Histidine switch controlling pH-dependent protein folding and DNA binding in a transcription factor at the core of synthetic network devices.

Therapeutic strategies have been reported that depend on synthetic network devices in which a urate-sensing transcriptional regulator detects pathological levels of urate and triggers production or release of urate oxidase. The transcription factor involved, HucR, is a member of the multiple antibiotic resistance (MarR) protein family. We show that protonation of stacked histidine residues at the pivot point of long helices that form the scaffold of the dimer interface leads to reversible formation of a molten globule state and significantly attenuated DNA binding at physiological temperatures. We also show that binding of urate to symmetrical sites in each protein lobe is communicated via the dimer interface. This is the first demonstration of regulation of a MarR family transcription factor by pH-dependent interconversion between a molten globule and a compact folded state. Our data further suggest that HucR may be utilized in synthetic devices that depend on detection of pH changes.

Effect of pH and salt bridges on structural assembly: molecular structures of the monomer and intertwined dimer of the Eps8 SH3 domain.

The SH3 domain of Eps8 was previously found to form an intertwined, domain-swapped dimer. We report here a monomeric structure of the EPS8 SH3 domain obtained from crystals grown at low pH, as well as an improved domain-swapped dimer structure at 1.8 A resolution. In the domain-swapped dimer the asymmetric unit contains two "hybrid-monomers." In the low pH form there are two independently folded SH3 molecules per asymmetric unit. The formation of intermolecular salt bridges is thought to be the reason for the formation of the dimer. On the basis of the monomer SH3 structure, it is argued that Eps8 SH3 should, in principle, bind to peptides containing a PxxP motif. Recently it was reported that Eps8 SH3 binds to a peptide with a PxxDY motif. Because the "SH3 fold" is conserved, alternate binding sites may be possible for the PxxDY motif to bind. The strand exchange or domain swap occurs at the n-src loops because the n-src loops are flexible. The thermal b-factors also indicate the flexible nature of n-src loops and a possible handle for domain swap initiation. Despite the loop swapping, the typical SH3 fold in both forms is conserved structurally. The interface of the acidic form of SH3 is stabilized by a tetragonal network of water molecules above hydrophobic residues. The intertwined dimer interface is stabilized by hydrophobic and aromatic stacking interactions in the core and by hydrophilic interactions on the surface.

A helical lid converts a sulfotransferase to a dehydratase.

We report here the crystal structure of retinol dehydratase, an enzyme that catalyzes the synthesis of anhydroretinol. The enzyme is a member of the sulfotransferase superfamily and its crystal structure reveals the insertion of a helical lid into a canonical sulfotransferase fold. Site-directed mutations demonstrate that this inserted lid is necessary for anhydroretinol production but not for sulfonation; thus, insertion of a helical lid can convert a sulfotransferase into a dehydratase.

Plasma retinol binding protein: structure and function of the prototypic lipocalin.

In terms of both structure and biological function, retinol binding protein (RBP) is one of the best characterized members of the lipocalin superfamily. The molecular interactions in which RBP participates are described herein.

Crystallization of retinol dehydratase from Spodoptera frugiperda: improvement of crystal quality by modification by ethylmercurythiosalicylate.

Retinol dehydratase is a sulfotransferase which is presumed to catalyze the dehydration of its substrate via a transient retinyl sulfate intermediate. Crystals (space group P2(1), unit-cell parameters a = 82.05, b = 66.61, c = 84.90 A, beta = 111.29 degrees ) are significantly improved by covalent modification of the protein with ethylmercury.

Protein and ligand adaptation in a retinoic acid binding protein.

A retinoic acid binding protein isolated from the lumen of the rat epididymis (ERABP) is a member of the lipocalin superfamily. ERABP binds both the all-trans and 9-cis isomers of retinoic acid, as well as the synthetic retinoid (E)-4-[2-(5,6,7,8)-tetrahydro-5,5,8,8-tetramethyl-2 napthalenyl-1 propenyl]-benzoic acid (TTNPB), a structural analog of all-trans retinoic acid. The structure of ERABP with a mixture of all-trans and 9-cis retinoic acid has previously been reported. To elucidate any structural differences in the protein when bound to the all-trans and 9-cis isomers, the structures of all-trans retinoic acid-ERABP and 9-cis retinoic acid ERABP were determined. Our results indicate that the all-trans isomer of retinoic acid adopts an 8-cis structure in the binding cavity with no concomitant conformational change in the protein. The structure of TTNPB-ERABP is also reported herein. To accommodate this all-trans analog, which cannot readily adopt a cis-like structure, alternative positioning of critical binding site side chains is required. Consequently, both protein and ligand adaption are observed in the formation of the various holo-proteins.

The structure of retinal dehydrogenase type II at 2.7 A resolution: implications for retinal specificity.

Retinoic acid, a hormonally active form of vitamin A, is produced in vivo in a two step process: retinol is oxidized to retinal and retinal is oxidized to retinoic acid. Retinal dehydrogenase type II (RalDH2) catalyzes this last step in the production of retinoic acid in the early embryo, possibly producing this putative morphogen to initiate pattern formation. The enzyme is also found in the adult animal, where it is expressed in the testis, lung, and brain among other tissues. The crystal structure of retinal dehydrogenase type II cocrystallized with nicotinamide adenine dinucleotide (NAD) has been determined at 2.7 A resolution. The structure was solved by molecular replacement using the crystal structure of a mitochondrial aldehyde dehydrogenase (ALDH2) as a model. Unlike what has been described for the structures of two aldehyde dehydrogenases involved in the metabolism of acetaldehyde, the substrate access channel is not a preformed cavity into which acetaldehyde can readily diffuse. Retinal dehydrogenase appears to utilize a disordered loop in the substrate access channel to discriminate between retinaldehyde and short-chain aldehydes.

The structure of human retinol-binding protein (RBP) with its carrier protein transthyretin reveals an interaction with the carboxy terminus of RBP.

Whether ultimately utilized as retinoic acid, retinal, or retinol, vitamin A is transported to the target cells as all-trans-retinol bound to retinol-binding protein (RBP). Circulating in the plasma, RBP itself is bound to transthyretin (TTR, previously referred to as thyroxine-binding prealbumin). In vitro one tetramer of TTR can bind two molecules of retinol-binding protein. However, the concentration of RBP in the plasma is limiting, and the complex isolated from serum is composed of TTR and RBP in a 1 to 1 stoichiometry. We report here the crystallographic structure at 3.2 A of the protein-protein complex of human RBP and TTR. RBP binds at a 2-fold axis of symmetry in the TTR tetramer, and consequently the recognition site itself has 2-fold symmetry: Four TTR amino acids (Arg-21, Val-20, Leu-82, and Ile-84) are contributed by two monomers. Amino acids Trp-67, Phe-96, and Leu-63 and -97 from RBP are flanked by the symmetry-related side chains from TTR. In addition, the structure reveals an interaction of the carboxy terminus of RBP at the protein-protein recognition interface. This interaction, which involves Leu-182 and Leu-183 of RBP, is consistent with the observation that naturally occurring truncated forms of the protein are more readily cleared from plasma than full-length RBP. Complex formation prevents extensive loss of RBP through glomerular filtration, and the loss of Leu-182 and Leu-183 would result in a decreased affinity of RBP for TTR.

Purification, crystallization and preliminary X-ray diffraction studies of retinal dehydrogenase type II.

One enzyme which catalyzes the last step of the formation of the hormone retinoic acid from vitamin A (retinol) is retinal dehydrogenase type II (Ra1DH2). Ra1DH2, expressed in the Escherichia coli BL21(DE3) strain, was purified and crystallized using ammonium sulfate as a precipitant. These crystals belong to the space group P212121 (a = 108, b = 150, c = 168 A, alpha = beta = gamma = 90 degrees).

Crystal structure of apo-glycine N-methyltransferase (GNMT).

The crystal structure of the recombinant apo-form of glycine N-methyltransferase (GNMT) has been determined at 2.5 A resolution. GNMT is a tetrameric enzyme (monomer Mr = 32,423Da, 292 amino acids) that catalyzes the transfer of a methyl group from S-adenosylmethionine (AdoMet) to glycine with the formation of S-adenosylhomocysteine (AdoHcy) and sarcosine (N-methylglycine). GNMT is a regulatory enzyme, which is inhibited by 5-methyltetrahydrofolate pentaglutamate and believed to control the ratio of AdoMet to AdoHcy in tissues. The crystals belong to the orthorhombic space group P2(1)2(1)2 (a = 85.39, b = 174.21, c = 44.71 A) and contain one dimer per asymmetric unit. The AdoMet-GNMT structure served as the starting model. The structure was refined to an R-factor of 21.9%. Each monomer is a three-domain structure with a large cavity enclosed by the three domains. The tetramer resembles a square with a central channel about which N-terminal domains are intertwined. Only localized changes of the residues involved in the binding pocket are observed for the apo-GNMT structure when compared to that determined in the presence of substrate and substrate analog.

Molecular cloning and hormonal regulation of a murine epididymal retinoic acid-binding protein messenger ribonucleic acid.

A complementary DNA encoding the mouse epididymal secretory protein MEP 10 (mouse epididymal protein 10) was cloned and is now renamed murine epididymal retinoic acid binding protein (mE-RABP). The analysis of the predicted primary amino acid sequence showed that mE-RABP has a 75% identity with rat ESP I (epididymal secretory protein I), another epididymal retinoic acid-binding protein. The homology strongly suggests that mE-RABP is the mouse orthologue of rat ESP I. A computer analysis of the predicted three-dimensional structure confirmed that mE-RABP can accommodate retinoic acid as ligand. In the rat, ESP I messenger RNA (mRNA) is expressed in the efferent ducts and in the entire caput epididymidis. However, in the mouse, the expression of a 950-bp mE-RABP mRNA was detected only in principal cells of the mid/distal caput epididymidis, suggesting that the regulation of region-specific expression is different in rat and mouse. Northern blot analyses showed that mE-RABP gene expression is no longer detected 10 days after castration but progressively rebounds between days 15 and 60. However, mE-RABP protein could not be detected by Western blot 30 days after castration. Androgen replacement, begun 5 days after castration and continued for 4 days restored significant expression of mE-RABP mRNA. Efferent duct ligation for 10 days did not affect gene expression. Taken together, these results indicate that mE-RABP mRNA expression is regulated by androgens but not by testicular factors. The overall similarity in the primary amino acid sequence of mE-RABP with ESP I and other members of the lipocalin superfamily suggests that they are evolutionarily related.

Structure and putative function of a murine epididymal retinoic acid-binding protein (mE-RABP).

Vitamin A is required to maintain the epididymal epithelium. In this report, the characterization and putative functions of a murine epididymal retinoic acid-binding protein (mE-RABP) that is secreted into the lumen from the mid-/distal caput epididymidis are discussed. The amino acid sequence analysis of the mE-RABP preprotein shows that mE-RABP is the mouse orthologue of the rat epididymal secretory protein I (ESPI). These proteins belong to the lipocalin superfamily and bind to active retinoids but not to retinol. Therefore, we propose that mE-RABP may function as an extracellular retinoid carrier-protein involved in the paracrine regulation of epididymal function by retinoids.

The SH3 domain of Eps8 exists as a novel intertwined dimer.

SH3 domains are structurally well-characterized as monomeric modular units of protein structure that mediate protein-protein recognition in numerous signal transduction proteins. The X-ray crystallographic structure of the Eps8 SH3 domain reveals a novel variation of the canonical SH3 fold: the SH3 domain from Eps8 is a dimer formed by strand interchange. In addition, co-immunoprecipitation experiments show that intact Eps8 is multimeric in vivo. Hence, the SH3 domain of Eps8 may represent a dimerization motif.

Test of the contribution of an amino-aromatic hydrogen bond to protein function.

Hydrogen bonds which form between a hydrogen bond donor and an aromatic ring as acceptor are thought to contribute to the stability and function of proteins. We have tested the function of such an interaction in a highly homologous pair of proteins, cellular retinol-binding protein (CRBP) and cellular retinol-binding protein, type II [CRBP(II)]. Both proteins bind the ligand all-trans-retinal with comparable affinities, but CRBP has an approximately 100-fold higher affinity for all-trans retinal. The greater affinity of CRBP for all-trans-retinol has been attributed to the presence of an amino-aromatic hydrogen bond, which is absent in CRBP(II). We have generated a pair of mutant proteins, in which the amino-aromatic interaction was removed from CRBP and introduced into CRBP(II). Spectral analyses of retinol when bound to the wild-type and mutant CRBP suggested that it adopted an identical conformation within both proteins, a conformation that was distinct from that of retinol bound to CRBP(II), both wild-type and mutant. Unexpectedly, the affinities of the mutant binding proteins for all-trans-retinol were indistinguishable from those of their corresponding wild-type proteins. Further, in ligand competition experiments, there were no observable differences between mutant and wild-type CRBP, or between mutant and wild-type CRBP(II), in their preferences for binding all-trans-retinol versus all-trans-retinal. The results of this direct test of the proposed function of an amino-aromatic hydrogen bond did not support a functional role for such bonds, at least in this system.

Retinoid-binding proteins: structural determinants important for function.

The transport and functions of biologically active naturally occurring retinoids (Vitamin A, retinol, and its metabolites) are mediated by extracellular, intracellular, and nuclear proteins. X-ray crystallographic studies to date on the extra- and intracellular proteins have helped to define distinct protein retinoid recognition mechanisms, each with a characteristic structural motif. The extracellular proteins (serum retinol-binding protein and a retinoic acid-binding protein from rat epididymis) bind retinoids with a hand-in-glove like fit in deep, hydrophobic-binding cavities. The intracellular proteins (cellular retinol-binding proteins types I and II) encapsulate the ligand in an aqueous internal cavity. The details of the mechanisms of retinoid recognition, and how they result as a consequence of the different protein structures, are described in this review.

Cellular retinoid-binding proteins: limited proteolysis reveals a conformational change upon ligand binding.

Intracellular retinoid-binding proteins are small, tightly folded, compact proteins, which appear to be involved in the delivery of retinoids to microsomal metabolic enzymes, among other potential roles. Recently, it has been demonstrated that two of these binding proteins, cellular retinol-binding protein (CRBP) and cellular retinol-binding protein type II [CRBP(II)], interact with the same microsomal enzyme but in different manners, depending on the absence or presence of ligand [Herr, F.M., & Ong, D.E. (1992) Biochemistry 31, 6748-6755]. The structural components of the binding proteins responsible for these differential interactions are presently unknown. In addition, it is not clear how the ligand is able to gain entry into the solvent-inaccessible interior binding cavity. Limited proteolysis of the apo and holo forms of CRBP and CRBP(II) was used to probe the conformational differences between the different states of these two proteins in solution. It was found that the apo forms of both proteins were significantly more susceptible to proteolysis, and probably adopted a more open conformation, than the holo forms. The initial cleavage site of endoproteinase Arg-C in the apo forms occurred at a conserved arginine residue near a possible site of ligand entry. Similar results were obtained by limited proteolysis of cellular retinoic acid-binding protein and heart fatty acid-binding protein, indicating that a common ligand-induced conformational change may occur for other members of this family of intracellular binding proteins.(ABSTRACT TRUNCATED AT 250 WORDS)