Biomedical subjects

J C Sacchettini

Publications and source records attributed to J C Sacchettini.

At least 73 records · Page 4Linked to original sources

PubMed1995

Expression, purification, and characterization of Escherichia coli dihydrodipicolinate reductase.

Dihydrodipicolinate reductase catalyzes the NAD(P)H-dependent reduction of the carbon-carbon double bond of the alpha,beta-unsaturated cyclic imine dihydrodipicolinate to form the cyclic imine tetrahydrodipicolinate. The enzyme is a component of the bacterial biosynthetic pathway forming L-lysine and diaminopimelate from L-aspartate. The gene encoding dihydrodipicolinate reductase, dapB, has been cloned and sequenced from Escherichia coli (Bouvier et al., 1984), and we have used this sequence information to generate an expression vector containing the dapB gene. Expression and purification of dihydrodipicolinate reductase to homogeneity have allowed us to characterize the kinetics, stereochemistry, and chemical mechanism of the enzymatic reaction. The kinetic mechanism is ordered, with reduced nucleotide binding preceding dihydrodipicolinate binding and presumably with tetrahydrodipicolinate dissociating prior to oxidized nucleotide release. The enzyme has a unique nucleotide specificity, with NADH being twice as effective as NADPH as a reductant. The enzyme catalyzes the stereospecific transfer of the 4R hydrogen atom of the reduced nucleotide, as a hydride ion, to the 4 position of dihydrodipicolinate. These results allow us to propose a chemical mechanism for the reaction catalyzed by dihydrodipicolinate reductase involving hydride transfer to the beta carbon of the unsaturated imine. The resonance-stabilized C3 carbanion is protonated to generate the reduced product, tetrahydrodipicolinate.

Chromatography, Gel↗

PubMed1995

Three-dimensional structure of Escherichia coli dihydrodipicolinate reductase.

Dihydrodipicolinate reductase is an enzyme found in bacteria and higher plants involved in the biosynthesis of diaminopimelic acid and lysine. Because these pathways are unique to bacteria and plants, they may represent attractive targets for new antimicrobial or herbicidal compounds. The three-dimensional structure of Escherichia coli dihydrodipicolinate reductase, complexed with NADPH, has been determined and refined to a crystallographic R-factor of 18.6% with diffraction data to 2.2 A resolution. The refined model contains the complete protein chain, the cofactor NADPH, and 55 water molecules. The enzyme is composed of two domains. The dinucleotide binding domain has a central seven-stranded parallel beta-sheet surrounded by four alpha-helices, with the cofactor binding site located at the carboxy-terminal edge of the sheet. The second domain contains four beta-strands and two alpha-helices that form an open mixed beta-sandwich. A possible binding site for dihydrodipicolinate has been identified in this second domain, about 12 A away from the dinucleotide binding site. This would imply that the protein must undergo some conformational change in order to perform catalysis. In the crystal, the native enzyme is a homotetramer generated by a 222 crystallographic axis. Implications of the tetrameric structure for the enzyme function are presented. Dihydrodipicolinate reductase uses both NADH and NADPH as cofactors, and analysis of its cofactor binding site allows for a molecular understanding of the enzyme's dual specificity.

Amino Acid Sequence↗

PubMed1995

Crystal structure and function of the isoniazid target of Mycobacterium tuberculosis.

Resistance to isoniazid in Mycobacterium tuberculosis can be mediated by substitution of alanine for serine 94 in the InhA protein, the drug's primary target. InhA was shown to catalyze the beta-nicotinamide adenine dinucleotide (NADH)-specific reduction of 2-trans-enoyl-acyl carrier protein, an essential step in fatty acid elongation. Kinetic analyses suggested that isoniazid resistance is due to a decreased affinity of the mutant protein for NADH. The three-dimensional structures of wild-type and mutant InhA, refined to 2.2 and 2.7 angstroms, respectively, revealed that drug resistance is directly related to a perturbation in the hydrogen-bonding network that stabilizes NADH binding.

Bacterial Proteins↗

PubMed1995

Structural studies of class I major histocompatibility complex proteins: insights into antigen presentation.

The three-dimensional structures of three human and two murine class I molecules, in complex with single peptides and with mixtures of endogenous peptides, have now been determined to high resolution. These structures have afforded important insights into the way in which antigenic peptides are bound by an MHC class I molecule, and how a given MHC molecule can bind a large number and variety of peptides, for presentation to a T cell receptor. Peptides are bound in a cleft located in the alpha 1/alpha 2 domain of a class I molecule. They are tethered by an array of hydrogen bonding interactions many of which are conserved among the different structures. Binding is also accomplished through van der Waals interactions between two or three peptide residues and complementary pockets in the cleft. The location and the characteristics of its pockets are unique to a given MHC class I molecule, and so determine the identity of the anchor residues of the set of peptides that bind. The antigenic epitope recognized by the TCR consists of residues on the MHC as well as the side chains of those peptide residues that point out from the cleft. The various strategies used to expand the repertoire of peptides bound and presented are discussed.

Amino Acid Sequence↗

PubMed1995

An amino acid substitution in the human intestinal fatty acid binding protein is associated with increased fatty acid binding, increased fat oxidation, and insulin resistance.

The intestinal fatty acid binding protein locus (FABP2) was investigated as a possible genetic factor in determining insulin action in the Pima Indian population. A polymorphism at codon 54 of FABP2 was identified that results in an alanine-encoding allele (frequency 0.71) and a threonine-encoding allele (frequency 0.29). Pimas who were homozygous or heterozygous for the threonine-encoding allele were found to have a higher mean fasting plasma insulin concentration, a lower mean insulin-stimulated glucose uptake rate, a higher mean insulin response to oral glucose and a mixed meal, and a higher mean fat oxidation rate compared with Pimas who were homozygous for the alanine-encoding allele. Since the FABP2 threonine-encoding allele was found to be associated with insulin resistance and increased fat oxidation in vivo, we further analyzed the FABP2 gene products for potential functional differences. Titration microcalorimetry studies with purified recombinant protein showed that the threonine-containing protein had a twofold greater affinity for long-chain fatty acids than the alanine-containing protein. We conclude that the threonine-containing protein may increase absorption and/or processing of dietary fatty acids by the intestine and thereby increase fat oxidation, which has been shown to reduce insulin action.

Adult↗

PubMed1995

MHC class I-peptide interactions and TCR recognition.

The recent crystal structure determinations of MHC class I molecules with single bound peptides have allowed us to understand the guidelines that govern peptide binding in a given MHC allele. Evolution has provided for MHC class I molecules whose antigen binding clefts possess distinct physical and chemical properties as a result of the particular arrangement of variable residues lining the binding cleft. As a result, a given molecule binds a unique set of peptides, the position and identity of whose anchor residues are dictated by the features of the cleft. In addition to the interactions between the anchor residues and the cleft, the peptide is held in the groove by a highly conserved array of hydrogen bonds. A tantalizing application of our newfound understanding of peptide binding will be in the design of model peptides that either block or enhance immune response, in order to achieve effective treatments for a variety of disorders of the immune system.

Alleles↗

PubMed1994

A general method for facilitating heterodimeric pairing between two proteins: application to expression of alpha and beta T-cell receptor extracellular segments.

Generation of soluble T-cell receptor (TCR) molecules by a variety of genetic engineering methods has been hampered by inefficient pairing of alpha and beta subunits in the absence of their respective transmembrane regions and associated CD3 components. To overcome this obstacle, we have added 30-amino acid-long segments to the carboxyl termini of alpha and beta extracellular domains via a cleavable flexible linker. These peptide segments (BASE-p1 for alpha and ACID-p1 for beta) have been previously shown to selectively associate to form a stable heterodimeric coiled coil termed a leucine zipper. Homodimeric structures are not permitted due to electrostatic repulsion among amino acid side chains. Expression of a representative TCR-leucine zipper fusion protein in a baculovirus expression system results in production of alpha beta TCR heterodimer at 0.6-1.4 mg/liter. This yield is 5- to 10-fold greater than that of the TCR expressed in the absence of the synthetic leucine zipper sequence. The structure of the TCR component of the fusion heterodimer was judged to be native when probed with a panel of 17 mAbs specific for alpha and beta constant and variable domains. A mAb specific for the isolated BASE-p1/ACID-p1 coiled coil was also generated and shown to react with the TCR fusion protein. The above technology should be broadly useful in the efficient production and purification of TCRs as well as other heterodimeric proteins.

Amino Acid Sequence↗

PubMed1994

Crystal structure of kappa-bungarotoxin at 2.3-A resolution.

kappa-Neurotoxins display a very low affinity for neuromuscular receptors, but bind tightly to, and inhibit, nicotinic acetylcholine receptors in neuronal tissue such as the chick ciliary ganglia. In contrast, alpha-neurotoxins bind with high affinity and inhibit nicotinic acetylcholine receptors at the neuromuscular junction. The origin of this difference in specificity has been a long-studied question in the field. Here we report the first crystal structure of a kappa-neurotoxin, kappa-bungarotoxin. Unlike the NMR structure previously reported [Sutcliffe, M. J., Dobson, C. M., & Oswald, R. E. (1992) Biochemistry 31, 2962-2970], the present crystal structure more accurately defines the polypeptide fold and the nature of the interaction between subunits in the active dimer, which is a unique feature of the kappa-neurotoxins. The structure has been refined to R = 19.6% with X-ray diffraction data extending to a resolution of 2.3 A. There are two independent protein molecules (66 amino acid residues each) in the asymmetric unit that are arranged as a dimer with the two subunits related by a rotation of 178.6 degrees. Each subunit consists of three main-chain loops. Three of the five beta-strands of each subunit form an antiparallel beta-sheet which becomes an extended six-stranded antiparallel beta-sheet, by virtue of the approximate 2-fold symmetry of the dimer. The interactions at the dimer interface consist of six main-chain-main-chain hydrogen bonds, as well as three other hydrogen-bonding interactions involving side chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1994

Crystal structure of recombinant farnesyl diphosphate synthase at 2.6-A resolution.

The synthesis of farnesyl diphosphate (FPP), a key intermediate in the isoprenoid biosynthetic pathway required for the synthesis of cholesterol and in the formation of prenylated proteins, is catalyzed by the enzyme farnesyl diphosphate synthase (FPS). The crystal structure of avian recombinant FPS, the first three-dimensional structure for any prenyltransferase, was determined to 2.6-A resolution. The enzyme exhibits a novel fold composed entirely of alpha-helices joined by connecting loops. The enzyme's most prominent structural feature is the arrangement of 10 core helices around a large central cavity. Two aspartate-rich sequences that are highly conserved among the isoprenyl diphosphate synthase family of prenyltransferases, and are essential for enzymatic activity, were found on opposite walls of this cavity, with the aspartate side chains approximately 12 A apart and facing each other. The location and metal ion binding properties of these sequences suggest that the conserved aspartate residues participate in substrate binding of catalysis.

Alkyl and Aryl Transferases↗

PubMed1994

The crystal structure of human hypoxanthine-guanine phosphoribosyltransferase with bound GMP.

The crystal structure of HGPRTase with bound GMP has been determined and refined to 2.5 A resolution. The enzyme has a core alpha/beta structure resembling the nucleotide-binding fold of dehydrogenases, and a second lobe composed of residues from the amino and carboxy termini. The GMP molecule binds in an anti conformation in a solvent-exposed cleft of the enzyme. Lys-165, which forms a hydrogen bond to O6 of GMP, appears to be critical for determining the specificity for guanine and hypoxanthine over adenine. The location of active site residues also provides evidence for a possible mechanism for general base-assisted HGPRTase catalysis. A rationalization of the effects on stability and activity of naturally occurring single amino acid mutations of HGPRTase is presented, including a discussion of several mutations at the active site that lead to Lesch-Nyhan syndrome.

Amino Acid Sequence↗

PubMed1994

Crystal structures of soybean beta-amylase reacted with beta-maltose and maltal: active site components and their apparent roles in catalysis.

The crystal structures of catalytically competent soybean beta-amylase, unliganded and bathed with small substrates (beta-maltose, maltal), were determined at 1.9-2.2-A resolution. Two molecules of beta-maltose substrate bind to the protein in tandem, with some maltotetraose enzymic condensation product sharing the same binding sites. The beta-amylase soaked with maltal shows a similar arrangement of two bound molecules of 2-deoxymaltose, the enzymic hydration product. In each case the nonreducing ends of the saccharide ligands are oriented toward the base of the protein's active site pocket. The catalytic center, located between the bound disaccharides and found deeper in the pocket than where the inhibitor alpha-cyclodextrin binds, is characterized by the presence of oppositely disposed carboxyl groups of two conserved glutamic acid residues. The OE2 carboxyl of Glu 186 is below the plane of the penultimate glucose residue (Glc 2) of bound maltotetraose, 2.6 A from the oxygen atom of that ligand's penultimate alpha-1,4-glucosidic linkage. The OE2 carboxyl of Glu 380 lies above the plane of Glc 2, 2.8 A from the O-1 atom of the more deeply bound beta-maltose. Saccharide binding does not alter the spatial coordinates of these two carboxyl groups or the overall conformation of the 57-kDa protein. However, the saccharide complexes of the active enzyme are associated with a significant (10 A) local conformational change in a peptide segment of a loop (L3) that borders the active site pocket.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Structural studies on human muscle fatty acid binding protein at 1.4 A resolution: binding interactions with three C18 fatty acids.

BACKGROUND: Muscle fatty acid binding protein (M-FABP) is one of a family of cytosolic lipid-binding proteins involved in fatty acid processing. In order to investigate the precise interactions between M-FABP and its ligands and to understand the structural basis of differential binding affinity, we have compared the structures of M-FABP in complex with three C18 fatty acids. RESULTS: We describe the crystal structures of M-FABP in complex with n-octadecanoate (stearate), trans-delta 9-octadecenoate (elaidate) and cis-delta 9-octadecenoate (oleate). These structures were refined using least-squares positional and anisotropic temperature factor refinement to final R-factors of 11.4%, 12.1% and 13.2% respectively for all the data between 8.0 A and 1.4 A resolution. CONCLUSIONS: Stearate, elaidate and oleate each adopt highly similar U-shaped conformations when they bind to M-FABP within a large interior binding cavity, which also contains 13 ordered water molecules. The atomic structure of the protein is virtually identical, regardless of the nature of the bound ligand. The fatty acid is thought to enter the interior cavity of the protein via a portal in its surface while interior solvent is released through a secondary opening. The ligand affinity can be correlated with the conformational energy and the solubility of the bound ligand.

Carrier Proteins↗

PubMed1994

Primary structure and binding characteristics of locust and human muscle fatty-acid-binding proteins.

The conservation between muscle fatty-acid-binding proteins (M-FABP) of Locusta migratoria flight muscle and human skeletal muscle was investigated. The locust M-FABP cDNA (632 bp) was isolated by 5' and 3' rapid amplification of cDNA ends. The identities of the locust and human M-FABP on the cDNA and protein levels were 54% and 42%, respectively. The predicted amino acid sequence of locust M-FABP indicated a molecular mass of 14935 Da and isoelectric point 6.1. The locust M-FABP was expressed in Escherichia coli, purified by (NH4)2SO4 precipitation, anion-exchange and gel-filtration chromatographies and compared with the recombinant human M-FABP with respect to immunological and binding properties. In spite of the high sequence similarity, the proteins did not show immunological cross-reactivity. The binding parameters of locust M-FABP were analyzed with radiolabeled oleic acid by the Lipidex assay and titration microcalorimetry. Both methods revealed a Kd for oleic acid of 0.5 microM and a binding stoichiometry of 1 mol fatty acid/mol FABP. The delta H, delta G and delta S for oleic acid binding were -146 kJ.mol-1 and -36 J.mol-1 and -369 J.mol-1.K-1 respectively. All the information obtained from binding, fluorescence and displacement studies indicated that locust M-FABP has binding characteristics similar to human M-FABP. Finally the recombinant locust M-FABP was crystallized with and without oleic acid. All crystals were trigonal in the P3(1)21 space group. The unit cell dimensions were a = b = 5.89 nm and c = 14.42 nm.

Amino Acid Sequence↗

PubMed1994

Crystal structure of orotate phosphoribosyltransferase.

Phosphoribosyltransferases (PRTases) are enzymes involved in the synthesis of purine, pyrimidine, and pyridine nucleotides. They utilize alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) and a nitrogenous base to form a beta-N-riboside monophosphate and pyrophosphate (PPi), and their functional significance in nucleotide homeostasis is evidenced by the devastating effects of inherited diseases associated with the decreased activity and/or stability of these enzymes. The 2.6-A structure of the Salmonella typhimurium orotate phosphoribosyltransferase (OPRTase) complexed with its product orotidine monophosphate (OMP) provides the first detailed image of a member of this group of enzymes. The OPRTase three-dimensional structure was solved using multiple isomorphous replacement methods and reveals two major features: a core five-stranded alpha/beta twisted sheet and an N-terminal region that partially covers the C-terminal portion of the core. PRTases show a very high degree of base specificity. In OPRTase, this is determined by steric constraints and the position of hydrogen bond donors/acceptors of a solvent-inaccessible crevice where the orotate ring of bound OMP resides. Crystalline OPRTase is a dimer, with catalytically important residues from each subunit available to the neighboring subunit, suggesting that oligomerization is necessary for its activity. On the basis of the presence of a common PRPP binding motif among PRTases and the similar chemistry these enzymes perform, we propose that the alpha/beta core found in OPRTase will represent a common feature for PRTases. This generality is demonstrated by construction of a model of the human hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) from secondary structure predictions for HGPRTase and the three-dimensional structure of OPRTase.

Amino Acid Sequence↗

PubMed1994

The three-dimensional structure of H-2Db at 2.4 A resolution: implications for antigen-determinant selection.

Solution at 2.4 A resolution of the structure of H-2Db with the influenza virus peptide NP366-374 (ASNEN-METM) and comparison with the H-2Kb-VSV (RGY-VYQGL) structure allow description of the molecular details of MHC class I peptide binding interactions for mice of the H-2b haplotype, revealing a strategy that maximizes the repertoire of peptides than can be presented. The H-2Db cleft has a mouse-specific hydrophobic ridge that causes a compensatory arch in the backbone of the peptide, exposing the arch residues to TCR contact and requiring the peptide to be at least 9 residues. This ridge occurs in about 40% of the known murine D and L allelic molecules, classifying them as a structural subgroup.

Amino Acid Sequence↗

PubMed1994

Evolutionary relationships and functional conservation among vertebrate Max-associated proteins: the zebra fish homolog of Mxi1.

In mammals, current evidence supports the view that Myc-responsive activities are regulated in part through an intracellular balance between levels of transcriptionally-active Myc/Max heterodimers and those of transcriptionally-inert Max/Max, Mad/Max and Mxi1/Max complexes. To gain insight into the roles of Mad and Mxi1 in cellular growth and differentiation and to fortify key structure-function relationships from an evolutionary standpoint, low stringency hybridization screens were used to identify potential homologs of these Max-associated proteins in the zebra fish genome. A single class of cDNA clones that cross-hybridized both to human mad and mxi1 probes was shown to encode a putative protein with significantly greater homology to mammalian Mxi1 than to Mad, particularly in the basic and helix-loop-helix (bHLH) regions. The high degree of structural relatedness between vertebrate Mxi1 proteins apparent in molecular modelling studies was consistent with the findings that the HLH/leucine zipper (LZ) region of zMxi1 exhibited the same profile of dimerization specificities as its mammalian counterpart in the two-hybrid system and that zmxi1 could, like human mxi1 (Lahoz et al., 1994), suppress the oncogenic potential of mouse c-myc in a mammalian cell. Finally, a comparison of steady-state zc-myc and zmxi1 mRNA levels during zebra fish embryogenesis demonstrated (i) high levels of zc-myc relative to zmxi1 mRNA during initiation of organogenesis, a period characterized by intense growth and active differentiation and (ii) rising levels of zmxi1 mRNA during progression towards the terminally differentiated state. These contrasting patterns of developmental expression together with the capacity of zmxi1 to repress myc-induced transformation support a model for the regulation, by Max-associated proteins, of Myc functions in the control of normal cell development and neoplastic growth.

Amino Acid Sequence↗

PubMed1993

Escherichia coli-derived rat intestinal fatty acid binding protein with bound myristate at 1.5 A resolution and I-FABPArg106-->Gln with bound oleate at 1.74 A resolution.

Rat intestinal fatty acid binding protein (I-FABP) is a 131-residue protein composed of two short alpha-helices (alpha I and alpha II) and 10 anti-parallel beta-strands organized into two nearly orthogonal beta-sheets. The structure of crystalline I-FABP with bound tetradecanoate (myristate) has been refined to a resolution of 1.5 A and compared to the 1.2 A structure of apo-I-FABP, the 1.9 A structure of I-FABP:hexadecanoate (palmitate) and the 1.75 A structure of I-FABP:9Z-octadecanoate (oleate) to determine how this model fatty acid receptor accommodates changes in the length of its fatty acid ligand. Myristate is located in the interior of the protein. A highly ordered, electrostatic network containing 7 hydrogen (H)-bonds links the OE1 and OE2 atoms of myristate's carboxylate group, the indole nitrogen of Trp82, NH1, and NH2 of Arg106, NE2, and OE1 of Gln115, and 2 interior ordered waters. The hydrocarbon chain of the bound fatty acid is slightly bent. Its convex face lies in a crevice, forming van der Waals contacts with the side chains of several hydrophobic and aromatic residues. Its concave face is exposed to an array of 8 interior ordered waters whose positions are stabilized by H-bond interactions with other waters, H-bond interactions with the side chains of polar/ionizable residues, and van der Waals contacts with the surface of the fatty acid. Addition of 2 or 4 methylenes to myristate produces remarkably little change in the positions of I-FABP's main chain and side chain atoms and interior ordered waters. The principal alterations are in the conformation of a surface opening (portal) connecting external and internal solvent and in the position of the benzene side chain of Phe55. Changes in the conformation of the portal reflect movement of two of its components: the backbone of alpha II and a type I turn (Ala73, Asp74) connecting two beta-strands. The positions of the main chain atoms of Ala73 and Asp74 appear to be determined by their ability to form van der Waals contacts with the omega-terminus of the fatty acid. The side chain of Phe55 appears to function as an adjustable aromatic lid, located over the portal, whose position is dependent on an ability to form van der Waals contacts with a fatty acid's omega-terminus.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Structural evidence for a pH-sensitive dilysine trigger in the hen ovotransferrin N-lobe: implications for transferrin iron release.

Members of the transferrin family of proteins are involved in Fe3+ transport (serum transferrins) and are also believed to possess antimicrobial activity (ovotransferrins and lactoferrins). The structure of the monoferric N-terminal half-molecule of hen ovotransferrin, reported here at 2.3-A resolution, reveals an unusual interdomain interaction formed between the side-chain NZ atoms of Lys 209 and Lys 301, which are 2.3 A apart. This strong interaction appears to be an example of a low-barrier hydrogen bond between the two lysine NZ atoms, both of which are also involved in a hydrogen-bonding interaction with the aromatic ring of a tyrosine residue. Crystals of the protein were grown at pH 5.9, which is well below the usual pKa approximately 10 for a lysine side chain. We suggest that the pKa of either one or both of these residues lies below the pH of the structure determination and is, therefore, not positively charged. This finding may serve to explain, on a molecular basis, the pH dependence of transferrin Fe3+ release. We propose that uptake of the Fe(3+)-transferrin complex into an acidic endosome (viz., pH approximately 5.0) via receptor-mediated endocytosis will result in the protonation of both lysine residues. The close proximity of the two resulting positive charges, and their location on opposite domains of the N-lobe, might well be the driving force that opens the two domains of the protein, exposing the Fe3+ ion and facilitating its release.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗