The Spo0F-Spo0B interaction appears to be a prototype for response regulator-histidine kinase interactions. The primary contact surface between these two proteins is formed by hydrophobic regions in both proteins. The Spo0F residues making up the hydrophobic patch are very similar in all response regulators suggesting that the binding is initiated through the same residues in all interacting response regulator-kinase pairs. The bulk of the interactions outside this patch are through nonconserved residues. Recognition specificity is proposed to arise from interactions of the nonconserved residues, especially the hypervariable residues of the beta4-alpha4 loop.

Sporulation in Bacillus species, the ultimate bacterial adaptive response, requires the precisely coordinated expression of a complex genetic pathway, and is initiated through the accumulation of the phosphorylated form of Spo0A, a pleiotropic response regulator transcription factor. Spo0A controls the transcription of several hundred genes in all spore-forming Bacilli including genes for sporulation and toxin regulation in pathogens such as Bacillus anthracis. The crystal structure of the effector domain of Spo0A from Bacillus subtilis in complex with its DNA target was determined. In the crystal lattice, two molecules form a tandem dimer upon binding to adjacent sites on DNA. The protein:protein and protein:DNA interfaces revealed in the crystal provide a basis for interpreting the transcription activation process and for the design of drugs to counter infections by these bacteria.

The structural analysis reveals that the overall topology and metal-binding coordination at the active site are similar to those of the bacterial chemotaxis response regulator CheY. Structural differences between Spo0F and CheY in the vicinity of the active site provide an insight into how similar molecular scaffolds can be adapted to perform different biological roles by the alteration of only a few amino acid residues. These differences may contribute to the observed stability of the phosphorylated species of Spo0F, a feature demanded by its role as a secondary messenger within the phosphorelay system which controls sporulation.

NMR has been employed for structural and dynamic studies of the bacterial response regulator, Spo0F. This 124-residue protein is an essential component of the sporulation phosphorelay signal transduction pathway in Bacillus subtilis. Three-dimensional 1H, 15N, and 13C experiments have been used to obtain full side chain assignments and the 1511 distance, 121 dihedral angle, and 80 hydrogen bonding restraints required for generating a family of structures (14 restraints per residue). The structures give a well-defined (alpha/beta)5 fold for residues 4-120 with average rms deviations of 0.59 A for backbone heavy atoms and 1.02 A for all heavy atoms. Analyses of backbone 15N relaxation measurements demonstrate relative rigidity in most regions of regular secondary structure with a generalized order parameter (S2) of 0.9 +/- 0.05 and a rotational correlation time (taum) of 7.0 +/- 0.5 ns. Loop regions near the site of phosphorylation have higher than average rms deviation values and T1/T2 ratios suggesting significant internal motion or chemical exchange at these sites. Additionally, multiple conformers are observed for the beta4-alpha4 loop and beta-strand 5 region. These conformers may be related to structural changes associated with phosphorylation and also indicative of the propensity this recognition surface has for differential protein interactions. Comparison of Spo0F structural features to those of other response regulators reveals subtle differences in the orientations of secondary structure in the putative recognition surfaces and the relative charge distribution of residues surrounding the site of phosphorylation. These may be important in providing specificity for protein-protein interactions and for determining the lifetimes of the phosphorylated state.

Amino acid Asp-351 in the ligand binding domain of estrogen receptor ␣ (ER␣) plays an important role in regulating the estrogen-like activity of selective estrogen receptor modulator-ER␣ complexes. 4-Hydroxytamoxifen is a full agonist at a transforming growth factor ␣ target gene in situ in MDA-MB-231 human breast cancer cells stably transfected with the wild-type ER␣. In contrast, raloxifene (Ral), which is also a selective estrogen receptor modulator, is a complete antiestrogen in this system. Because D351G ER␣ allosterically silences activation function-1 activity in the 4-hydroxytamoxifen-ER␣ complex with the complete loss of estrogenlike activity, we examined the converse interaction of amino acid 351 and the piperidine ring of the antiestrogen side chain of raloxifene to enhance estrogen-like action. MDA-MB-231 cells were either transiently or stably transfected with Asp-351 (the wild type), D351E, D351Y, or D351F ER␣ expression vectors. Profound differences in the agonist and antagonist actions of Ral⅐ER␣ complexes were noted only in stable transfectants. The agonist activity of the Ral⅐ER␣ complex was enhanced with D351E and D351Y ER␣, but raloxifene lost its agonist activity with D351F ER␣. The distance between the piperidine nitrogen of raloxifene and the negative charge of amino acid 351 was critical for estrogen-like actions. The role of the piperidine ring in neutralizing Asp-351 was addressed using compound R1h, a raloxifene derivative replacing the nitrogen on its piperidine ring with a carbon to form cyclohexane. The derivative was a potent agonist with wild type ER␣. These results support the concept that the side chain of raloxifene shields and neutralizes the Asp-351 to produce an antiestrogenic ER␣ complex. Alteration of either the side chain or its relationship with the negative charge at amino acid 351 controls the estrogen-like action at activating function 2b of the selective estrogen receptor modulator ER␣ complex.Raloxifene (Ral) 1 (see Fig. 1) is a polyhydroxyphenyl benzothiophene antiestrogen that has low estrogen agonist activity in the rodent uterus (1). The compound originally referred to as Ly156758 or keoxifene was abandoned for development as a treatment for breast cancer (2), because its bioavailability was less than 2% administered dose (3). However, the recognition that raloxifene maintains bone density (4, 5) and inhibits mammary carcinogenesis in the rat (6, 7) illustrates the concept of selective estrogen receptor modulation. Raloxifene is used for the prevention of osteoporosis in postmenopausal women (8), and treatment is associated with a reduced incidence of breast cancer (9).Tamoxifen is the prototype selective estrogen receptor modulator (SERM) that is used clinically for the treatment and prevention of breast cancer (10, 11) with the ability to maintain bone density in postmenopausal women (12). However, tamoxifen therapy is also associated with estrogen-like effects in the uterus with an increased incidence of endometrial cancer (13). Raloxifene is currently being ...

SpoOF, sporulation stage 0 F protein, a 124‐residue protein responsible, in part, for regulating the transition of Bacillus subtilis from a vegetative state to a dormant endospore, has been studied by high‐resolution NMR. The 1H, 15N, and 13C chemical shift assignments for the backbone residues have been determined from analyses of 3D spectra, 15N TOCSY‐HSQC, 15N NOESY‐HSQC, HNCA, and HN(CO)CA. Assignments for many side‐chain proton resonances are also reported. The secondary structure, inferred from short‐ and medium‐range NOEs, 3JHNα coupling constants, and hydrogen exchange patterns, define a topology consistent with a doubly wound (α/β)5 fold. Interestingly, comparison of the secondary structure of SpoOF to the structure of the Escherichia coli response regulator, chemotaxis Y protein (CheY) (Volz K, Matsumura P, 1991, J Biol Chem 266:15511–15519; Bruix M et al., 1993, Eur J Biochem 2/5:573–585), show differences in the relative length of secondary structure elements that map onto a single face of the tertiary structure of CheY. This surface may define a region of binding specificity for response regulators. Magnesium titration of SpoOF, followed by amide chemical shift changes, gives an equilibrium dissociation constant of 20 ± 5 mM. Amide resonances most perturbed by magnesium binding are near the putative site of phosphorylation, Asp 54.

Transient phosphorylation at an aspartate residue on the Spo0F protein is a central step in the phosphorelay signal transduction pathway controlling sporulation in Bacilli. The response regulator Spo0F-P is stable to hydrolysis (t1/2 > 24 h at 23 degrees C in the absence of Mg2+), allowing the use of nondenaturing PAGE to separate the phosphorylated and non-phosphorylated forms of Spo0F. Using this novel assay, phosphoramidate containing compounds were found to specifically phosphorylate Spo0F, a reaction that requires the presence of a divalent metal, but mixed phosphate-carboxylate compounds did not act as phospho donors. Rapid hydrolysis of Spo0F-P generated with phosphoramidate by proteins downstream in the phosphorelay (Spo0B and Spo0A) is consistent with phosphorylation at the active site of Spo0F. The initial rate of Spo0F-P formation from phosphoramidate displays Michaelis-Menten kinetics, providing evidence for the proposal that response regulators, such as Spo0F, function as phosphoryl transferase enzymes (McCleary et al., 1993). The results establish that Spo0F functions as a phosphoryl transferase that uses exclusively a phosphoramidate rather than an acyl phosphate as substrate during autophosphorylation.

Two-component signaling systems are used by bacteria, plants, and lower eukaryotes to adapt to environmental changes. The first component, a protein kinase, responds to a signal by phosphorylating the second component; a response regulator protein that often acts by inducing the expression of specific genes. Response regulators also have an autophosphatase activity that ensures that the proteins are not permanently activated by phosphorylation. The magnitude of this activity varies by at least 1000-fold between various response regulators, and the molecular features responsible for this varied autophosphatase activity have not been clearly defined. Using wild-type and mutant derivatives of the sporulation response regulator Spo0F, it has been demonstrated that a key residue in determining the magnitude of this activity is that at position 56 of Spo0F approximately P; this residue is adjacent to the site of phosphorylation, Asp 54. For example, Spo0F approximately P K56N has a 23-fold greater autophosphatase activity (t1/2 = 8 min) than wild-type Spo0F approximately P (t1/2 = 180 min). It is suggested that, by analogy to the GTPase activity of p21(ras) and by examining the crystallographic structure of Spo0F, that the carboxyamide of the mutant Asn 56 may favorably position a catalytic water near the protein acyl phosphate to promote Spo0F approximately P K56N hydrolysis. It is also deduced that Lys 56 in the wild-type protein is critical for the efficient interaction and phosphoryl transfer between Spo0F and it's cognate protein kinase, KinA. Comparison of the known response regulators shows that inefficient autophosphatases (t1/2 on the order of hours) typically contain an amino acid residue with a long side chain at the position equivalent to 56 in Spo0F, whereas efficient autophosphatases (t1/2 on the order of minutes) frequently contain a residue with a carboxyamide or carboxylate side chain at this position. It appears that, by altering residues adjacent to the active site, the autophosphatase activity of response regulator proteins has been attenuated to match the diverse biological roles played by these proteins.