News and Publications

Bacterial Response to Environmental Stimuli

K.I. Varughese, Madhusudan, J. Zapf, U. Sen, J.M. Whiteley, J.A. Hoch

Gene activation in response to environmental stimuli is characteristic of all life forms, from bacteria to humans. Bacteria respond to environmental stress by activating transcription of genes that code for products that enable the bacteria to adapt to the new environment. Common stresses include osmotic shock; starvation of nitrogen, phosphate, and various carbon sources; and responses to oxygen concentration. These and common bacterial processes such as motility and chemotaxis; secretion of enzymes; transport of hexoses, dicarboxylates, and tricarboxylates; and the capacity for virulence of some human and plant pathogens involve the induction of specific metabolic pathways and biochemical systems so that the organisms can cope with the new environmental status.

In all these systems, a signal induces a transcriptional change via a so-called 2-component regulatory switch. These switches consist of a histidine protein kinase and a response regulator. The signal activates the kinase to autophosphorylate, and then transfer of this phosphoryl group to the amino-terminal domain of the regulatory protein activates the transcription of genes. The amino-terminal domains of the phosphoreceptor proteins are highly conserved. The 2-component system was initially thought to occur only in bacteria; however, similar systems have been found in species of yeast and certain other eukaryotes.

Bacteria such as Bacillus subtilis also respond to environmental stress by forming spores, an action that is regulated by a somewhat more complex phosphorelay system. This system contains 4 major components--a kinase (KinA), a receptor (Spo0F), a transfer protein (Spo0B), and a transcription activator (Spo0A)--and some additional phosphatase-regulating features. The genes for each of these proteins have been expressed in Escherichia coli, and the proteins have been isolated and characterized by chromatographic procedures.

Of note, the phosphate group in the phosphorelay is passed from histidine to aspartate to histidine to aspartate in a uniquely responsive low-energy transfer system. In addition, the smaller molecular weight Spo0F correlates with the amino-terminal region of Spo0A. This last observation coupled with the remarkable stability of Spo0F and its phosphorylated derivative (Spo0F~P) suggested that examination of the molecule by x-ray crystallography and high-resolution nuclear magnetic resonance techniques would be feasible. Both a Y13S mutant and the wild-type Spo0F have been crystallized, and their structures have been analyzed at high resolution.

The 3-dimensional structure of Spo0F is similar to that of CheY, a response regulatory protein in the chemotaxis pathway. Despite structural and sequence similarities between these 2 response regulators, Spo0F~P was nearly 3 orders of magnitude more stable than phosphorylated CheY to phosphate hydrolysis. Response regulator proteins are activated by phosphorylation, and phosphate hydrolysis or protein dephosphorylation is thought to be due to an autophosphatase activity that ensures that these proteins do not remain permanently activated by phosphorylation. The "stability" or the magnitude of the autophosphatase activities of Spo0F~P and phosphorylated CheY are appropriately matched to suit the respective biological roles of the 2 regulators. Sporulation is a slow process that occurs over an hour or hours, whereas chemotaxis is a rapid response that occurs in a matter of seconds.

Subtle differences between the structures of Spo0F and CheY in the regions adjacent to the phosphorylation active site may account for the divergent phosphorylation properties of these 2 response regulators. A possible model for Spo0F~P designed by using computer graphics highlighted the importance of nonconserved residues located adjacent to the phosphorylation active site. This model showed that the side chains of Lys56 and Gln12 could interact with the phosphoryl oxygens and stabilize the phosphoryl group. Indeed, mutation of Lys56 to Asn56, the residue occurring at the equivalent position in CheY, increased the autophosphatase activity 23-fold. Response regulators naturally containing an asparagine residue at the position equivalent to 56 in Spo0 F also hydrolyze phosphate in a matter of minutes. These results suggest that the type of residue at the position equivalent to 56 in Spo0F "tunes" the response regulator autophosphatase activity to suit a particular biological role.

Spo0B is 1 of 4 components of the phosphorelay that controls sporulation in B subtilis. Spo0B transfers a phosphoryl moiety from an aspartate in Spo0F to an aspartate in Spo0A by undergoing transient phosphorylation on a histidine residue. The crystal structure of Spo0B has been determined at 2.6-Å resolution and contains 2 domains: an amino-terminal -helical hairpin and a carboxy-terminal /ß structure. The hairpins from 2 monomers associate in a parallel manner to form a novel type of 4-helix bundle at the interface of a dimer. The residue that undergoes phosphorylation, His30, is located in the middle of a helix in the 4-helix bundle and is exposed to solvent. The 2 active sites per dimer are formed by residues from both monomers.

Docking studies showed that Spo0F can fit into the active site without either the Spo0B dimer or Spo0F undergoing any global changes to bring Asp-P close to His30 for phosphate transfer (Fig. 1). A comparison of the current model of Spo0B and Spo0F complex with the structure of the complex of CheY and the P2 domain of CheA show that different surfaces of the response regulators may be involved in molecular recognition of the regulators' cognate phosphotransfer domains.

PUBLICATIONS

Madhusudan, Zapf, J., Hoch, J.A., Whiteley, J.M., Xuong, N.H., Varughese, K.I. A response regulatory protein with the site of phosphorylation blocked by an arginine interaction: Crystal structure of Spo0F from Bacillus subtilis. Biochemistry 36:12739, 1997.

Zapf, J., Madhusudan, Grimshaw, C.E., Hoch, J.A., Varughese, K.I., Whiteley, J.M. A source of response regulator autophosphatase activity: The critical role of a residue adjacent to the Spo0F autophosphorylation active site. Biochemistry 37:7725, 1998.

Structural Studies of the von Willebrand Factor A1 Domain

K.I. Varughese, R. Celikel, S. Vasudevan, Z.M. Ruggeri

Normal platelet function needed to arrest hemorrhage requires binding of the membrane glycoprotein (GP) Ib, a component of the GP Ib-IX-V receptor complex, to the A1 domain of von Willebrand factor (vWF). This binding is the sole adhesive interaction capable of mediating the initial tethering of circulating platelets to thrombogenic surfaces exposed to rapid blood flow. The phenotypes of von Willebrand disease, the most prevalent congenital bleeding disorder in humans, and of the less common Bernard-Soulier syndrome clearly indicate the physiologic hemostatic function mediated by GP Ib and the A1 domain. The interaction of GP Ib and the A1 domain, however, may contribute to acute thrombotic occlusion of atherosclerotic stenosed arteries, causing catastrophic organ damage, as in myocardial infarction in patients with coronary artery disease.

We obtained crystals of the active A1 domain in complex with the Fab fragment of NMC-4, a monoclonal antibody that binds to the domain with high affinity, and used x-ray diffraction data to generate a structural model to 2.2-Å resolution. The complex is formed through interactions of the helix 4 of the A1 domain with loops L3, H2, and H3 of the Fab complementarity-determining regions.

Soluble vWF in blood does not interact with GP Ib to any significant degree. The interaction between the A1 domain and GP Ib is enhanced by the type IIB mutations in the A1 domain that cause type IIB von Willebrand disease. These mutations alter the function of vWF to the point that it can bind to GP Ib even when the factor is in solution. Of the 10 such mutations detected so far, 7 are on the surface of the A1 domain, 1 (Ile546) is only partly exposed, and 2 (Val551 and Val553) are buried. The exposed residues appear to regulate the function of vWF indirectly by interfacing with other parts of intact vWF; the buried residues appear to alter the dissociation properties by having an indirect effect on the surface shape and flexibility. Structural characterization of these mutants is expected to provide insight into interactions between the A1 domain and GP Ib.

PUBLICATIONS

Celikel, R., Varughese, K.I., Madhusudan, Yoshioka, A., Ware, J., Ruggeri, Z.M. Crystal structure of the von Willebrand factor A1 domain in complex with the function blocking NMC-4 Fab. Nature Struct. Biol. 5:189, 1998.