The zeolite ZSM-5 has been synthesized from amorphous aluminosilicate gels in a vapour of ethylenediamine, triethylamine, and water and characterized by XRD and chemical analysis.

An emerging theme in cell signaling is that membrane-bound channels and receptors are organized into supramolecular signaling complexes for optimum function and cross-talk. In this study, we determined how protein kinase C (PKC) phosphorylation influences the scaffolding protein Na ؉ /H ؉ exchanger regulatory factor 1 (NHERF) to assemble protein complexes of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel that controls fluid and electrolyte transport across cell membranes. NHERF directs polarized expression of receptors and ion transport proteins in epithelial cells, as well as organizes the homo-and hetero-association of these cell surface proteins. NHERF contains two modular PDZ domains that are modular protein-protein interaction motifs, and a C-terminal domain. Previous studies have shown that NHERF is a phosphoprotein, but how phosphorylation affects NHERF to assemble macromolecular complexes is unknown. We show that PKC phosphorylates two amino acid residues Ser-339 and Ser-340 in the C-terminal domain of NHERF, but a serine 162 of PDZ2 is specifically protected from being phosphorylated by the intact C-terminal domain. PKC phosphorylation-mimicking mutant S339D/S340D of NHERF has increased affinity and stoichiometry when binding to C-CFTR. Moreover, solution small angle x-ray scattering indicates that the PDZ2 and C-terminal domains contact each other in NHERF, but such intramolecular domain-domain interactions are released in the PKC phosphorylation-mimicking mutant indicating that PKC phosphorylation disrupts the autoinhibition interactions in NHERF. The results demonstrate that the C-terminal domain of NHERF functions as an intramolecular switch that regulates the binding capability of PDZ2, and thus controls the stoichiometry of NHERF to assemble protein complexes.

Na؉ /H ؉ exchanger regulatory factor (NHERF) is an adapter protein that is responsible for organizing a number of cell receptors and channels. NHERF contains two amino-terminal PDZ (postsynaptic density 95/disk-large/zonula occluden-1) domains that bind to the cytoplasmic domains of a number of membrane channels or receptors. The carboxyl terminus of NHERF interacts with the FERM domain (a domain shared by protein 4.1, ezrin, radixin, and moesin) of a family of actin-binding proteins, ezrin-radixin-moesin. NHERF was shown previously to be capable of enhancing the channel activities of cystic fibrosis transmembrane conductance regulator (CFTR). Here we show that binding of the FERM domain of ezrin to NHERF regulates the cooperative binding of NHERF to bring two cytoplasmic tails of CFTR into spatial proximity to each other. We find that ezrin binding activates the second PDZ domain of NHERF to interact with the cytoplasmic tails of CFTR (C-CFTR), so as to form a specific 2:1:1 (C-CFTR) 2 ⅐NHERF⅐ezrin ternary complex. Without ezrin binding, the cytoplasmic tail of CFTR only interacts strongly with the first amino-terminal PDZ domain to form a 1:1 C-CFTR⅐NHERF complex. Immunoprecipitation and immunoblotting confirm the specific interactions of NHERF with the full-length CFTR and with ezrin in vivo. Because of the concentrated distribution of ezrin and NHERF in the apical membrane regions of epithelial cells and the diverse binding partners for the NHERF PDZ domains, the regulation of NHERF by ezrin may be employed as a general mechanism to assemble channels and receptors in the membrane cytoskeleton.Na ϩ /H ϩ exchanger regulator factor (NHERF) 2 is an adaptor protein that is responsible for organizing membrane channels and receptors (1, 2). NHERF was originally identified as an essential cofactor for inhibiting a transmembrane transporter sodium-hydrogen exchanger isoform 3 (NHE3) by the cAMP-dependent protein kinase A in the kidney proximal tubule cells (1). However, subsequent studies find that NHERF is densely distributed in the apical membranes of polarized epithelial cells of several mammalian tissues (2, 3) and that NHERF participates in organizing the trafficking, localization, and membrane targeting of a large number of membrane receptors and channels to which NHERF binds (3)(4)(5)(6)(7)(8)(9)(10)(11)(12).NHERF is a multidomain and multivalent protein that recruits different signaling partners. The amino terminus of NHERF contains two modular PDZ (name derived from the first three proteins that this domain was identified postsynaptic density 95/disk-large/zonula occluden-1) domains, PDZ1 and PDZ2 (see Fig. 1). The NHERF PDZ domains bind to the consensus PDZ-binding motif D(S/T)X(V/I/L) (X denoting any amino acid residue) at the carboxyl termini of a number of membrane channels or receptors (13)(14)(15)(16)(17)(18)(19). The carboxyl terminus of NHERF recognizes the FERM domain (a conserved domain that is shared by protein 4.1, ezrin, radixin, and moesin) of a family of cytoskeletal actin-binding proteins, ez...

In recent years, it has become clear that in many proteins, significant regions are encoded by amino acid sequences that do not automatically fold into their fully condensed, functional structures. Characterization of the conformational propensities and function of the nonglobular protein sequences represents a major challenge. Striking among proteins with unfolded regions are numbers of transcription factors, including steroid receptors. In many cases, the unfolded or partially folded regions of such proteins take shape when the protein interacts with its proper binding partner(s), that is, the molecules to which it must bind to carry out its function. The AF1 domain of the androgen receptor (AR) shows little structure, when expressed as a recombinant peptide. It has been shown previously that AF1 interacts with transcription factor TFIIF in vitro. Using Fourier transform infrared (FTIR), we tested whether this interaction can induce structure in the AR AF1. Our results demonstrate that the recombinant AR AF1 can acquire significantly higher helical content after interacting with RAP74, a subunit of the TFIIF complex. We further show that this induced conformation in the AR AF1 is well-suited for its interaction with SRC-1.

Amino acid substitutions at distant sites in the Escherichia coli cyclic AMP receptor protein (CRP) have been shown to affect both the nature and magnitude of the energetics of cooperativity of cAMP binding, ranging from negative to positive. In addition, the binding to DNA is concomitantly affected. To correlate the effects of amino acid substitutions on the functional energetics and global structural properties in CRP, the partial specific volume (v(o)), the coefficient of adiabatic compressibility (beta(s)(o)), and the rate of amide proton exchange were determined for the wild-type and eight mutant CRPs (K52N, D53H, S62F, T127L, G141Q, L148R, H159L, and K52N/H159L) by using sound velocity, density measurements, and hydrogen-deuterium exchange as monitored by Fourier transform infrared spectroscopy at 25 degrees C. These mutations induced large changes in v(o) (0.747-0.756 mL/g) and beta(s)(o) (6.89-9.68 Mbar(-1)) compared to the corresponding values for wild-type CRP (v(o)= 0.750 mL/g and beta(s)(o)= 7.98 Mbar(-1)). These changes in global structural properties correlated with the rate of amide proton exchange. A linear correlation was established between beta(s)(o) and the energetics of cooperativity of binding of cAMP to the high-affinity sites, regardless of the nature of cooperativity, be it negative or positive. This linear correlation indicates that the nature and magnitude of cooperativity are a continuum. A similar linear correlation was established between compressibility and DNA binding affinity. In addition, linear correlations were also found among the dynamics of CRP and functional energetics. Double mutation (K52N/H159L) at positions 52 and 159, whose alpha-carbons are separated by 34.6 A, showed nonadditive effects on v(o) and beta(s)(o). These results demonstrate that a small alteration in the local structure due to amino acid substitution is dramatically magnified in the overall protein dynamics which plays an important role in modulating the allosteric behavior of CRP.

The mammalian Na؉ /H ؉ exchange regulatory factor 1 (NHERF1) is a multidomain scaffolding protein essential for regulating the intracellular trafficking and macromolecular assembly of transmembrane ion channels and receptors. NHERF1 consists of tandem PDZ-1, PDZ-2 domains that interact with the cytoplasmic domains of membrane proteins and a C-terminal (CT) domain that binds the membrane-cytoskeleton linker protein ezrin. NHERF1 is held in an autoinhibited state through intramolecular interactions between PDZ2 and the CT domain that also includes a C-terminal PDZ-binding motif (-SNL). We have determined the structures of the isolated and tandem PDZ2CT domains by high resolution NMR using small angle x-ray scattering as constraints. The PDZ2CT structure shows weak intramolecular interactions between the largely disordered CT domain and the PDZ ligand binding site. The structure reveals a novel helix-turn-helix subdomain that is allosterically coupled to the putative PDZ2 domain by a network of hydrophobic interactions. This helical subdomain increases both the stability and the binding affinity of the extended PDZ structure. Using NMR and small angle neutron scattering for joint structure refinement, we demonstrate the release of intramolecular domain-domain interactions in PDZ2CT upon binding to ezrin. Based on the structural information, we show that human disease-causing mutations in PDZ2, R153Q and E225K, have significantly reduced protein stability. Loss of NHERF1 expressed in cells could result in failure to assemble membrane complexes that are important for normal physiological functions.

A number of transcription factor proteins contain domains that are fully or partially unstructured. The means by which such proteins acquire naturally folded conformations are not well understood. When they encounter their proper binding partner(s), several of these proteins adopt a folded conformation through an induced-fit mechanism. The glucocorticoid receptor (GR) is a ligand-activated transcription factor. Expressed independently as a recombinant peptide, the N-terminal transactivation domain (AF1) of the GR shows little structure and appears to exist as a collection of random coil configurations. The GR AF1 is known to interact with other transcription factors, including a critical component of the general transcription machinery proteins, the TATA box binding protein (TBP). We tested whether this interaction can lead to acquisition of structure in the GR AF1. Our results show that recombinant GR AF1 acquires a significant amount of helical content when it interacts with TBP. These structural changes were monitored by Fourier transform infrared and NMR spectroscopies, and by proteolytic digestions. Our results support a model in which TBP binding interaction with the GR AF1 induces significantly greater helical structure in the AF1 domain. This increased helical content in the GR AF1 appears to come mostly at the expense of random coil conformation. These results are in accordance with the hypothesis that an induced-fit mechanism gives structure to the GR AF1 when it encounters TBP.glucocorticoid receptor ͉ N-terminal activation function ͉ coregulatory protein ͉ protein folding T he glucocorticoid receptor (GR) is a ligand-activated transcription factor with the domain structural arrangement typical of the nuclear hormone receptors superfamily (1-4). The GR regulates transcription of target genes by binding DNA at specific hormone response elements and͞or by interacting with other transcription factors (5-8). Although the structural organization of steroid receptors into N-terminal domain (NTD), DNA binding domain (DBD), and ligand binding domain is well characterized (9-14), precisely how transcription is regulated by them is largely unknown. For all steroid receptors, this is in part due to the lack of information about their transcription activating domain AF1, located in the NTD (9, 10). The GR AF1 sequence resembles those of acidic transactivation domains of several transcription factors (15, 16). When expressed independently, the GR AF1 shows little structure and seems to exist as an ensemble of largely unstructured conformers (17, 18). The GR AF1 is known to interact with other transcription factors, and conditional folding has been suggested to be the key for these interactions (19)(20)(21)(22).It has been reported that in the presence of trifluoroethanol, the ''core'' of AF1 (AF1 c , amino acids 187-242), located toward the C-terminal end of AF1, adopts three helical segments (17). Independent experiments have shown that substitution of the ␣-helixbreaking amino acid proline for natural residues a...

Scaffolding proteins are molecular switches that control diverse signaling events. The scaffolding protein NHERF1 assembles macromolecular signaling complexes and regulates the macromolecular assembly, localization and intracellular trafficking of a number of membrane ion transport proteins, receptors, and adhesion/antiadhesion proteins. NHERF1 begins with two modular protein-protein interaction domains—PDZ1 and PDZ2—and ends with a C-terminal domain. This C-terminal domain binds to ezrin, which in turn interacts with cytosekeletal actin. Remarkably, ezrin binding to NHERF1 increases the binding capabilities of both PDZ domains. Here we use deuterium labeling and contrast variation neutron scattering experiments to determine the conformational changes in NHERF1 when it forms a complex with ezrin. Upon binding to ezrin, NHERF1 undergoes significant conformational changes in the region linking PDZ2 and its C-terminal ezrin-binding domain, as well as in the region linking PDZ1 and PDZ2, involving very long-range interactions over 120 Å. The results provide a structural explanation, at mesoscopic scales, of the allosteric control of NHERF1 by ezrin as it assembles protein complexes. Because of the essential roles of NHERF1 and ezrin in intracellular trafficking in epithelial cells, we hypothesize that this long-range allosteric regulation of NHERF1 by ezrin enables the membrane-cytoskeleton to assemble protein complexes that control cross-talk and regulate the strength and duration of signaling.