Crystal structure of the FRP and identification of the active site for modulation of OCP-mediated photoprotection in cyanobacteria.

Photosynthetic reaction centers are sensitive to high light conditions, which can cause damage because of the formation of reactive oxygen species. To prevent high-light induced damage, cyanobacteria have developed photoprotective mechanisms. One involves a photoactive carotenoid protein that decreases the transfer of excess energy to the reaction centers. This protein, the orange carotenoid protein (OCP), is present in most cyanobacterial strains; it is activated by high light conditions and able to dissipate excess energy at the site of the light-harvesting antennae, the phycobilisomes. Restoration of normal antenna capacity involves the fluorescence recovery protein (FRP). The FRP acts to dissociate the OCP from the phycobilisomes by accelerating the conversion of the active red OCP to the inactive orange form. We have determined the 3D crystal structure of the FRP at 2.5 Å resolution. Remarkably, the FRP is found in two very different conformational and oligomeric states in the same crystal. Based on amino acid conservation analysis, activity assays of FRP mutants, FRP:OCP docking simulations, and coimmunoprecipitation experiments, we conclude that the dimer is the active form. The second form, a tetramer, may be an inactive form of FRP. In addition, we have identified a surface patch of highly conserved residues and shown that those residues are essential to FRP activity.

Elucidating essential role of conserved carboxysomal protein CcmN reveals common feature of bacterial microcompartment assembly.

Bacterial microcompartments are organelles composed of a protein shell that surrounds functionally related proteins. Bioinformatic analysis of sequenced genomes indicates that homologs to shell protein genes are widespread among bacteria and suggests that the shell proteins are capable of encapsulating diverse enzymes. The carboxysome is a bacterial microcompartment that enhances CO(2) fixation in cyanobacteria and some chemoautotrophs by sequestering ribulose-1,5-bisphosphate carboxylase/oxygenase and carbonic anhydrase in the microcompartment shell. Here, we report the in vitro and in vivo characterization of CcmN, a protein of previously unknown function that is absolutely conserved in ß-carboxysomal gene clusters. We show that CcmN localizes to the carboxysome and is essential for carboxysome biogenesis. CcmN has two functionally distinct regions separated by a poorly conserved linker. The N-terminal portion of the protein is important for interaction with CcmM and, by extension, ribulose-1,5-bisphosphate carboxylase/oxygenase and the carbonic anhydrase CcaA, whereas the C-terminal peptide is essential for interaction with the carboxysome shell. Deletion of the peptide abolishes carboxysome formation, indicating that its interaction with the shell is an essential step in microcompartment formation. Peptides with similar length and sequence properties to those in CcmN can be bioinformatically detected in a large number of diverse proteins proposed to be encapsulated in functionally distinct microcompartments, suggesting that this peptide and its interaction with its cognate shell proteins are common features of microcompartment assembly.

Conformation-sensing antibodies stabilize the oxidized form of PTP1B and inhibit its phosphatase activity.

Protein tyrosine phosphatase 1B (PTP1B) plays important roles in downregulation of insulin and leptin signaling and is an established therapeutic target for diabetes and obesity. PTP1B is regulated by reactive oxygen species (ROS) produced in response to various stimuli, including insulin. The reversibly oxidized form of the enzyme (PTP1B-OX) is inactive and undergoes profound conformational changes at the active site. We generated conformation-sensor antibodies, in the form of single-chain variable fragments (scFvs), that stabilize PTP1B-OX and thereby inhibit its phosphatase function. Expression of conformation-sensor scFvs as intracellular antibodies (intrabodies) enhanced insulin-induced tyrosyl phosphorylation of the ß subunit of the insulin receptor and its substrate IRS-1 and increased insulin-induced phosphorylation of PKB/AKT. Our data suggest that stabilization of the oxidized, inactive form of PTP1B with appropriate therapeutic molecules may offer a paradigm for phosphatase drug development.

Methods for preparing crystals of reversibly oxidized proteins: crystallization of protein tyrosine phosphatase 1B as an example.

Regulation of protein activity through the oxidation and reduction of cysteines is emerging as an important mechanism in the control of cell-signaling pathways. Protein tyrosine phosphatase 1B (PTP1B), for example, is reversibly inhibited by oxidation at the catalytic cysteine in response to stimulation of cells by insulin or epidermal growth factor. We have conducted structural studies on the redox regulation of PTP1B and have demonstrated that the oxidation of the catalytic cysteine results in the formation of a bond between the sulfur atom of the catalytic cysteine and the amide nitrogen of the neighboring serine. This bond, referred to here as a sulfenamide bond, is reversible upon the addition of glutathione, indicating that this sulfenamide intermediate could function within signaling pathways to protect the cysteine from overoxidation to less readily reducible states. Formation of the sulfenamide bond is accompanied by changes in the tertiary structure at the catalytic site, and these changes may be important for additional regulation of the enzyme. Here, we present methods for preparing crystals ofPTP1B with a sulfenamide bond at the catalytic cysteine. The methods may be adaptable for other proteins that are subject to redox regulation.

Functions and mechanisms of redox regulation of cysteine-based phosphatases.

Reactive oxygen species (ROS) have been implicated as mediators of cell-signaling responses, particularly in pathways involving protein tyrosine phosphorylation. One mechanism by which ROS are thought to exert their effects is through the reversible regulation of cysteine-based phosphatases (CBPs). The CBPs, which include protein tyrosine phosphatases (PTPs), dual-specificity phosphatases, low-molecular-weight PTPs, and the lipid phosphatase PTEN, all contain a nucleophilic catalytic cysteine within a conserved motif that enables these enzymes to dephosphorylate phosphoproteins or phospholipids. In addition to enabling phosphatase activity, the nucleophilic catalytic cysteines of CBPs are also highly susceptible to oxidation, a property that permits redox regulation of this enzyme family. In this review, we discuss the evidence implicating ROS as mediators of CBP activity within signaling pathways and discuss how specificity of ROS-dependent signaling involving CBPs may be achieved. We also discuss the molecular mechanisms that facilitate the stabilization of a reversibly oxidized form of the catalytic cysteine. These mechanisms include the formation of disulfide bonds or the formation of a sulfenamide bond, a novel mechanism that was identified for PTP1B. Formation of either type of covalent bond may be accompanied by dramatic structural rearrangements that can affect downstream signaling events and allow for multitiered enzyme regulation.

Redox regulation of protein tyrosine phosphatase 1B involves a sulphenyl-amide intermediate.

The second messenger hydrogen peroxide is required for optimal activation of numerous signal transduction pathways, particularly those mediated by protein tyrosine kinases. One mechanism by which hydrogen peroxide regulates cellular processes is the transient inhibition of protein tyrosine phosphatases through the reversible oxidization of their catalytic cysteine, which suppresses protein dephosphorylation. Here we describe a structural analysis of the redox-dependent regulation of protein tyrosine phosphatase 1B (PTP1B), which is reversibly inhibited by oxidation after cells are stimulated with insulin and epidermal growth factor. The sulphenic acid intermediate produced in response to PTP1B oxidation is rapidly converted into a previously unknown sulphenyl-amide species, in which the sulphur atom of the catalytic cysteine is covalently linked to the main chain nitrogen of an adjacent residue. Oxidation of PTP1B to the sulphenyl-amide form is accompanied by large conformational changes in the catalytic site that inhibit substrate binding. We propose that this unusual protein modification both protects the active-site cysteine residue of PTP1B from irreversible oxidation to sulphonic acid and permits redox regulation of the enzyme by promoting its reversible reduction by thiols.