beta-Lactamase binds to GroEL in a conformation highly protected against hydrogen/deuterium exchange.

Escherichia coli RTEM beta-lactamase reversibly forms a stable complex with GroEL, devoid of any enzymatic activity, at 48 degrees C. When beta-lactamase is diluted from this complex into denaturant solution, its unfolding rate is identical to that from the native state, while the unfolding rate from the molten globule state is too fast to be measured. Electrospray mass spectrometry shows that the rate of proton exchange in beta-lactamase in the complex at 48 degrees C is slower than in the absence of GroEL at the same temperature, and resembles the exchange of the native state at 25 degrees C. Similarly, the final number of protected deuterons is higher in the presence of GroEL than in its absence. We conclude that, for beta-lactamase, a state with significant native structure is bound to GroEL. Thus, different proteins are recognized by GroEL in very different states, ranging from totally unfolded to native-like, and this recognition may depend on which state can provide sufficient accessible hydrophobic amino acids in a suitably clustered arrangement. Reversible binding of native-like states with hydrophobic patches may be an important property of GroEL to protect the cell from aggregating protein after heat-shock.

Cdc45p assembles into a complex with Cdc46p/Mcm5p, is required for minichromosome maintenance, and is essential for chromosomal DNA replication.

We report the isolation and characterization of CDC45, which encodes a polypeptide of 650 amino acids that is essential for the initiation of chromosomal DNA replication in the budding yeast, Saccharomyces cerevisiae. CDC45 genetically interacts with at least two members of the MCM (minichromosome maintenance) family of replication genes, CDC46 and CDC47, which are proposed to perform a role in restricting initiation of DNA replication to once per cell cycle. Like mutants in several MCM genes, alleles of CDC45 also show a severe minichromosome maintenance defect. Together, these observations imply that Cdc45p performs a role in the control of initiation events at chromosomal replication origins. We investigated this possibility further and present evidence demonstrating that Cdc45p is assembled into complexes with one MCM family member, Cdc46p/Mcm5p. These observations point to a role for Cdc45p in controlling the early steps of chromosomal DNA replication in conjunction with MCM polypeptide complexes. Unlike the MCMs, however, the subcellular localization of Cdc45p does not vary with the cell cycle, making it likely that Cdc45p interacts with MCMs only during the nuclear phase of MCM localization in G1.

Direct evidence for modified solvent structure within the hydration shell of a hydrophobic amino acid.

Neutron scattering experiments are used to determine scattering profiles for aqueous solutions of hydrophobic and hydrophilic amino acid analogs. Solutions of hydrophobic solutes show a shift in the main diffraction peak to smaller angle as compared with pure water, whereas solutions of hydrophilic solutes do not. The same difference for solutions of hydrophobic and hydrophilic side chains is also predicted by molecular dynamics simulations. The neutron scattering curves of aqueous solutions of hydrophobic amino acids at room temperature are qualitatively similar to differences between the liquid molecular structure functions measured for ambient and supercooled water. The nonpolar solute-induced expansion of water structure reported here is also complementary to recent neutron experiments where compression of aqueous solvent structure has been observed at high salt concentration.

Mapping rat megalin: the second cluster of ligand binding repeats contains a 46-amino acid pathogenic epitope involved in the formation of immune deposits in Heymann nephritis.

Megalin (gp330), an epithelial endocytic receptor, is a major target antigen of Heymann nephritis (HN), an autoimmune disease in rats. To elucidate the mechanisms of HN, we have mapped a pathogenic epitope in megalin that binds anti-megalin antibodies. We focused our attention on four clusters of cysteine-rich, low density lipoprotein receptor (LDLR) ligand binding repeats in the extracellular domain of megalin because they represent putative ligand binding regions and therefore would be expected to be exposed in vivo and to be able to bind circulating antibodies. Rat megalin cDNA fragments I through IV encoding the first through fourth clusters of ligand-binding repeats, respectively, were expressed in a baculovirus system. All four expression products were detected by immunoblotting with two antisera capable of inducing passive HN (pHN). When antibodies eluted from glomeruli of rats with pHN were used for immunoblotting, only the expression product encoded by fragment II was detected. This indicates that the second cluster of LDLR ligand binding repeats is directly involved in binding anti-megalin antibodies and in the induction of pHN. To narrow the major epitope in this domain, fragment II was used to prepare proteins sequentially truncated from the C- and N-terminal ends by in vitro translation. Analysis of the truncated translation products by immunoprecipitation with anti-megalin IgG revealed that the fifth ligand-binding repeat (amino acids 1160-1205) contains the major epitope recognized. This suggests that a 46-amino acid sequence in the second cluster of LDLR ligand binding repeats contains a major pathogenic epitope that plays a key role in pHN. Identification of this epitope will facilitate studies on the pathogenesis of HN.

Crystal structure of D-amino acid oxidase: a case of active site mirror-image convergent evolution with flavocytochrome b2.

D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of D-amino acids to the corresponding alpha-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that of p-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate alpha-hydrogen to the flavin during the enzyme reductive half-reaction.The active site Of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups (if these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalvze similar reactions (oxidation of alpha-amino acids or alpha-hydroxy acids), although with opposite stereochemistry.

Changes in voltage activation, Cs+ sensitivity, and ion permeability in H5 mutants of the plant K+ channel KAT1.

KAT1 is a voltage-dependent inward rectifying K+ channel cloned from the higher plant Arabidopsis thaliana [Anderson, J. A., Huprikar, S. S., Kochian, L. V., Lucas, W. J. & Gaber, R. F. (1992) Proc. Natl. Acad. Sci. USA 89, 3736-3740]. It is related to the Shaker superfamily of K+ channels characterized by six transmembrane spanning domains (S1-S6) and a putative pore-forming region between S5 and S6 (H5). The 115 region between Pro-247 and Pro-271 in KAT1 contains 14 additional amino acids when compared with Shaker [Aldrich, R. W. (1993) Nature (London) 362, 107-108]. We studied various point mutations introduced into H5 to determine whether voltage-dependent plant and animal K+ channels share similar pore structures. Through heterologous expression in Xenopus oocytes and voltage-clamp analysis combined with phenotypic analysis involving a potassium transport-defective Saccharomyces cerevisiae strain, we investigated the selectivity filter of the mutants and their susceptibility toward inhibition by cesium and calcium ions. With respect to electrophysiological properties, KAT1 mutants segregated into three groups: (i) wild-type-like channels, (ii) channels modified in selectivity and Cs+ or Ca2+ sensitivity, and (iii) a group that was additionally affected in its voltage dependence. Despite the additional 14 amino acids in H5, this motif in KAT1 is also involved in the formation of the ion-conducting pore because amino acid substitutions at Leu-251, Thr-256, Thr-259, and Thr-260 resulted in functional channels with modified ionic selectivity and inhibition. Creation of Ca2+ sensitivity and an increased susceptibility to Cs+ block through mutations within the narrow pore might indicate that both blockers move deeply into the channel. Furthermore, mutations close to the rim of the pore affecting the half-activation potential (U1/2) indicate that amino acids within the pore either interact with the voltage sensor or ion permeation feeds back on gating.

Molecular characterization of a FKBP-type immunophilin from higher plants.

Immunophilins are intracellular receptors for the immunosuppressants cyclosporin A, FK506, and rapamycin. In addition to their use in organ transplantation, these natural products have been used to investigate signaling pathways in yeast, plant, and mammalian cells. We have recently described the identification of an immunosuppressant-sensitive signaling pathway in and the purification of several immunophilins from Vicia faba plants. We now report the molecular characterization of a 15 kDa FK506- and rapamycin-binding protein from V. faba (VfFKBP15). The amino acid sequence deduced from the cDNA starts with a signal peptide of 22 hydrophobic amino acids. The core region of VfFKBP15 is most similar to yeast and mammalian FKBP13 localized in the endoplasmic reticulum (ER). In addition, VfFKBP15 has a carboxyl-terminal sequence that is ended with SSEL, a putative ER retention signal. These findings suggest that VfFKBP15 is a functional homolog of FKBP13 from other organisms. Interestingly, two distinct cDNAs corresponding to two isoforms of FKBP15 have been cloned from Arabidopsis and also identified from rice data base, suggesting that pFKBP15 (plant FKBP15) is encoded by a small gene family in plants. This adds to the diversity of plant FKBP members even with the same subcellular localization and is in contrast with the situation in mammalian and yeast systems in which only one FKBP13 gene has been found. Like the mammalian and yeast FKBP13, the recombinant VfFKBP15 protein has rotamase activity that is inhibited by both FK506 and rapamycin with a Ki value of 30 nM and 0.9 nM, respectively, illustrating that VfFKBP15 binds rapamycin in preference over FK506. The mRNA of VfFKBP15 is ubiquitously expressed in various plant tissues including leaves, stems, and roots, consistent with the ER localization of the protein. Levels of VfFKBP15 mRNA are elevated by heat shock, suggesting a possible role for this FKBP member under stress conditions.

Posttranslational amino acid epimerization: enzyme-catalyzed isomerization of amino acid residues in peptide chains.

Since ribosomally mediated protein biosynthesis is confined to the L-amino acid pool, the presence of D-amino acids in peptides was considered for many years to be restricted to proteins of prokaryotic origin. Unicellular microorganisms have been responsible for the generation of a host of D-amino acid-containing peptide antibiotics (gramicidin, actinomycin, bacitracin, polymyxins). Recently, a series of mu and delta opioid receptor agonists [dermorphins and deltorphins] and neuroactive tetrapeptides containing a D-amino acid residue have been isolated from amphibian (frog) skin and mollusks. Amino acid sequences obtained from the cDNA libraries coincide with the observed dermorphin and deltorphin sequences, suggesting a stereospecific posttranslational amino acid isomerization of unknown mechanism. A cofactor-independent serine isomerase found in the venom of the Agelenopsis aperta spider provides the first major clue to explain how multicellular organisms are capable of incorporating single D-amino acid residues into these and other eukaryotic peptides. The enzyme is capable of isomerizing serine, cysteine, O-methylserine, and alanine residues in the middle of peptide chains, thereby providing a biochemical capability that, until now, had not been observed. Both D- and L-amino acid residues are susceptible to isomerization. The substrates share a common Leu-Xaa-Phe-Ala recognition site. Early in the reaction sequence, solvent-derived deuterium resides solely with the epimerized product (not substrate) in isomerizations carried out in 2H2O. Significant deuterium isotope effects are obtained in these reactions in addition to isomerizations of isotopically labeled substrates (2H at the epimerizeable serine alpha-carbon atom). The combined kinetic and structural data suggests a two-base mechanism in which abstraction of a proton from one face is concomitant with delivery from the opposite face by the conjugate acid of the second enzymic base.

Aromatic amino acid transamination and methionine recycling in trypanosomatids.

Although trypanosomatids are known to rapidly transaminate exogenous aromatic amino acids in vitro and in vivo, the physiological significance of this reaction is not understood. In postmitochondrial supernatants prepared from Trypanosoma brucei brucei and Crithidia fasciculata, we have found that aromatic amino acids were the preferred amino donors for the transamination of alpha-ketomethiobutyrate to methionine. Intact C. fasciculata grown in the presence of [15N]tyrosine were found to contain detectable [15N]methionine, demonstrating that this reaction occurs in situ in viable cells. This process is the final step in the recycling of methionine from methylthioadenosine, a product of decarboxylated S-adenosylmethionine from the polyamine synthetic pathway. Mammalian liver, in contrast, preferentially used glutamine for this reaction and utilized a narrower range of amino donors than seen with the trypanosomatids. Studies with methylthioadenosine showed that this compound was readily converted to methionine, demonstrating a fully functional methionine-recycling pathway in trypanosomatids.

A new high molecular weight immunoglobulin class from the carcharhine shark: implications for the properties of the primordial immunoglobulin.

All immunoglobulins and T-cell receptors throughout phylogeny share regions of highly conserved amino acid sequence. To identify possible primitive immunoglobulins and immunoglobulin-like molecules, we utilized 3' RACE (rapid amplification of cDNA ends) and a highly conserved constant region consensus amino acid sequence to isolate a new immunoglobulin class from the sandbar shark Carcharhinus plumbeus. The immunoglobulin, termed IgW, in its secreted form consists of 782 amino acids and is expressed in both the thymus and the spleen. The molecule overall most closely resembles mu chains of the skate and human and a new putative antigen binding molecule isolated from the nurse shark (NAR). The full-length IgW chain has a variable region resembling human and shark heavy-chain (VH) sequences and a novel joining segment containing the WGXGT motif characteristic of H chains. However, unlike any other H-chain-type molecule, it contains six constant (C) domains. The first C domain contains the cysteine residue characteristic of C mu1 that would allow dimerization with a light (L) chain. The fourth and sixth domains also contain comparable cysteines that would enable dimerization with other H chains or homodimerization. Comparison of the sequences of IgW V and C domains shows homology greater than that found in comparisons among VH and C mu or VL, or CL thereby suggesting that IgW may retain features of the primordial immunoglobulin in evolution.

Mass spectrometric amino acid sequencing of a mixture of seed storage proteins (napin) from Brassica napus, products of a multigene family.

The amino acid sequences of a number of closely related proteins ("napin") isolated from Brassica napus were determined by mass spectrometry without prior separation into individual components. Some of these proteins correspond to those previously deduced (napA, BngNAP1, and gNa), chiefly from DNA sequences. Others were found to differ to a varying extent (BngNAP1', BngNAP1A, BngNAP1B, BngNAP1C, gNa', and gNaA). The short chains of gNa and gNa' and of BngNAP1 and BngNAP1' differ by the replacement of N-terminal proline by pyroglutamic acid; the long chains of gNaA and BngNAP1B contain a six amino acid stretch, MQGQQM, which is present in gNa (according to its DNA sequence) but absent from BngNAP1 and BngNAP1C. These alternations of sequences between napin isoforms are most likely due to homologous recombination of the genetic material, but some of the changes may also be due to RNA editing. The amino acids that follow the untruncated C termini of those napin chains for which the DNA sequences are known (napA, BngNAP1, and gNa) are aromatic amino acids. This suggests that the processing of the proprotein leading to the C termini of the two chains is due to the action of a protease that specifically cleaves a G/S-F/Y/W bond.

Protein synthesis editing by a DNA aptamer.

Potential errors in decoding genetic information are corrected by tRNA-dependent amino acid recognition processes manifested through editing reactions. One example is the rejection of difficult-to-discriminate misactivated amino acids by tRNA synthetases through hydrolytic reactions. Although several crystal structures of tRNA synthetases and synthetase-tRNA complexes exist, none of them have provided insight into the editing reactions. Other work suggested that editing required active amino acid acceptor hydroxyl groups at the 3' end of a tRNA effector. We describe here the isolation of a DNA aptamer that specifically induced hydrolysis of a misactivated amino acid bound to a tRNA synthetase. The aptamer had no effect on the stability of the correctly activated amino acid and was almost as efficient as the tRNA for inducing editing activity. The aptamer has no sequence similarity to that of the tRNA effector and cannot be folded into a tRNA-like structure. These and additional data show that active acceptor hydroxyl groups in a tRNA effector and a tRNA-like structure are not essential for editing. Thus, specific bases in a nucleic acid effector trigger the editing response.

Thermal unfolding of human high-density apolipoprotein A-1: implications for a lipid-free molten globular state.

Apolipoprotein A-1 (apoA-1) in complex with high-density lipoprotein is critically involved in the transport and metabolism of cholesterol and in the pathogenesis of atherosclerosis. We reexamined the thermal unfolding of lipid-free apoA-1 in low-salt solution at pH approximately 7, by using differential scanning calorimetry and circular dichroism. At protein concentrations <5 mg/ml, thermal unfolding of apoA-1 is resolved as an extended peak (25 degrees C-90 degrees C) that can be largely accounted for by a single reversible non-two-state transition with midpoint Tm 57 +/- 1 degree C, calorimetric enthalpy deltaH(Tm)= 200 +/- 20 kcal/mol (1 kcal = 4.18 kJ), van't Hoff enthalpy deltaHv(Tm) approximately 32.5 kcal/mol, and cooperativity deltaHv(Tm)/deltaH(Tm) approximately 0.16. The enthalpy deltaH(Tm) can be accounted for by melting of the alpha-helical structure that is inferred by CD to constitute approximately 60% of apoA-1 amino acids. Farand near-UV CD spectra reveal noncoincident melting of the secondary and tertiary structural elements and indicate a well-defined secondary structure but a largely melted tertiary structure for apoA-1 at approximately 37 degrees C and pH 7. This suggests a molten globular-like state for lipid-free apoA-1 under near-physiological conditions. Our results suggest that in vivo lipid binding by apoA-1 may be mediated via the molten globular apolipoprotein state in plasma.

Human stanniocalcin: a possible hormonal regulator of mineral metabolism.

We have isolated a human cDNA clone encoding the mammalian homolog of stanniocalcin (STC), a calcium- and phosphate-regulating hormone that was first described in fishes where it functions in preventing hypercalcemia. STC has a unique amino acid sequence and, until now, has remained one of the few polypeptide hormones never described in higher vertebrates. Human STC (hSTC) was found to be 247 amino acids long and to share 73% amino acid sequence similarity with fish STC. Polyclonal antibodies to recombinant hSTC localized to a distinct cell type in the nephron tubule, suggesting kidney as a possible site of synthesis. Recombinant hSTC inhibited the gill transport of calcium when administered to fish and stimulated renal phosphate reabsorption in the rat. The evidence suggests that mammalian STC, like its piscine counterpart, is a regulator of mineral homeostasis.

Single-amino acid substitutions eliminate lysine inhibition of maize dihydrodipicolinate synthase.

Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) catalyzes the first step in biosynthesis of lysine in plants and bacteria. DHPS in plants is highly sensitive to end-product inhibition by lysine and, therefore, has an important role in regulating metabolite flux into lysine. To better understand the feedback inhibition properties of the plant enzyme, we transformed a maize cDNA for lysine-sensitive DHPS into an Escherichia coli strain lacking DHPS activity. Cells were mutagenized with ethylmethanesulfonate, and potential DHPS mutants were selected by growth on minimal medium containing the inhibitory lysine analogue S-2-aminoethyl-L-cysteine. DHPS assays identified surviving colonies expressing lysine-insensitive DHPS activity. Ten single-base-pair mutations were identified in the maize DHPS cDNA sequence; these mutations were specific to one of three amino acid residues (amino acids 157, 162, and 166) localized within a short region of the polypeptide. No other mutations were present in the remaining DHPS cDNA sequence, indicating that altering only one of the three residues suffices to eliminate lysine inhibition of maize DHPS. Identification of these specific mutations that change the highly sensitive maize DHPS to a lysine-insensitive isoform will help resolve the lysine-binding mechanism and the resultant conformational changes involved in inhibition of DHPS activity. The plant-derived mutant DHPS genes may also be used to improve nutritional quality of maize or other cereal grains that have inadequate lysine content when fed to animals such as poultry, swine, or humans.