Marcia E. Newcomer

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.

Insights from the X-ray crystal structure of coral 8R-lipoxygenase: calcium activation via a C2-like domain and a structural basis of product chirality.

Lipoxygenases (LOXs) catalyze the regio- and stereospecific dioxygenation of polyunsaturated membrane-embedded fatty acids. We report here the 3.2 Å resolution structure of 8R-LOX from the Caribbean sea whip coral Plexaura homomalla, a LOX isozyme with calcium dependence and the uncommon R chiral stereospecificity. Structural and spectroscopic analyses demonstrated calcium binding in a C2-like membrane-binding domain, illuminating the function of similar amino acids in calcium-activated mammalian 5-LOX, the key enzyme in the pathway to the pro-inflammatory leukotrienes. Mutation of Ca2+-ligating amino acids in 8R-LOX resulted not only in a diminished capacity to bind membranes, as monitored by fluorescence resonance energy transfer, but also in an associated loss of Ca2+-regulated enzyme activity. Moreover, a structural basis for R chiral specificity is also revealed; creation of a small oxygen pocket next to Gly428 (Ala in all S-LOX isozymes) promoted C-8 oxygenation with R chirality on the activated fatty acid substrate.

Structure of the epididymal retinoic acid binding protein at 2.1 Å resolution

BACKGROUND Androgen-dependent proteins in the lumen of the epididymis are required for sperm maturation. One of these is a retinoic acid binding protein, E-RABP, which binds both all-trans and 9-cis retinoic acid. The other retinoid-binding proteins whose structures are known do not bind 9-cis retinoids. RESULTS We describe the X-ray structure determination of E-RABP with and without bound ligand. The ligand binds deep in the beta-barrel of the protein, the beta-ionone ring innermost. The binding site, like the ligand, is amphipathic and the deepest part of the cavity is formed by a ring of aromatic amino acids. The isoprene tail of all-trans retinoic acid is bound in a folded conformation which resembles that of the 9-cis isomer. CONCLUSION E-RABP achieves high-affinity binding of both all-trans and 9-cis isomers of retinoic acid by forcing the all-trans form to bind in a folded conformation. The RAR family of nuclear receptors for retinoic acid also binds both isomers, and their binding sites may therefore be similar.

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 Å 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.

Structure and Function of Retinoid-Binding Proteins

Biological processes which require compounds derived from vitamin A (retinol) include morphogenesis, spermatogenesis, the maintenance of epithelial tissue, and vision. For the last process the light-activated cis-trans isomerization of rhodopsin-bound retinaldehyde triggers a cascade of events which ultimately results in the hydrolysis of cyclic GMP. For morphogenesis, spermatogenesis, and the maintenance of epithelial tissue it is presumably retinoic acid that is the active metabolite. Both 9-cis and all-trans retinoic acid are transcriptional activators which exert their effects through binding to nuclear proteins that are members of the superfamily that includes the steroid hormone receptors. Various proteins mediate the transport and metabolism of the hydrophobic vitamin and consequently modulate the concentration of retinoic acid available to the nucleus. Both extracellular and intracellular retinoid-binding proteins have been identified in numerous cell types. In addition, the eye has retinoid binding proteins which are distinct for that organ: a cellular retinaldehyde-binding protein (CRalBP) and a interstitial/interphotoreceptor retinoid-binding protein (I-RBP).

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.

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).

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.

Detection of Conformational Changes in Cellular Retinoid-Binding Proteins by Limited Proteolysis

Partial proteolysis of an undenatured protein is a wtdely used, powerful technique to probe protein conformation in the native state. The basts for thts technique is that the more exposed an amino-acid residue is to the solvent, the easier it 1s for a protease to cleave a peptide bond at that site (1,2). Therefore, regions of a protein with an extended conformation, such as those found in large multidomain proteins, are better substrates for proteolysis than are more tightly folded motifs. Using thus technique, it 1s possible to define protein domains, because the flexible regions between them are more susceptible to proteolysis. It is possible to further define these domains by obtaining N-terminal amino acid sequence of the resulting fragments. Likewise, by momtoring altered susceptibility to proteolysis, changes in protein conformation may be detected. If partial sequence of the resulting proteolytic fragments is obtained, the regions of the protein involved in these conformational changes can be mapped. Generally, the smaller the protein, the more resistant rt is to proteolysts. Changes in protein structure may expose (or hide) residues from attack. A number of factors may induce these structural changes, such as the binding of substrates or cofactors, heat, denaturants, and stabihzmg compounds such as DMSO and glycerol. Protease specificity can also affect the rate of proteolysis. Enzymes that recognize sites that include hydrophobic residues are less likely to cleave native structures than proteases that cleave bonds adjacent to charged side chains. The size of both the proteinase and its active site also influence the