Direct evidence for calcineurin binding to the exon-7 loop of the sodium-bicarbonate cotransporter NBCn1.

The NaHCO3 cotransporter NBCn1 plays a role in neutralizing intracellular acid loads at the basolateral membrane in cells of the medullary thick ascending limb (mTAL). Calcineurin inhibitors (Cn-Is) are known to both downregulate NBCn1 expression in the distal nephron and cause renal tubular acidosis (RTA), a risk factor for nephrocalcinosis and nephrolithiasis. In this report, we provide a new perspective on concurrent studies of NBCn1 in various tissues by using cell-free binding assays to investigate the interaction of NBCn1 with the calcineurin (Cn) isoform PPP3CA. Surface plasmon resonance (SPR) analyses show that the protein domain Exon 7 (translated from cassette II of NBCn1) binds Cn with an equilibrium dissociation constant (KD) of 30 +/- 15 nm. Linked-reaction tests suggest that the binding involves a conformational change. Nested PCR reactions were used to show that NBCn1-Exon 7 splice variants with alternative N-termini regions are expressed in the kidney, as well as other tissues. Additionally, we discuss NBCn1-Exon 7 implication in acid-base balance and calcium crystallization in the kidney.

Kinetic characterization of Salmonella FliK-FlhB interactions demonstrates complexity of the Type III secretion substrate-specificity switch.

The bacterial flagellum is a complex macromolecular machine consisting of more than 20 000 proteins, most of which must be exported from the cell via a dedicated Type III secretion apparatus. At a defined point in flagellar morphogenesis, hook completion is sensed and the apparatus switches substrate specificity type from rod and hook proteins to filament ones. How the switch works is a subject of intense interest. FliK and FlhB play central roles. In the present study, two optical biosensing methods were used to characterize FliK-FlhB interactions using wild-type and two variant FlhBs from mutants with severe flagellar structural defects. Binding was found to be complex with fast and slow association and dissociation components. Surprisingly, wild-type and variant FlhBs had similar kinetic profiles and apparent affinities, which ranged between 1 and 10.5 microM, suggesting that the specificity switch is more complex than presently understood. Other binding experiments provided evidence for a conformational change after binding. Liquid chromatography-mass spectrometry (LC-MS) and NMR experiments were performed to identify a cyclic intermediate product whose existence supports the mechanism of autocatalytic cleavage at FlhB residue N269. The present results show that while autocatalytic cleavage is necessary for proper substrate specificity switching, it does not result in an altered interaction with FliK, strongly suggesting the involvement of other proteins in the mechanism.

Evolution reversed: the ability to bind iron restored to the N-lobe of the murine inhibitor of carbonic anhydrase by strategic mutagenesis.

The murine inhibitor of carbonic anhydrase (mICA) is a member of the superfamily related to the bilobal iron transport protein transferrin (TF), which binds a ferric ion within a cleft in each lobe. Although the gene encoding ICA in humans is classified as a pseudogene, an apparently functional ICA gene has been annotated in mice, rats, cows, pigs, and dogs. All ICAs lack one (or more) of the amino acid ligands in each lobe essential for high-affinity coordination of iron and the requisite synergistic anion, carbonate. The reason why ICA family members have lost the ability to bind iron is potentially related to acquiring a new function(s), one of which is inhibition of certain carbonic anhydrase (CA) isoforms. A recombinant mutant of the mICA (W124R/S188Y) was created with the goal of restoring the ligands required for both anion (Arg124) and iron (Tyr188) binding in the N-lobe. Absorption and fluorescence spectra definitively show that the mutant binds ferric iron in the N-lobe. Electrospray ionization mass spectrometry confirms the presence of both ferric iron and carbonate. At the putative endosomal pH of 5.6, iron is released by two slow processes indicative of high-affinity coordination. Induction of specific iron binding implies that (1) the structure of mICA resembles those of other TF family members and (2) the N-lobe can adopt a conformation in which the cleft closes when iron binds. Because the conformational change in the N-lobe indicated by metal binding does not impact the inhibitory activity of mICA, inhibition of CA was tentatively assigned to the C-lobe. Proof of this assignment is provided by limited trypsin proteolysis of porcine ICA.

Direct binding of visual arrestin to a rhodopsin carboxyl terminal synthetic phosphopeptide.

PURPOSE: The phosphorylated carboxyl terminus of rhodopsin is required for the stable binding of visual arrestin to the full length rhodopsin molecule. Phosphorylation of the carboxyl terminus has been shown to induce conformational changes in arrestin, which promote its binding to the cytoplasmic loops of rhodopsin. However, it has not been determined whether phosphorylation is also responsible for the direct binding of the rhodopsin carboxyl terminus to arrestin. To further investigate the role of rhodopsin phosphorylation on arrestin binding, surface plasmon resonance was used to measure the interaction between a synthetic phosphopeptide corresponding to the carboxyl terminus of rhodopsin and visual arrestin in real time. METHODS: Synthetic peptides were generated that correspond to the phosphorylated and nonphosphorylated carboxyl terminus of bovine rhodopsin. These peptides were immobilized on a biosensor chip and their interaction with purified visual arrestin was monitored by surface plasmon resonance on a BIAcore 2000 or 3000. RESULTS: A synthetic peptide phosphorylated on residues corresponding to Ser-338, Thr-340, Thr-342 and Ser-343 of bovine rhodopsin was sufficient for direct binding to visual arrestin. In contrast, a second phosphopeptide phosphorylated on Thr-340 and Thr-342 and a nonphosphorylated synthetic peptide were not able to bind arrestin. A peptide fully substituted at all serine and threonine residues with glutamic acid was unable to substitute for phosphorylation. CONCLUSIONS: Surface plasmon resonance is a sensitive method for detecting small differences in affinity. We were successful in using this technique to detect differences in the affinity of phosphorylated and nonphosphorylated rhodopsin peptides for visual arrestin. The data suggest that these are low-affinity interactions and indicate that phosphorylation is responsible for the direct binding of the rhodopsin carboxyl terminus to visual arrestin. Four phosphorylated residues are sufficient for this interaction. Because the affinity of the synthetic phosphopeptide for arrestin is substantially lower than the full length rhodopsin molecule, the cytoplasmic loops and rhodopsin carboxyl terminus appear to interact in a cooperative manner to stably bind arrestin.

Human substance P receptor undergoes agonist-dependent phosphorylation by G protein-coupled receptor kinase 5 in vitro.

G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied G protein-coupled receptors, leading to receptor desensitization. Seven GRKs, designated GRK1 through 7, have been characterized. GRK5 is negatively regulated by protein kinase C. We investigated whether human substance P receptor (hSPR) is a substrate of GRK5. We report that membrane-bound hSPR is phosphorylated by purified GRK5, and that both the rate and extent of phosphorylation increase dramatically in the presence of substance P. The phosphorylation has a high stoichiometry (20+/-4 mol phosphate/mol hSPR) and a low K(m) (1.7+/-0.1 nM). These data provide the first evidence that hSPR is a substrate of GRK5.