Florante A. Quiocho

Sulfate-binding protein dislikes protonated oxyacids. A molecular explanation.

We have determined the effect of pH on the binding affinities of the conjugate bases of four different tetrahedral oxyacids to the sulfate-binding protein. The equilibrium dissociation constants of the binding of sulfate (Kd = 0.12 microM) and selenate (Kd = 5 microM) were found to be pH independent over the range pH 5 to pH 8.1, whereas chromate binding exhibited a pH dependence that is approximately attributable to the pK2 of the chromic acid. Phosphate was bound with an affinity five orders of magnitude weaker than that of sulfate. In light of the highly refined 2 A structure of the complex of the sulfate-binding protein with sulfate, and considering the protonation state and net charge of the various oxyacids, we conclude that the pH dependence of chromate binding and the extremely low affinity of phosphate are attributable mainly to a lack of hydrogen bond acceptors in the binding site. These studies demonstrate that the sulfate-binding site is stringently designed to bind tightly tetrahedral, fully ionized, oxyacid dianions. The presence of a donatable proton on the ligand reduces binding energy by approximately 7 kcal/mol.

Structure of the l-arabinose-binding protein from Escherichia coli at 2.4 Å resolution

The structure of l-arabinose-binding protein (Mr 33, 100), an essential component of the osmotic shock-sensitive, high-affinity l-arabinose transport system in Escherichia coli, has been determined at 2.4 A resolution. The phases were solved by the method of multiple isomorphous replacement, using four derivatives, p-chloromercuribenzenesulfonate and CdI2 (data to 2.4 A resolution), and p-chloromercurinitrophenol and (NH4)2PtCl4 to 3.5 A resolution. A final mean figure of merit of 0.65 was obtained for 9628 reflections. With the aid of the amino acid sequence determined by Hogg & Hermodson (1977), a complete model of the protein molecule has been determined using initially an optical comparator. The entire model was subsequently examined in detail using a computer graphic system. The protein molecule is ellipsoidal (axial ratio of 2:1), and consists of two globular domains (designated P and Q). Each domain is made from two separate polypeptide chain segments. Despite the discontinuity in the folding, the arrangements of the secondary structure in the two domains are very similar. Both domains contain a six-stranded parallel β-sheet (with the exception of the sixth anti-parallel strand in the Q domain) flanked by two α-helices on either side. The packing topology is α/β. A C-terminal helix is shared by both domains. The two domains show significant conformational similarity but lack sequence homology. A comparison of the two domains revealed that of the 139 α-carbons in the P domain and 152 in the Q domain, 92 were found to be equivalent with a root-mean-square distance of 2.6 A. The cleft formed by the packing of the two domains is predominantly lined with hydrophilic residues. The sugar-binding site is located in this cleft.

Preliminary crystallographic data for a leucine, isoleucine, valine-binding protein from Escherichia coli K12.

Single crystals of a leucine, isoleuoine, valine-binding protein, an essential component of the high affinity transport system for these amino acids in Escherichia coli K12, that are suitable for high resolution structural analysis have been obtained. The crystals belong to the space group P212121, unit cell dimensions: 40.1 A × 71.2 A × 115.9 A. The asymmetric unit contains one binding protein molecule of 36,000 molecular weight.

Dinucleotide fold proteins: Interaction of arabinose binding protein with Cibacron Blue 3G-A

Abstract Cibacron Blue 3G-A, the dye moiety of Blue dextran-Sepharose, has been shown to not specifically bind a protein with a dinucleotide fold-like supersecondary structure, L -arabinose binding protein from Escherichia coli. This shows that Cibacron Blue 3G-A is not suitable as a definitive probe for the dinucleotide fold as suggested earlier ( Thompson et al., 1975 ; Stellwagen, 1977 ). An explanation for the large predominance of proteins containing this protein supersecondary structure that bind to this dye is presented.

Sterol biosynthesis: Establishment of the structure of 3β-p-bromobenzoyloxy-5α-cholest-8(14)-en-15β-ol

Abstract Previous studies have established that hydride reduction of 3β-benzoyloxy-5α-cholest-8(14)-en-15-one yields two epimers (at C-15) of 5α-cholest-8(14)-en-3β,15-diol which were designated as diol A and B. Efficient enzymatic conversion of both compounds to cholesterol was observed. To determine the absolute configuration of the 15-OH function in the two compounds, the 3β- p -bromobenzoyl ester of diol B was prepared from 3β- p -bromobenzoyloxy-5α-cholest-8(14)-en-15-one by reduction with sodium borohydride. Crystals of the derivative were found to belong to the space group P1, with unit cell parameters; a = 9.24 A , b = 12.61 A , c = 7.03 A , α = 93.05°, β = 100.27°, γ = 90.82°, and one molecule per unit cell. Least-squares refinement of the structure was carried out to final R value of 0.14. The configuration of the hydroxyl group at the 15 position of diol B has been determined to be β.

Synthesis and crystal structure of 3β-p-bromobenzoyloxy-14α, 15α-epoxy-5α-cholest-7-ene

The chemical synthesis of 3beta-bromobenzoyloxy-14alpha, 15alpha-epoxy-5alpha-cholest-7-ene is described. Single crystal structral analysis was employed to unambiguously determine the location and absolute configuration of the epoxide moiety in the 3beta-p-bromobenzoyloxy-14alpha, 15alpha-epoxy-5alpha-cholest-7-ene. The space group of the crystal was P1, with unit cell parameters: a=10.873 A, b=13.841 A, c=11.037 A, alpha=75.19 degrees, beta=78.79 degrees, gamma=101.57 degrees, and two molecules per unit cell. Intitial phases were derived from the two bromine atoms. Least squares refinements on all non-hydrogen atoms were carried out to a final unweighted R value of 0.10 and weighted R value of 0.04. The epoxide ring was located at the 14, 15 position and was found to extend to the alpha side of the molecule. Molecular measurements for asymmetry parameters of the sterol nuceus indicate that ring A has a symmetrical chair conformation and ring B has a half chair conformation due to the double bond at C(7). Ring C has a fairly distorted chair conformation due to the trigonal C(8) on one sie and the almost planar 5-membered ring on the other. Ring D has the 17alpha-envelope conformation.

Crystallographic data of an l-arabinose-binding protein from Escherichia coli

Abstract Single crystals of an l -arabinose-binding protein from Escherichia coli B/r suitable for a high-resolution structural analysis have been obtained. An X-ray examination of the orthorhombic crystals shows the space group is P212121, with unit cell dimensions: a = 55.3 A , b = 71.4 A and c = 77.5 A . The asymmetric unit contains one protein molecule with a molecular weight of 38,000.

Preliminary crystallographic data of receptors for transport and chemotaxis in Escherichia coli: d-galactose and maltose-binding proteins

Abstract We have obtained single crystals of maltose-binding protein ( M r = 40,500) and d -galactose-binding protein ( M r = 32,000), chemoreceptors for active transport and chemotaxis in Escherichia coli . This brings to a total of five the binding proteins that we have thus far crystallized; they include the l -arabinose-binding protein, the leucine, isoleucine, valine-binding protein from Escherichia coli , and a sulfate-binding protein from Salmonella typhimurium . The crystal structure of the l -arabinose-binding protein has been determined at 2.8 A resolution (Quiocho et al. , 1977 a ).

Concerning the structure of 3β-benzoyloxy-5β-cholesta-8,14-diene, a major byproduct in the chemical synthesis of 5α-cholest-8(14)-en-3β-ol-15-one

The X-ray crystal structure of 3 beta-(p-bromobenzoyloxy)-5 beta-cholesta-8,14-diene (space group P21, a = 10.698 A, b = 9.487 A, c = 15.024 A, beta = 96.05 degrees, Z = 2) was determined by the heavy atom method and refined to R = 0.075. This heavy atom derivative was synthesized from 5 beta-cholesta-8,14-diene-3 beta-ol, the benzoate ester of which was previously shown to be the major byproduct in the low-temperature isomerization of 7-dehydrocholesteryl benzoate in HCl/chloroform. The work presented here establishes unequivocally that the configuration of this isomerization byproduct at C-5 is 5 beta-H and that the configuration at C-17 was unchanged.

Inhibitors of sterol synthesis. Concerning the structure of 15β-methyl-5α,14β-cholest-7-ene-3β,15α-diol, an inhibitor of cholesterol biosynthesis

The chemical synthesis of 3β-p-bromobenzoyloxy-15β-methyl-5α,14β-cholest-7-en-15α-ol from 15β-methyl-5α, l4β-cholest-7-ene-3β,15α-diol is described. The structure of the former compound was unambiguously determined by X-ray crystallographic analysis. The space group of the crystal was P21 with unit cell parameters a = 12.611 A, b = 9.826 A, c = 13.221 A, b = 91.71° and Z = 2. The structure was solved by the heavy atom method and refined to a final R of 0.041. Asymmetry parameters indicated that ring A is a symmetrical chair, that rings B and C are half chairs, and that ring D is a 15α-envelope.