Identification of a ligand-dependent switch within a muscarinic receptor.

G-protein-coupled receptors spontaneously switch between active and inactive conformations. Agonists stabilize the active conformation, whereas antagonists stabilize the inactive conformation. In a systematic search for residues that participate in receptor function, several regions of the m5 muscarinic receptor were randomly mutated and tested for their functional properties. Mutations spanning one face of transmembrane 6 (TM6) were found to induce high levels of receptor activity in the absence of agonists (constitutive activity). The same face of TM6 contained several residues crucial for receptor activation by agonists and one residue identified as a contact site for both agonists and antagonists. In addition, one mutation induced agonist-like responses from the receptor when exposed to classical antagonists. These results suggest that TM6 is a switch that defines the activation state of the receptor, and that ligand interactions with TM6 stabilize the receptor in either an active or an inactive conformation.

Monomer-heptamer equilibrium of the Escherichia coli chaperonin GroES.

In an effort to clarify the role of GroES in chaperonin-facilitated protein folding, a plasmid-encoding expression system for GroES incorporating a histidine-tagged, thrombin-cleavable, N-terminal sequence was constructed. This approach facilitated the rapid purification of native-like, histidine-cleaved GroES (HC-GroES). The addition of NaSCN to purification buffers to mildly promote subunit dissociation enabled the complete separation of chromosomally encoded, wild-type GroES chains from recombinant chains, allowing the production of homogeneous mutant variants of GroES. A substitution of histidine-7 to tryptophan in GroES was used to demonstrate the concentration-dependent modulation of the heptameric quaternary structure of the chaperonin. Fluorescence and light scattering studies of this mutant suggest that GroES heptamers dissociate to monomers upon dilution with half-times of 2-4 min. Sedimentation equilibrium experiments using either wild-type or HC-GroES can best be described by a monomer--heptamer equilibrium, yielding dissociation constants of 1 x 10(-38) M6 for native GroES and 2 x 10(-32) M6 for HC-GroES. These results are supported by subunit exchange experiments using mixtures of native or HC-GroES and GroES containing the complete N-terminal histidine tail. Native polyacrylamide gel electrophoresis demonstrates that these mixtures form an eight-membered hybrid set within minutes. The studies described here suggest a dynamic equilibrium for the quaternary structure of GroES, which may be an important feature for its role in GroEL-mediated protein folding reactions.

An expanded two-state model accounts for homotropic cooperativity in biosynthetic threonine deaminase from Escherichia coli.

The linkage between substrate and regulatory effector binding to separate sites on allosteric enzymes results in shifts in their sigmoidal kinetics to regulate metabolism. Control of branched chain amino acid biosynthesis in Escherichia coli occurs in part through shifts in the sigmoidal dependence of alpha-ketobutyrate production promoted by isoleucine and valine binding to biosynthetic threonine deaminase. The structural similarity of threonine, valine, and isoleucine have given rise to suggestions that there may be competition among different ligands for the same sites on this tetrameric enzyme, resulting in a complex pattern of regulation. In an effort to provide a coherent interpretation of the cooperative association of ligands to the active sites and to the effector sites of threonine deaminase, binding studies using single amino acid variants were undertaken. A previously-isolated, feedback-resistant mutant identified in Salmonella typhimurium, ilvA219, has been cloned and sequenced. The phenotype is attributable to a single amino acid substitution in the regulatory domain of the enzyme in which leucine at position 447 is substituted with phenylalanine. The mutant exhibits hyperbolic saturation curves in both ligand binding and steady-state kinetics. These results, in addition to calorimetric and spectroscopic measurements of isoleucine and valine binding, indicate that the low affinity (T) state is destabilized in the mutant and that it exists predominantly in the high affinity (R) conformation in the absence of ligands, providing an explanation for its resistance to isoleucine. Chemical and spectroscopic analyses of another mutant, in which alanine has replaced an essential lysine at position 62 that forms a Schiff base with pyridoxal phosphate, indicate that the cofactor is complexed to exogenous threonine and is therefore unable to bind additional amino acids at the active sites. Isoleucine and valine binding to this inactive, active site-saturated enzyme revealed that it too was stabilized in the R state, yielding binding constants in excellent agreement with the leucine to phenylalanine mutant. The lysine to alanine mutant was further utilized to demonstrate that both threonine and 2-aminobutyrate bind with stronger affinity to the regulatory sites than to the active sites. A direct consequence of these results is that substrates and analogs have a synergistic effect on the allosteric transition since, in effect, they act as both homotropic and heterotropic effectors.(ABSTRACT TRUNCATED AT 250 WORDS)