James Zapf

A transient interaction between two phosphorelay proteins trapped in a crystal lattice reveals the mechanism of molecular recognition and phosphotransfer in signal transduction.

BACKGROUND Spo0F and Spo0B specifically exchange a phosphoryl group in a central step of the phosphorelay signal transduction system that controls sporulation in Bacilli. Spo0F belongs to the superfamily of response regulator proteins and is one of 34 such proteins in Bacillus subtilis. Spo0B is structurally similar to the phosphohistidine domain of histidine kinases, such as EnvZ, and exchanges a phosphoryl group between His30 and Asp54 on Spo0F. Information at the molecular level on the interaction between response regulators and phosphohistidine domains is necessary to develop a rationale for how phospho-signaling fidelity is maintained in two-component systems. RESULTS Structural analysis of a co-crystal of the Spo0F response regulator interacting with the Spo0B phosphotransferase of the phosphorelay signal transduction system of B. subtilis was carried out using X-ray crystallographic techniques. The association of the two molecules brings the catalytic residues from both proteins into precise alignment for phosphoryltransfer. Upon complex formation, the Spo0B conformation remains unchanged. Spo0F also retains the overall conformation; however, two loops around the active site show significant deviations. CONCLUSIONS 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.

Crystal structure of a phosphatase-resistant mutant of sporulation response regulator Spo0F from Bacillus subtilis.

BACKGROUND Spo0F, a phosphotransferase containing an aspartyl pocket, is involved in the signaling pathway (phosphorelay) controlling sporulation in Bacillus subtilis. It belongs to the superfamily of bacterial response regulatory proteins, which are activated upon phosphorylation of an invariant aspartate residue. This phosphorylation is carried out in a divalent cation dependent reaction catalyzed by cognate histidine kinases. Knowledge of the Spo0F structure would provide valuable information that would enable the elucidation of its function as a secondary messenger in a system in which a phosphate is donated from Spo0F to Spo0B, the third of four main proteins that constitute the phosphorelay. RESULTS We have determined the crystal structure of a Rap phosphatase resistant mutant, Spo0F Tyr13-->Ser, at 1.9 A resolution. The structure was solved by single isomorphous replacement and anomalous scattering techniques. The overall structural fold is (beta/alpha)5 and contains a central beta sheet. The active site of the molecule is formed by three aspartate residues and a lysine residue which come together at the C terminus of the beta sheet. The active site accommodates a calcium ion. CONCLUSIONS 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.

DNA Complexed Structure of the Key Transcription Factor Initiating Development in Sporulating Bacteria

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.

Metals in the sporulation phosphorelay: manganese binding by the response regulator Spo0F

As a part of studies on the structural characterization of the components of the sporulation phosphorelay in Bacillus subtilis, the crystal structure of the manganese derivative of an intermediate signal transducer, Spo0F, has been elucidated at 2.25 A resolution. The calcium complex and the apo structures have been analyzed previously. In apo Spo0F, the active-site cation cavity is only partially formed and it only becomes completed upon metal coordination. The carbonyl of Lys56 is coordinated to the metal and interestingly the side chain of Lys56 exists in a variety of conformations in the three crystal structures of Spo0F. The affinity of the magnesium ion for Spo0F is in fact low; however, it binds Spo0F when it is in complex with Spo0B. It is proposed that the existence of a deep pocket which extends from the surface to the metal site could attract and direct the metal, thereby facilitating the metal binding of the complex.

Crystallization and preliminary X-ray analysis of a Y13S mutant of Spo0F from Bacillus subtilis.

Spo0F, a member of a superfamily of bacterial response regulatory proteins, is crucial to the regulation of sporulation in Bacillus subtilis. As there were difficulties in reproducing crystals of wild-type Spo0F, we report here the crystallization and preliminary studies of a mutant, Y13S protein, which gave well diffracting reproducible crystals. The crystals of the mutant obtained by the hanging-drop method belong to the tetragonal space group P4(1)2(1)2 (P4(3)2(1)2) a = b = 105.1, c = 85.9 A. Diffraction data were collected at 2.8 A at the laboratory source and subsequently 2.05. A data were collected upon flash freezing the crystal at the Stanford Synchrotron Radiation Laboratory. This mutant participates in the phosphorelay in a similar manner to the wild-type protein. The presence of divalent cations are essential for wild-type phosphorylation and the present mutant crystal form is obtained in the presence of calcium.

Aspartyl phosphates in the regulatory control of bacterial response.

SummaryAcyl phosphates represent a mixed anhydride class of compounds whose lability allows the phosphorylation of an aspartyl carboxyl contained in a protein to reversibly induce changes in structure that may have biological significance, particularly in prokaryotic systems. In this report the phosphorelay system that regulates sporulation inBacillus subtilis is described briefly and its analogy to other regulatory systems is outlined. The structural properties of the aspartate containing second component of the phosphorelay system SpoOF is examined by multidimensional NMR techniques and comparison is made with a known sequence analog, CheY. Distinct differences are apparent that are reflected by the extended half life of phosphorylated SpoOF relative to the CheY analog. It is probable that in a general way the distinct half life characteristics may be related to the differing functions of the various regulatory aspartate containing proteins in the cell.

Sporulation Phosphorelay Proteins and Their Complexes: Crystallographic Characterization

Bacteria use two-component systems to adapt to changes in environmental conditions. In response to deteriorating conditions of growth, certain types of bacteria form spores instead of proceeding with cell division. The formation of spores is controlled by an expanded version of two-component systems called the phosphorelay. The phosphorelay comprises a primary kinase that receives the signal/stimulus and undergoes autophosphorylation, followed by two intermediate messengers that regulate the flow of the phosphoryl group to the ultimate response regulator/transcription factor. Sporulation is initiated when the level of phosphorylation of the transcription factor reaches a critical point. This chapter describes efforts to understand the mechanism of initiation of sporulation at the molecular level using X-ray crystallography as a tool. Structural analyses of individual members, as well as their complexes, provide insight into the mechanism of phosphoryl transfer and the origin of specificity in signal transduction.