Dissecting cooperative and additive binding energetics in the affinity maturation pathway of a protein-protein interface

When two proteins associate they form a molecular interface that is a structural and energetic mosaic. Within such interfaces, individual amino acid residues contribute distinct binding energies to the complex. In combination, these energies are not necessarily additive, and significant positive or negative cooperative effects often exist. The basis of reliable algorithms to predict the specificities and energies of protein-protein interactions depends critically on a quantitative understanding of this cooperativity. We have used a model protein-protein system defined by an affinity maturation pathway, comprising variants of a T cell receptor Vbeta domain that exhibit an overall affinity range of approximately 1500-fold for binding to the superantigen staphylococcal enterotoxin C3, in order to dissect the cooperative and additive energetic contributions of residues within an interface. This molecular interaction has been well characterized previously both structurally, by x-ray crystallographic analysis, and energetically, by scanning alanine mutagenesis. Through analysis of group and individual maturation and reversion mutations using surface plasmon resonance spectroscopy, we have identified energetically important interfacial residues, determined their cooperative and additive energetic properties, and elucidated the kinetic and thermodynamic bases for molecular evolution in this system. The summation of the binding free energy changes associated with the individual mutations that define this affinity maturation pathway is greater than that of the fully matured variant, even though the affinity gap between the end point variants is relatively large. Two mutations in particular, both located in the complementarity determining region 2 loop of the Vbeta domain, exhibit negative cooperativity.     

Mice with targeted mutation of peroxiredoxin 6 develop normally but are susceptible to oxidative stress

Reactive oxygen species, especially hydrogen peroxide, are important in cellular signal transduction. However, excessive amounts of these species damage tissues and cells by oxidizing virtually all important biomolecules. Peroxiredoxin 6 (PRDX6) (also called antioxidant protein 2, or AOP2) is a novel peroxiredoxin family member whose function in vivo is unknown. Through immunohistochemistry, we have determined that the PRDX6 protein was widely expressed in every tissue examined, most abundantly in epithelial cells. It was found in cytosol, but not in membranes, organelles, and nuclei fractions. Prdx6 mRNA was also expressed in every tissue examined. The widespread expression of Prdx6 suggested that its functions were quite important. To determine these functions, we generated Prdx6-targeted mutant (Prdx6-/-) mice, confirmed the gene disruption by Southern blots, PCR, RT-PCR, Western blots, and immunohistochemistry, and compared the effects of paraquat, hydrogen peroxide, and t-butyl hydroperoxide on Prdx6-/- and wild-type (Prdx6+/+) macrophages, and of paraquat on Prdx6-/- and Prdx6+/+ mice. Prdx6-/- macrophages had higher hydrogen peroxide levels, and lower survival rates; Prdx6-/- mice had significantly lower survival rates, more severe tissue damage, and higher protein oxidation levels. Additionally, there were no differences in the mRNA expression levels of other peroxiredoxins, glutathione peroxidases, catalase, superoxide dismutases, thioredoxins, and glutaredoxins between normal Prdx6-/- and Prdx6+/+ mice and those injected with paraquat. Our study provides in vivo evidence that PRDX6 is a unique non-redundant antioxidant that functions independently of other peroxiredoxins and antioxidant proteins.     

Specific binding of dehydroepiandrosterone to the N terminus of the microtubule-associated protein MAP2

The effect of neurosteroids is mediated through their membrane or nuclear receptors. However, no dehydroepiandrosterone (DHEA)-specific receptors have been evidenced so far in the brain. In this paper, we showed by isothermal titration calorimetry that the DHEA specifically binds to the dendritic brain microtubule-associated protein MAP2C with an association constant of 2.7 x 10(7) m-1 and at a molar ratio of 1:1. By partial tryptic digestions and mass spectrometry analysis, we found that the binding involved the N-terminal region of MAP2C. Interestingly, MAP2C displays homologies with 17 beta-hydroxysteroid dehydrogenase 1, an enzyme required for estrogen synthesis. Based on these sequence homologies and on the x-ray structure of the DHEA-binding pocket of 17 beta-hydroxysteroid dehydrogenase 1, we modeled the complex of DHEA with MAP2C. The binding of DHEA to MAP2C involved specific hydrogen bonds that orient the steroid into the pocket. This work suggests that DHEA can directly influence brain plasticity via MAP2C binding. It opens interesting ways for understanding the role of DHEA in the brain.     

Characterization of the interaction of the stress kinase SPAK with the Na+-K+-2Cl- cotransporter in the nervous system: evidence for a scaffolding role of the kinase

Activity of heterologously expressed NKCC1 was analyzed under basal and activated conditions in the presence and absence of binding of Ste20-related proline-alanine-rich kinase (SPAK). Mutant NKCC1 that lacks the ability to bind to this kinase showed K+ transport function identical to wild-type NKCC1. Thus, preventing the binding of the kinase to the cotransporter does not affect cotransporter function. In contrast, several experiments suggest a possible role for SPAK as a scaffolding protein. First, Western blot analysis revealed the presence, and in some tissues abundance, of truncated forms of SPAK and OSR1 in which the kinase domains are affected and thus lack kinase activity. Second, a yeast two-hybrid screen of proteins that interact with the regulatory (binding) domain of SPAK identified several proteins all involved in cellular stress pathways. Third, p38, one of the three major MAPKs, can be coimmunoprecipitated with SPAK and with NKCC1 in an activity-dependent manner. The amount of p38 coimmunoprecipitated with the kinase and the cotransporter significantly decreases upon cellular stress, whereas the interaction of the kinase with NKCC1 remains unchanged. These findings suggest that cation-chloride cotransporters might act as "sensors" for cellular stress, and SPAK, by interacting with the cotransporter, serves as an intermediate in the response to cellular stress.     

The cationic amphipathic alpha-helix of HIV-1 viral protein R (Vpr) binds to nucleic acids,permeabilizes membranes,and efficiently transfects cells

Viral protein R (Vpr) is a small protein of 96 amino acids that is conserved among the lentiviruses human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus. We recently sought to determine whether the karyophilic properties of Vpr, as well as its ability to bind nucleic acids, could be used to deliver DNA into cells. We have found that the C-terminal domain of Vpr-(52-96) is able to efficiently transfect various cell lines. Here, we show that the shortest active sequence for gene transfer corresponds to the domain that adopts a alpha-helix conformation. DNA binding studies and permeabilization assays performed on cells demonstrated that the peptides that are efficient in transfection condense plasmid DNA and are membranolytic. Electron microscopy studies and transfection experiments performed in the presence of inhibitors of the endocytic processes indicated that the major entry pathway of Vpr-DNA complexes is through endocytosis. Taken together, the results show that the cationic C-terminal alpha-helix of Vpr has DNA-condensing as well as membrane-destabilizing capabilities, both properties that are indispensable for efficient DNA transfection.     

N-formyl peptide receptors internalize but do not recycle in the absence of arrestins

Arrestins mediate phosphorylation-dependent desensitization, internalization, and initiation of signaling cascades for the majority of G protein-coupled receptors (GPCRs). Many GPCRs undergo agonist-mediated internalization through arrestin-dependent mechanisms, wherein arrestin serves as an adapter between the receptor and endocytic proteins. To understand the role of arrestins in N-formyl peptide receptor (FPR) trafficking, we stably expressed the FPR in a mouse embryonic fibroblast cell line (MEF) that lacked endogenous arrestin 2 and arrestin 3 (arrestin-deficient). We compared FPR internalization and recycling kinetics in these cells to congenic wild type MEF cell lines. Internalization of the FPR was not altered in the absence of arrestins. Since the FPR remains associated with arrestins following internalization, we investigated whether the rate of FPR recycling was altered in arrestin-deficient cells. While the FPR was able to recycle in the wild type cells, receptor recycling was largely absent in the arrestin double knockout cells. Reconstitution of the arrestin-deficient line with either arrestin 2 or arrestin 3 restored receptor recycling. Confocal fluorescence microscopy studies demonstrated that in arrestin-deficient cells the FPR may become trapped in the perinuclear recycling compartment. These observations indicate that, although the FPR can internalize in the absence of arrestins, recycling of internalized receptors to the cell surface is prevented. Our results suggest a novel role for arrestins in the post-endocytic trafficking of GPCRs.     

A maurotoxin with constrained standard disulfide bridging: innovative strategy of chemical synthesis,pharmacology, and docking on K+ channels

Maurotoxin (MTX) is a 34-residue toxin that has been isolated initially from the venom of the scorpion Scorpio maurus palmatus. It presents a large number of pharmacological targets, including small conductance Ca2+-activated and voltage-gated K+ channels. Contrary to other toxins of the alpha-KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C1-C5, C2-C6, C3-C4, and C7-C8 (instead of the conventional C1-C5, C2-C6, C3-C7, and C4-C8, herein referred to as Pi1-like) that does not prevent its folding along the classic alpha/beta scaffold of scorpion toxins. Here, we developed an innovative strategy of chemical peptide synthesis to produce an MTX variant (MTXPi1) with a conventional pattern of disulfide bridging without any alteration of the toxin chemical structure. This strategy was used solely to address the impact of half-cystine pairings on MTX structural properties and pharmacology. The data indicate that MTXPi1 displays some marked changes in affinities toward the target K+ channels. Computed docking analyses using molecular models of both MTXPi1 and the various voltage-gated K+ channel subtypes (Shaker B, Kv1.2, and Kv1.3) were found to correlate with MTXPi1 pharmacology. A functional map detailing the interaction between MTXPi1 and Shaker B channel was generated in line with docking experiments.     

Alpha4beta1 integrin affinity changes govern cell adhesion

Integrin alpha4beta1 is a receptor for vascular cell adhesion molecule-1 and fibronectin. It is important in lymphopoiesis, inflammatory recruitment of leukocytes, and other situations that require cell adhesion to the vascular endothelium. The avidity of the cells expressing alpha4beta1 integrin can be rapidly changed by chemokines and chemoattractants. Different mechanisms, including changes in the number of interacting molecules due to the alteration of the receptor topology or changes in the affinity of the individual bonds, have been proposed to explain the nature of these fast changes in avidity. Recently, we described a fluorescent LDV-containing small molecule, which we used to monitor the affinity changes on live cells in real time (Chigaev, A., Blenc, A. M., Braaten, J. V., Kumaraswamy, N., Kepley, C. L., Andrews, R. P., Oliver, J. M., Edwards, B. S., Prossnitz, E. R., Larson, R. S. et al. (2001) J. Biol. Chem. 276, 48670-48678). Here we show that the affinity of the small molecule probe as well as the native ligand vascular cell adhesion molecule-1 varies in parallel when the integrin is modulated with divalent cations and that the affinity modulation leads to the changes in cell avidity. Using formyl peptide receptor-transfected U937 cells, we further show that the time course of avidity changes in response to the receptor activation coincides with the time course of the affinity changes. Taken together, these data are consistent with the idea that affinity regulation is a major factor that governs the avidity of cell adhesion mediated by the alpha4 integrin.     

Rib72, a conserved protein associated with the ribbon compartment of flagellar A-microtubules and potentially involved in the linkage between outer doublet microtubules

Ciliary and flagellar axonemes are basically composed of nine outer doublet microtubules and several functional components, e.g. dynein arms, radial spokes, and interdoublet links. Each A-tubule of the doublet contains a specialized "ribbon" of three protofilaments composed of tubulin and other proteins postulated to specify the three-dimensional arrangement of the various axonemal components. The interdoublet links hold the doublet microtubules together and limit their sliding during the flagellar beat. In this study on Chlamydomonas reinhardtii, we cloned a cDNA encoding a 71,985-Da polypeptide with three DM10 repeats, two C-terminal EF-hand motifs, and homologs extending to humans. This polypeptide, designated as Rib72, is a novel component of the ribbon compartment of flagellar microtubules. It remained associated with 9-fold arrays of doublet tubules following extraction under high and low ionic conditions, and anti-Rib72 antibodies revealed an approximately 96-nm periodicity along axonemes, consistent with Rib72 associating with interdoublet links. Following proteolysis- and ATP-dependent disintegration of axonemes, the rate of cleavage of Rib72 correlated closely with the rate of sliding disintegration. These observations identify a ribbon-associated protein that may function in the structural assembly of the axoneme and in the mechanism and regulation of ciliary and flagellar motility.