A Leu----His substitution at residue 93 in human corticosteroid binding globulin results in reduced affinity for cortisol.

A steroid binding capacity assay and a radioimmunoassay were both used to measure corticosteroid binding globulin (CBG) in serum samples from 22 patients with sepsis. An approximately 50% discordancy between the two values in one patient suggested the presence of a CBG variant with reduced affinity for cortisol, and this was confirmed by Scatchard analysis. We therefore used the polymerase chain reaction to amplify exons that encode for human CBG from the genomic DNA of this patient. This revealed two mutations within the coding sequences: one of which results in a Leu----His substitution at residue 93 and another which encodes a Ser----Ala substitution at residue 224 of the human CBG polypeptide. To assess the impact of each substitution on the steroid binding affinity of CBG, each mutation was introduced separately into a normal human CBG cDNA, and the normal and mutated cDNAs were expressed in Chinese hamster ovary cells. Scatchard analysis of the CBG produced in culture indicated that the His93 mutation (Kd = 2.24 +/- 1.75 nM) reduced the cortisol binding affinity of CBG (mean +/- SD) significantly (P less than 0.024) when compared to normal CBG (Kd = 0.64 +/- 0.31 nM), while the Ala224 mutation (Kd = 0.63 +/- 0.33 nM) did not influence cortisol binding affinity. We therefore conclude that residue 93 may play an important role in determining the structure of the CBG steroid binding site.     

Single amino acid substitution of rat MRP2 results in acquired transport activity for taurocholate

Multidrug resistance-associated protein 3 (MRP3), unlike other MRPs, transports taurocholate (TC). The difference in TC transport activity between rat MRP2 and MRP3 was studied, focusing on the cationic amino acids in the transmembrane domains. For analysis, transport into membrane vesicles from Sf9 cells expressing wild-type and mutated MRP2 was examined. Substitution of Arg at position 586 with Leu and Ile and substitution of Arg at position 1096 with Lys, Leu, and Met resulted in the acquisition of TC transport activity, while retaining transport activity for glutathione and glucuronide conjugates. Substitution of Leu at position 1084 of rat MRP3 (which corresponds to Arg-1096 in rat MRP2) with Lys, but not with Val or Met, resulted in the loss of transport activity for TC and glucuronide conjugates. These results suggest that the presence of the cationic charge at Arg-586 and Arg-1096 in rat MRP2 prevents the transport of TC, whereas the presence of neutral amino acids at the corresponding position of rat MRP3 is required for the transport of substrates.     

Interaction between corticosteroid binding globulin and activated leukocytes in vitro

The interaction between human corticosteroid binding globulin and activated leukocytes is restricted to the granulocyte population, and is characterized by specific proteolytic cleavage of corticosteroid binding globulin which markedly reduces its steroid binding activity. A direct interaction between corticosteroid binding globulin and the activated cells appears to enhance this event, and does not involve cellular internalization of corticosteroid binding globulin or its proteolytic degradation products, which resemble those obtained after incubation of corticosteroid binding globulin with neutrophil elastase. These data suggest that corticosteroid binding globulin interacts with elastase on the surface of activated neutrophils, and may promote glucocorticoid delivery to these cells during inflammation.     

An amino acid substitution matrix for protein conformation identification

Amino acid substitution matrices play an essential role in protein sequence alignment, a fundamental task in bioinformatics. Most widely used matrices, such as PAM matrices derived from homologous sequences and BLOSUM matrices derived from aligned segments of PROSITE, did not integrate conformation information in their construction. There are a few structure-based matrices, which are derived from limited data of structure alignment. Using databases PDB_SELECT and DSSP, we create a database of sequence-conformation blocks which explicitly represent sequence-structure relationship. Members in a block are identical in conformation and are highly similar in sequence. From this block database, we derive a conformation-specific amino acid substitution matrix CBSM60. The matrix shows an improved performance in conformational segment search and homolog detection.     

The G464S amino acid substitution in Candida albicans sterol 14alpha-demethylase causes fluconazole resistance in the clinic through reduced affinity.

Fluconazole selectively inhibits fungal sterol 14alpha-demethylase, a cytochrome P450 enzyme found in plants, animals, fungi, and Mycobacteria. The mutation G464S, observed in the heme-binding domain of sterol 14alpha-demethylase in clinical strains of fluconazole-resistant Candida albicans, is shown here to cause resistance through substantially reducing the inhibitory effect of fluconazole and is associated with perturbation of the heme environment as indicated by spectral data. The protein exhibits 42% of the maximal enzymatic rate of the wild-type protein allowing continued production of the end product of fungal sterol biosynthesis, ergosterol, in resistant strains. This mutation may cause these phenotypes through altering the heme location, thus changing the ability of residues above the heme to bind the drug effectively. This perturbation would also account for the observation of reduced sterol demethylase catalytic activity by changing the location of the 14alpha-methyl group in relation to oxygen-bound heme during the catalytic cycle.     

An advanced affinity membrane for covalent binding of amino ligands

We report here an advanced, chemically active and yet hydrolytically stable microporous membrane which allows permanent covalent binding of amino ligands such as proteins. Rapid, single-step immobilizations produce a high density of immobilized ligands. Surface chemistry of the membrane is specifically designed to have extremely low nonspecific binding. Binding characteristics of the UltraBind membrane, various immobilization techniques and optimum immobilization conditions for diagnostic immunoassays are described.     

Homology model of human corticosteroid binding globulin: a study of its steroid binding ability and a plausible mechanism of steroid hormone release at the site of inflammation

Corticosteroid binding globulin (CBG) and thyroxin binding globulin (TBG) both belong to the same SERPIN superfamily of serine-proteinase inhibitors but in the course of evolution CBG has adapted to its new role as a transport agent of insoluble hormones. CBG binds corticosteroids in plasma, delivering them to sites of inflammation to modify the inflammatory response. CBG is an effective drug carrier for genetic manipulation, and hence there is immense biological interest in the location of the hormone binding site. The crystal structure of human CBG (hCBG) has not been determined, but sequence alignment with other SERPINs suggests that it conforms as a whole to the tertiary structure shared by the superfamily. Human CBG shares 52.15% and 55.50% sequence similarity with alpha1-antitrypsin and alpha1-antichymotrypsin, respectively. Multiple sequence alignment among the three sequences shows 73 conserved regions. The molecular structures of alpha1-antitrypsin and alpha1-antichymotrypsin, the archetype of the SERPIN superfamily, obtained by X-ray diffraction methods are used to develop a homology model of hCBG. Energy minimization was applied to the model to refine the structure further. The homology model of hCBG contains 371 residues (His13 to Val383 ). The secondary structure comprises 11 helices, 15 turns and 11 sheets. The putative corticosteroid binding region is found to exist in a pocket between beta-sheets S4, S10, S11 and alpha helix H10. Both cortisol and aldosterone are docked to the elongated hydrophobic ligand binding pocket with the polar residues at the two extremities. A difference accessible surface area (DASA) study revealed that cortisol binds with the native hCBG more tightly than aldosterone. Cleavage at the Val379-Met380 peptide bond causes a deformation of hCBG (also revealed through a DASA study). This deformation could probably trigger the release of the bound hormone. Figure Stereoscopic view of the ribbon diagram of hCBG complexed with cortisol. The bound cortisol is shown in space filling model in blue. Helices and sheets are shown in red and magenta respectively. Turns are shown in yellow.     

Hyporesponsiveness to glucocorticoids in mice genetically deficient for the corticosteroid binding globulin

Corticosteroid binding globulin (CBG) is the carrier for glucocorticoids in plasma. The protein is believed to keep the steroids inactive and to regulate the amount of free hormone acting on target tissues (free hormone hypothesis). Here, we generated a mouse model genetically deficient for CBG to test the contribution of the carrier to glucocorticoid action and adrenocortical stress response. The absence of CBG resulted in a lack of corticosterone binding activity in serum and in an approximately 10-fold increase in free corticosterone levels in CBG-null mice, consistent with its role in regulation of circulating free hormone levels. Surprisingly, cbg(-/-) animals did not exhibit features seen in organisms with enhanced glucocorticoid signaling. Rather, the mice exhibited increased activity of the pituitary axis of hormonal control, normal levels of gluconeogenetic enzymes, and fatigue, as well as an aggravated response to septic shock, indicating an inability to appropriately respond to the excess free corticosterone in the absence of CBG. Thus, our data suggest an active role for CBG in bioavailability, local delivery, and/or cellular signal transduction of glucocorticoids that extends beyond a function as a mere cargo transporter.     

Conversion of interleukin-13 into a high affinity agonist by a single amino acid substitution

We created a novel mutated form of human interleukin-13 (IL-13) in which a positively charged arginine (R) at position 112 was substituted to a negatively charged aspartic acid (D). This mutant, termed IL-13R112D, was expressed in Escherichia coli and purified to near homogeneity. IL-13R112D was found to be a potent IL-13 agonist with 5-10-fold improved binding affinity to IL-13 receptors compared with wild-type IL-13 (wtIL-13). The conclusion of IL-13 agonist activity was drawn on the basis of approximately 10-fold improved activity over wtIL-13 in several assays: (a) inhibition of CD14 expression in primary monocytes; (b) proliferation of TF-1 and B9 cell lines; and (c) activation of STAT6 in Epstein-Barr virus-immortalized B cells, primary monocytes, and THP-1 monocytic cell line. Furthermore, mutant IL-13R112D neutralized the cytotoxic activity of a chimeric fusion protein composed of wtIL-13 and a Pseudomonas exotoxin A (IL-13-PE38) approximately 10 times better than wtIL-13. Based on these results, it was concluded that IL-13R112D interacts with much stronger affinity than wtIL-13 on all cell types tested and that Arg-112 plays an important role in the interaction with its receptors (IL-13R). Thus, these results suggest that IL-13R112D may be a useful ligand for the study of IL-13 interaction with its receptors or, alternatively, in designing specific targeted agents for IL-13R-positive malignancies.     

A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity

The receptor binding specificity of influenza viruses may be important for host restriction of human and avian viruses. Here, we show that the hemagglutinin (HA) of the virus that caused the 1918 influenza pandemic has strain-specific differences in its receptor binding specificity. The A/South Carolina/1/18 HA preferentially binds the alpha2,6 sialic acid (human) cellular receptor, whereas the A/New York/1/18 HA, which differs by only one amino acid, binds both the alpha2,6 and the alpha2,3 sialic acid (avian) cellular receptors. Compared to the conserved consensus sequence in the receptor binding site of avian HAs, only a single amino acid at position 190 was changed in the A/New York/1/18 HA. Mutation of this single amino acid back to the avian consensus resulted in a preference for the avian receptor.     

An amino acid substitution determining G3m(g)

An allotypic G3m(g) marker-specific substitution was studied by sequence analysis of glycopeptides derived from myeloma proteins Ba (G3m(g+)) and Bu (G3m(g-)). The experimental results indicate that glutamic acid at position 295 is responsible for the specificity. Based on the results of chemical modification (Arg, Tyr, and Glu), this antigenic epitope is presumed to involve five sequential residues from Arg-292 to Tyr-296.     

A single amino acid substitution in yeast eIF-5A results in mRNA stabilization.

Most factors known to function in mRNA turnover are not essential for cell viability. To identify essential factors, approximately 4000 temperature-sensitive yeast strains were screened for an increase in the level of the unstable CYH2 pre-mRNA. At the non-permissive temperature, five mutants exhibited decreased decay rates of the CYH2 pre-mRNA and mRNA, and the STE2, URA5 and PAB1 mRNAs. Of these, the mutant ts1159 had the most extensive phenotype. Expression of the TIF51A gene (encoding eIF-5A) complemented the temperature-sensitive growth and mRNA decay phenotypes of ts1159. The tif51A allele was rescued from these cells and shown to encode a serine to proline change within a predicted alpha-helical segment of the protein. ts1159 also exhibited an approximately 30% decrease in protein synthesis at the restrictive temperature. Measurement of amino acid incorporation in wild-type cells incubated with increasing amounts of cycloheximide demonstrated that a decrease in protein synthesis of this magnitude could not account for the full extent of the mRNA decay defects observed in ts1159. Interestingly, the ts1159 cells accumulated uncapped mRNAs at the non-permissive temperature. These results suggest that eIF-5A plays a role in mRNA turnover, perhaps acting downstream of decapping.     

A single amino acid change (substitution of glutamate 3 with alanine) in the N-terminal region of rat liver carnitine palmitoyltransferase I abolishes malonyl-CoA inhibition and high affinity binding

We have recently shown by deletion mutation analysis that the conserved first 18 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) are essential for malonyl-CoA inhibition and binding (Shi, J., Zhu, H., Arvidson, D. N. , Cregg, J. M., and Woldegiorgis, G. (1998) Biochemistry 37, 11033-11038). To identify specific residue(s) involved in malonyl-CoA binding and inhibition of L-CPTI, we constructed two more deletion mutants, Delta12 and Delta6, and three substitution mutations within the conserved first six amino acid residues. Mutant L-CPTI, lacking either the first six N-terminal amino acid residues or with a change of glutamic acid 3 to alanine, was expressed at steady-state levels similar to wild type and had near wild type catalytic activity. However, malonyl-CoA inhibition of these mutant enzymes was reduced 100-fold, and high affinity malonyl-CoA binding was lost. A mutant L-CPTI with a change of histidine 5 to alanine caused only partial loss of malonyl-CoA inhibition, whereas a mutant L-CPTI with a change of glutamine 6 to alanine had wild type properties. These results demonstrate that glutamic acid 3 and histidine 5 are necessary for malonyl-CoA binding and inhibition of L-CPTI by malonyl-CoA but are not required for catalysis.     

Rat corticosteroid binding globulin: primary structure and messenger ribonucleic acid levels in the liver under different physiological conditions

Rat corticosteroid binding globulin (CBG) cDNAs were isolated from a lambda gt11 liver cDNA library. When rat hepatic mRNA was hybrid selected and translated in vitro, a major product reacted with antibodies against rat CBG and its Mr (approximately 43,000) was consistent with a nonglycosylated, CBG precursor polypeptide. Two overlapping cDNAs produced a 1,432 nucleotide sequence with an open reading frame comprising 396 amino acids. This includes a potential signal peptide of 22 residues followed by the amino terminus of purified rat CBG. Rat CBG therefore contains 374 amino acids (Mr = 42,196), and has six consensus sites for N-glycosylation. There is 60% identity in the primary structures of rat and human CBG over 383 residues that comprise the human sequence. Furthermore, the single cysteine in rat CBG corresponds to one of two cysteines in human CBG, and this may be significant because a cysteine is located in the human CBG steroid binding site. Northern analysis of RNA from various rat tissues revealed an approximate 1.8 kilobase CBG mRNA only in the liver. Its relative abundance in a pregnant rat was only 30% higher than in an adult female; approximately 3-fold higher than in an adult male, and 25-fold higher than in the fetuses from the same animal. Southern analysis of rat genomic DNA suggests the presence of a single gene for CBG.     

Antithrombin Budapest 3 An antithrombin variant with reduced heparin affinity resulting from the substitution L99F

The molecular basis and functional properties of a variant antithrombin (AT) protein. AT Budapest 3, were studied. A single base substitution was identified in codon 99, TC→TC, altering the normal leucine to phenylalanine. The proband presented with a history of venous thrombotic disease and was found to be homozygous for the mutation. The variant protein demonstrated reduced heparin affinity and reduced antiproteinase activity in the presence of either unfractionated heparin or the AT-binding heparin pentasaccharide, when compared to normal AT. A small change in the isoelectric point was also identified. The substituted amino acid residue of AT Budapest 3 is located near to the proposed AT heparin binding site, and it is suggested that reduced heparin affinity of the variant protein may result from substitution-induced distortion of positive charge geometry in the binding site and/or changes in its position relative to the rest of the inhibitor molecule.     

Host-selected amino acid changes at the sialic acid binding pocket of the parvovirus capsid modulate cell binding affinity and determine virulence

The role of receptor recognition in the emergence of virulent viruses was investigated in the infection of severe combined immunodeficient (SCID) mice by the apathogenic prototype strain of the parvovirus minute virus of mice (MVMp). Genetic analysis of isolated MVMp viral clones (n = 48) emerging in mice, including lethal variants, showed only one of three single changes (V325M, I362S, or K368R) in the common sequence of the two capsid proteins. As was found for the parental isolates, the constructed recombinant viruses harboring the I362S or the K368R single substitutions in the capsid sequence, or mutations at both sites, showed a large-plaque phenotype and lower avidity than the wild type for cells in the cytotoxic interaction with two permissive fibroblast cell lines in vitro and caused a lethal disease in SCID mice when inoculated by the natural oronasal route. Significantly, the productive adsorption of MVMp variants carrying any of the three mutations selected through parallel evolution in mice showed higher sensitivity to the treatment of cells by neuraminidase than that of the wild type, indicating a lower affinity of the viral particle for the sialic acid component of the receptor. Consistent with this, the X-ray crystal structure of the MVMp capsids soaked with sialic acid (N-acetyl neuraminic acid) showed the sugar allocated in the depression at the twofold axis of symmetry (termed the dimple), immediately adjacent to residues I362 and K368, which are located on the wall of the dimple, and approximately 22 A away from V325 in a threefold-related monomer. This is the first reported crystal structure identifying an infectious receptor attachment site on a parvovirus capsid. We conclude that the affinity of the interactions of sialic-acid-containing receptors with residues at or surrounding the dimple can evolutionarily regulate parvovirus pathogenicity and adaptation to new hosts.     

Cooperative binding of initiator protein to replication origin conferred by single amino acid substitution.

The replication initiator protein pi of plasmid R6K binds seven 22 bp direct repeats (DR) in the gamma origin. The pi protein also binds to an inverted repeat (IR) in the operator of its own gene, pir, which lies outside the gamma origin sequences. A genetic system was devised to select for pi protein mutants which discriminate between IR and DR (York et al., Gene (Amst.) 116, 7-12, 1992; York and Filutowicz, J. Biol. Chem. 268, 21854-21861, 1993). From this selection the mutant pi S87N protein was isolated which is deficient in repressing the pir gene's expression because it cannot bind to IR at the pir gene operator. Remarkably, we discovered that pi S87N binds to DR cooperatively under conditions where wt pi binds independently. Moreover, the pi S87N is more active as a replication initiator in vivo when supplied at the same level as wt pi. Quantitative binding assays showed that both wt pi and pi S87N bind a DNA fragment containing a single DR unit with a similar affinity (Kd = 0.3 x 10(-12) M). Thus, cooperativity of pi S87N is most likely achieved through altered interactions between promoters bound at adjacent DR units.