Most prokaryotic signal-transduction systems and a few eukaryotic pathways use phosphotransfer schemes involving two conserved components, a histidine protein kinase and a response regulator protein. The histidine protein kinase, which is regulated by environmental stimuli, autophosphorylates at a histidine residue, creating a high-energy phosphoryl group that is subsequently transferred to an aspartate residue in the response regulator protein. Phosphorylation induces a conformational change in the regulatory domain that results in activation of an associated domain that effects the response. The basic scheme is highly adaptable, and numerous variations have provided optimization within specific signaling systems. The domains of two-component proteins are modular and can be integrated into proteins and pathways in a variety of ways, but the core structures and activities are maintained. Thus detailed analyses of a relatively small number of representative proteins provide a foundation for understanding this large family of signaling proteins.

We report the x-ray crystal structure of the methylesterase CheB, a phosphorylation-activated response regulator involved in reversible modification of bacterial chemotaxis receptors. Methylesterase CheB and methyltransferase CheR modulate signaling output of the chemotaxis receptors by controlling the level of receptor methylation. The structure of CheB, which consists of an N-terminal regulatory domain and a C-terminal catalytic domain joined by a linker, was solved by molecular replacement methods using independent search models for the two domains. In unphosphorylated CheB, the N-terminal domain packs against the active site of the C-terminal domain and thus inhibits methylesterase activity by directly restricting access to the active site. We propose that phosphorylation of CheB induces a conformational change in the regulatory domain that disrupts the domain interface, resulting in a repositioning of the domains and allowing access to the active site. Structural similarity between the two companion receptor modification enzymes, CheB and CheR, suggests an evolutionary and͞or functional relationship. Specifically, the phosphorylated N-terminal domain of CheB may facilitate interaction with the receptors, similar to the postulated role of the N-terminal domain of CheR. Examination of surfaces in the N-terminal regulatory domain of CheB suggests that despite a common fold throughout the response regulator family, surfaces used for proteinprotein interactions differ significantly. Comparison between CheB and other response regulators indicates that analogous surfaces are used for different functions and conversely, similar functions are mediated by different molecular surfaces.

The response regulator CheB functions within the bacterial chemotaxis system together with the methyltransferase CheR to control the level of chemoreceptor methylation, influencing the signaling activities of the receptors. CheB catalyzes demethylation of specific methylglutamate residues introduced into the chemoreceptors by CheR. CheB has a two-domain architecture consisting of an N-terminal regulatory domain joined by a linker to a C-terminal effector domain. In the unphosphorylated state of the response regulator, the regulatory domain inhibits the methylesterase activity of the effector domain. Upon phosphorylation of a specific aspartate residue within the regulatory domain, the C-terminal methylesterase activity is stimulated, resulting in the subsequent demethylation of the chemoreceptors. We have investigated the mechanism of regulation of CheB activity by the N-terminal regulatory domain. First, we have found that phosphorylation of the N-terminal domain not only relieves inhibition of the C-terminal methylesterase activity but also provides an enhancement of this activity above that seen for the C-terminal effector domain alone. Second, we have identified mutations in CheB that show an enhancement of methylesterase activity in the absence of phosphorylation. Most of these single-site mutations are localized in the linker region joining the regulatory and effector domains. On the basis of these observations, we propose a model for activation of CheB in which phosphorylation of the regulatory domain results in a reorganization of the domain interface, allowing exposure of the active site to the receptor substrate and simultaneously stimulating methylesterase activity.

The binding stoichiometry of gene V protein from bacteriophage ft to several oligonucleotides was studied using electrospray ionization-mass spectrometry (ESI-MS). Using mild mass spectrometer interface conditions that preserve noncovalent associations in solution, gene V protein was observed as dimer ions from a 10 mM NH4OAc solution.Addition of oligonucleotides resulted in formation of proteinoligonucleotide complexes with stoichiometry of approximately four nucleotides (nt) per protein monomer. A 16-mer oligonucleotide gave predominantly a 4:1 (protein monomer: oligonucleotide) complex while oligonucleotides shorter than 15 nt showed stoichiometries of 2:1. Stoichiometries and relative binding constants for a mixture of oligonucleotides were readily measured using mass spectrometry. The binding stoichiometry of the protein with the 16-mer oligonucleotide was measured independently using size-exclusion chromatography and the results were consistent with the mass spectrometric data. These results demonstrate, for the first time, the observation and stoichiometric measurement of proteinoligonucleotide complexes using ESI-MS. The sensitivity and high resolution of ESI-MS should make it a useful tool in the study of protein-DNA interactions.Protein-DNA interactions are involved in many cellular processes and thus are fundamentally important for the functioning of biological systems. The stoichiometry of binding is an important characteristic of protein-DNA interactions. Determination of binding stoichiometries can be difficult when there are mixed stoichiometries present in a sample or when molecular mass (Mr) differences between possible alternative stoichiometries are small (1). Mass spectrometry (MS) is the most accurate method for Mr measurement, and thus is potentially useful for accurate determination of protein-DNA binding stoichiometry. However, the application of MS to the direct characterization of protein-DNA complexes has been precluded due to the difficulty of transferring these complexes intact from solution into the mass spectrometer using conventional ionization methods. Electrospray ionization (ESI) allows ionization of non-volatile samples from solution and the development of this technique has greatly expanded the application of mass spectrometry in the analysis of biomolecules (2-5). Recently, a number of groups have reported observations of specific noncovalent complexes of biomolecules from solution using ESI-MS (6-11). These reports prompted us to investigate the application of this technique to intact protein-DNA noncovalent complexes.We chose gene V protein from bacteriophage fl for our study due to its well characterized structure (12-16) and the extensive studies on the DNA binding stoichiometry and cooperatively (17)(18)(19)(20)(21)(22). The gene V protein stabilizes singlestranded DNA (ssDNA) during phage replication, and shifts the synthesis of phage DNA from double-stranded form to single-stranded form, in the. later stage of phage infection, by preventing the synthesis of th...