Kottayil I. Varughese

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.

Formation of a novel four-helix bundle and molecular recognition sites by dimerization of a response regulator phosphotransferase.

A basis for understanding specificity of molecular recognition between phosphorelay proteins has been deduced from the 2.6 A structure of the Spo0B phosphotransferase of the phosphorelay regulating sporulation initiation. Spo0B consists of two domains: an N-terminal alpha-helical hairpin domain and a C-terminal alpha/beta domain. Two subunits of Spo0B dimerize by a parallel association of helical hairpins to form a novel four-helix bundle from which the active histidine protrudes. Docking studies show that both the monomers interact with a Spo0F molecule at the region surrounding the active site aspartate to position it for phosphotransfer. It is apparent that different surfaces of response regulators may be involved in recognition of the protein partners to which they are paired.

von Willebrand factor conformation and adhesive function is modulated by an internalized water molecule.

Platelet participation in hemostasis and arterial thrombosis requires the binding of glycoprotein (GP) Ibα to von Willebrand factor (vWF). Hemodynamic forces enhance this interaction, an effect mimicked by the substitution I546V in the vWF A1 domain. A water molecule becomes internalized near the deleted Ile methyl group. The change in hydrophobicity of the local environment causes positional changes propagated over a distance of 27 Å. As a consequence, a major reorientation of a peptide plane occurs in a surface loop involved in GP Ibα binding. This distinct vWF conformation shows increased platelet adhesion and provides a structural model for the initial regulation of thrombus formation.

Interactions between the YycFG and PhoPR two‐component systems in Bacillus subtilis: the PhoR kinase phosphorylates the non‐cognate YycF response regulator upon phosphate limitation

Two‐component signal transduction systems (TCS) are an important mechanism by which bacteria sense and respond to their environment. Although each two‐component system appears to detect and respond to a specific signal(s), it is now evident that they do not always act independently of each other. In this paper we present data indicating regulatory links between the PhoPR two‐component system that participates in the cellular response to phosphate limitation, and the essential YycFG two‐component system in Bacillus subtilis. We show that the PhoR sensor kinase can activate the YycF response regulator during a phosphate limitation‐induced stationary phase, and that this reaction occurs in the presence of the cognate YycG sensor kinase. Phosphorylation of YycF by PhoR also occurs in vitro, albeit at a reduced level. However, the reciprocal cross‐phosphorylation does not occur. A second level of interaction between PhoPR and YycFG is indicated by the fact that cells depleted for YycFG have a severely deficient PhoPR‐dependent phosphate limitation response and that YycF can bind directly to the promoter of the phoPR operon. YycFG‐depleted cells neither activate expression of phoA and phoPR nor repress expression of the essential tagAB and tagDEF operons upon phosphate limitation. This effect is specific to the PhoPR‐dependent phosphate limitation response because PhoPR‐independent phosphate limitation responses can be initiated in YycFG‐depleted cells.

Platelet Dysfunction and a High Bone Mass Phenotype in a Murine Model of Platelet-Type von Willebrand Disease

The platelet glycoprotein Ib-IX receptor binds surface-bound von Willebrand factor and supports platelet adhesion to damaged vascular surfaces. A limited number of mutations within the glycoprotein Ib-IX complex have been described that permit a structurally altered receptor to interact with soluble von Willebrand factor, and this is the molecular basis of platelet-type von Willebrand disease. We have developed and characterized a mouse model of platelet-type von Willebrand disease (G233V) and have confirmed a platelet phenotype mimicking the human disorder. The mice have a dramatic increase in splenic megakaryocytes and splenomegaly. Recent studies have demonstrated that hematopoetic cells can influence the differentiation of osteogenic cells. Thus, we examined the skeletal phenotype of mice expressing the G233V variant complex. At 6 months of age, G233V mice exhibit a high bone mass phenotype with an approximate doubling of trabecular bone volume in both the tibia and femur. Serum measures of bone resorption were significantly decreased in G233V animals. With decreased bone resorption, cortical thickness was increased, medullary area decreased, and consequently, the mechanical strength of the femur was significantly increased. Using ex vivo bone marrow cultures, osteoclast-specific staining in the G233V mutant marrow was diminished, whereas osteoblastogenesis was unaffected. These studies provide new insights into the relationship between the regulation of megakaryocytopoiesis and bone mass.

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.

Molecular recognition of bacterial phosphorelay proteins.

The transfer of the phosphoryl group from a histidine kinase to a response regulator forms the basis of bacterial signal transduction. The critical question of how a component of a signal transduction system specifically associates with its partner to produce the ideal environment for phosphotransfer is addressed in this review in the light of the structure of the Spo0F-Spo0B complex in Bacillus subtilis.

Binding of α-thrombin to surface-anchored platelet glycoprotein Ibα sulfotyrosines through a two-site mechanism involving exosite I

The involvement of exosite I in α-thrombin (FIIa) binding to platelet glycoprotein Ibα (GPIbα), which could influence interactions with other substrates, remains undefined. To address the problem, we generated the GPIbα amino terminal domain (GPIbα-N) fully sulfated on three tyrosine residues and solved the structure of its complex with FIIa. We found that sulfotyrosine (Tys) 278 enhances the interaction mainly by establishing contacts with exosite I. We then evaluated how substituting tyrosine with phenylalanine, which cannot be sulfated, affects FIIa binding to soluble or surface-immobilized GPIbα-N. Mutating Tyr276, which mostly contacts exosite II residues, markedly reduced FIIa interaction with both soluble and immobilized GPIbα-N; mutating Tyr278 or Tyr279, which mostly contact exosite I residues, reduced FIIa complexing in solution by 0–20% but affinity for immobilized GPIbα-N 2 to 6-fold, respectively. Moreover, three exosite I ligands—aptamer HD1, hirugen, and lepirudin—did not interfere with soluble FIIa complexing to GPIbα-N, excluding that their binding caused allosteric effects influencing the interaction; nonetheless, all impaired FIIa binding to immobilized GPIbα-N and platelet GPIb nearly as much as aptamer HD22 and heparin, both exosite II ligands. Bound HD1 and hirugen alter Trp148 orientation in a loop near exosite I preventing contacts with the sulfate oxygen atoms of Tys279. These results support a mechanism in which binding occurs when the two exosites of one FIIa molecule independently interact with two immobilized GPIbα molecules. Through exosite engagement, GPIbα may influence FIIa-dependent processes relevant to hemostasis and thrombosis.

The crystal structure of YycH involved in the regulation of the essential YycFG two-component system in Bacillus subtilis reveals a novel tertiary structure.

The Bacillus subtilis YycFG two‐component signal transduction system is essential for cell viability, and the YycH protein is part of the regulatory circuit that controls its activity. The crystal structure of YycH was solved by two‐wavelength selenium anomalous dispersion data, and was refined using 2.3 Å data to an R‐factor of 25.2%. The molecule is made up of three domains, and has a novel three‐dimensional structure. The N‐terminal domain features a calcium binding site and the central domain contains two conserved loop regions.

Crystal structures of a therapeutic single chain antibody in complex with two drugs of abuse-Methamphetamine and 3,4-methylenedioxymethamphetamine.

Methamphetamine (METH) is a major drug threat in the United States and worldwide. Monoclonal antibody (mAb) therapy for treating METH abuse is showing exciting promise and the understanding of how mAb structure relates to function will be essential for future development of these important therapies. We have determined crystal structures of a high affinity anti‐(+)‐METH therapeutic single chain antibody fragment (scFv6H4, KD= 10 nM) derived from one of our candidate mAb in complex with METH and the (+) stereoisomer of another abused drug, 3,4‐methylenedioxymethamphetamine (MDMA), known by the street name “ecstasy.” The crystal structures revealed that scFv6H4 binds to METH and MDMA in a deep pocket that almost completely encases the drugs mostly through aromatic interactions. In addition, the cationic nitrogen of METH and MDMA forms a salt bridge with the carboxylate group of a glutamic acid residue and a hydrogen bond with a histidine side chain. Interestingly, there are two water molecules in the binding pocket and one of them is positioned for a CH ⃛O interaction with the aromatic ring of METH. These first crystal structures of a high affinity therapeutic antibody fragment against METH and MDMA (resolution = 1.9 Å, and 2.4 Å, respectively) provide a structural basis for designing the next generation of higher affinity antibodies and also for carrying out rational humanization.