buffer, pH 3.9, I 0.2. From the extent of enhancement of the apparent dimerization constant in the presence of 0.05-0.16 M sucrose, it is concluded that this effect of thermodynamic nonideality finds quantitative explanation in terms of excluded volume. However, the suggested approximation that the radius of an inert small solute would be sufficiently small to be neglected in the calculation of covolumes (D.J. Winzor and P.R. Wills, Biophys. Chem. 25 (1986) 243) has not withstood the more stringent test afforded by the present study of alpha-chymotrypsin dimerization. A value of 0.34 nm for the effective thermodynamic radius of sucrose was inferred from the covolume for self-interaction obtained by frontal gel chromatography on Sephadex G-10 under the conditions of the ultracentrifugal studies. Finally, results of sedimentation equilibrium experiments on alpha-chymotrypsin in the presence of 0.1 M glycerol were also shown to be consistent with interpretation in terms of the model of space-filling effects entailing complete exclusion of small solute from the hydrated protein domain.

Effects of ligand multivalency in binding studies: a general counterpart of the Scatchard analysis

A general counterpart of the Scatchard analysis has been developed which takes into account the valence of the ligand. Its use is first demonstrated by application to binding data obtained by exclusion chromatography of mixtures of Dextran T2000 and concanavalin A (a bivalent ligand) on a column of porous glass beads (Glyceryl-CPG 170) equilibrated at 5 degrees C with phosphate-chloride buffer (pH 5.5), I 0.5. A recycling partition equilibrium study with Sephadex G-100 as gel phase then provides a quantitative evaluation of the interaction between haemoglobin and a monoclonal mouse antihaemoglobin antibody preparation in 0.1 M phosphate (pH 7.0) in order to emphasize the ability of the present analysis to consider collectively binding results obtained with a range of acceptor concentrations. Finally, the use of the generalized Scatchard analysis to assess acceptor site homogeneity is illustrated by reappraisal of results for the binding of glyceraldehyde-3-phosphate dehydrogenase to erythrocyte membranes.