Ligand binding to the periplasmic domain of the transmembrane aspartate receptor generates an intramolecular conformational change which spans the bilayer and ultimately signals the cytoplasmic CheA histidine kinase, thereby triggering chemotaxis. The receptor is a homodimer stabilized by the interface between its two identical subunits: the present study investigates the role of the periplasmic and transmembrane regions of this interface in the mechanism of transmembrane signaling. Free cysteines and disulfide bonds are engineered into selected interfacial positions, and the resulting effects on the transmembrane signal are assayed by monitoring in vitro regulation of kinase activity. Three of the 14 engineered cysteine pairs examined, as well as six of the 14 engineered disulfides, cause perturbations of the interface structure which essentially destroy transmembrane regulation of the kinase. The remaining 11 cysteine pairs, and eight engineered disulfides covalently linking the two subunits at locations spanning positions 18-75, are observed to retain significant transmembrane kinase regulation. The eight functional disulfides positively identify adjacent faces of the two N-terminal helices in the native receptor dimer and indicate that large regions of the periplasmic and transmembrane subunit interface remain effectively static during the transmembrane signal. The results are consistent with a model in which the subunit interface plays a structural role, while the second membrane-spanning helix transmits the ligand-induced signal across the bilayer to the kinase binding domain. The effects of engineered cysteines and disulfides on receptor methylation in vitro are also measured, enabling direct comparison of the in vitro methylation and phosphorylation assays.

The kinetics of interaction between TCR and MHC-peptide show a general relationship between affinity and the biological response, but the reported kinetic differences between antigenic and antagonistic peptides are very small. Here, we show a remarkable difference in the kinetics of TCR interactions with strong agonist ligands at 37 degrees C compared to 25 degrees C. This difference is not seen with antagonist/positive selecting ligands. The interaction at 37 degrees C shows biphasic binding kinetics best described by a model of TCR dimerization. The altered kinetics greatly increase the stability of complexes with agonist ligands, accounting for the large differences in biological response compared to other ligands. Thus, there may be an allosteric, as well as a kinetic, component to the discrimination between agonists and antagonists.

Protein phosphorylation is one of the major regulatory mechanisms involved in signal-induced cellular events, including cell proliferation, apoptosis, and metabolism. Because many facets of biology are regulated by protein phosphorylation, aberrant kinase and/or phosphatase activity forms the basis for many different types of pathology. The disease relevance of protein kinases and phosphatases has led many pharmaceutical and biotechnology companies to expend significant resources in lead discovery programs for these two target classes. The existence of >500 kinases and phosphatases encoded by the human genome necessitates development of methodologies for the rapid screening for novel and specific compound inhibitors. We describe here a fluorescence-based, molecular assay platform that is compatible with robotic, ultra-high throughput screening systems and can be applied to virtually all tyrosine and serine/threonine protein kinases and phosphatases. The assay has a coupled-enzyme format, utilizing the differential protease sensitivity of phosphorylated versus nonphosphorylated peptide substrates. In addition to screening individual kinases, the assay can be formatted such that kinase pathways are re-created in vitro to identify compounds that specifically interact with inactive kinases. Miniaturization of this assay format to the 1-microl scale allows for the rapid and accurate compound screening of a host of kinase and phosphatase targets, thereby facilitating the hunt for new leads for these target classes.

The kinetics of the interaction between T cell receptor (TCR) and major histocompatibility complex (MHC) has an important role in determining thymocyte-positive and -negative selection in the thymus, as well as in T cell activation. The alpha chain of the TCR is the major player in determining how the TCR fits onto the MHC ligand, and thus has a major role in determining whether a T cell develops as class I or class II restricted. In this article, we summarize recent data from our laboratory and others on the role of polymorphism in the Valpha combining site in determining MHC class restriction, and on kinetic parameters in thymocyte selection.

Bacterial superantigens such as Staphylococcus aureus enterotoxin A (SEA) are very potent stimulators of T cells. They bind to the Vβ region of the TCR and to MHC class II, stimulating T cells at nanomolar concentrations. Using surface plasmon resonance measurements, we find that binding between the individual components of the complex (TCR-class II, TCR-SEA, SEA-class II) is very weak, but that the stability of the trimolecular complex is considerably enhanced, reaching an affinity similar to that found for TCR interactions with MHC:peptide ligand. Thus, the potency of SEA in stimulation of T cells is not due to particularly strong affinities between the proteins, but to a cooperative effect of interactions in the TCR-SEA-MHC class II trimolecular complex that brings the kinetics into a similar range to binding of conventional Ags. This range may be the optimum for T cell activation.