Biomedical subjects

D E Timm

Publications and source records attributed to D E Timm.

At least 19 recordsLinked to original sources

PubMed2001

Crystal structure of thiamin pyrophosphokinase.

Thiamin pyrophosphate (TPP) is a coenzyme derived from vitamin B1 (thiamin). TPP synthesis in eukaryotes requires thiamin pyrophosphokinase (TPK), which catalyzes the transfer of a pyrophosphate group from ATP to thiamin. TPP is essential for central metabolic processes, including the formation of acetyl CoA from glucose and the Krebs cycle. Deficiencies in human thiamin metabolism result in beriberi and Wernicke encephalopathy. The crystal structure of mouse TPK was determined by multiwavelength anomalous diffraction at 2.4 A resolution, and the structure of TPK complexed with thiamin has been refined at 1.9 A resolution. The TPK polypeptide folds as an alpha/beta-domain and a beta-sandwich domain, which share a central ten-stranded mixed beta-sheet. TPK subunits associate as a dimer, and thiamin is bound in the dimer interface. Despite lacking apparent sequence homology with other proteins, the alpha/beta-domain resembles the Rossman fold and is similar to other kinase structures, including another pyrophosphokinase and a thiamin biosynthetic enzyme. Comparison of mouse and yeast TPK structures reveals differences that could be exploited in developing species-specific inhibitors of potential use as antimicrobial agents.

Animals↗

PubMed2001

Structural and functional analysis of missense mutations in fumarylacetoacetate hydrolase, the gene deficient in hereditary tyrosinemia type 1.

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease caused by a deficiency of the enzyme involved in the last step of tyrosine degradation, fumarylacetoacetate hydrolase (FAH). Thus far, 34 mutations in the FAH gene have been reported in various HT1 patients. Site-directed mutagenesis of the FAH cDNA was used to investigate the effects of eight missense mutations found in HTI patients on the structure and activity of FAH. Mutated FAH proteins were expressed in Escherichia coli and in mammalian CV-1 cells. Mutations N16I, F62C, A134D, C193R, D233V, and W234G lead to enzymatically inactive FAH proteins. Two mutations (R341W, associated with the pseudo-deficiency phenotype, and Q279R) produced proteins with a level of activity comparable to the wild-type enzyme. The N16I, F62C, C193R, and W234G variants were enriched in an insoluble cellular fraction, suggesting that these amino acid substitutions interfere with the proper folding of the enzyme. Based on the tertiary structure of FAH, on circular dichroism data, and on solubility measurements, we propose that the studied missense mutations cause three types of structural effects on the enzyme: 1) gross structural perturbations, 2) limited conformational changes in the active site, and 3) conformational modifications with no significant effect on enzymatic activity.

Animals↗

PubMed2001

Mechanistic inferences from the crystal structure of fumarylacetoacetate hydrolase with a bound phosphorus-based inhibitor.

Fumarylacetoacetate hydrolase (FAH) catalyzes the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate to yield fumarate and acetoacetate as the final step of Phe and Tyr degradation. This unusual reaction is an essential human metabolic function, with loss of FAH activity causing the fatal metabolic disease hereditary tyrosinemia type I (HT1). An enzymatic mechanism involving a catalytic metal ion, a Glu/His catalytic dyad, and a charged oxyanion hole was previously proposed based on recently determined FAH crystal structures. Here we report the development and characterization of an FAH inhibitor, 4-(hydroxymethylphosphinoyl)-3-oxo-butanoic acid (HMPOBA), that competes with the physiological substrate with a K(i) of 85 microM. The crystal structure of FAH complexed with HMPOBA refined at 1.3-A resolution reveals the molecular basis for the competitive inhibition, supports the proposed formation of a tetrahedral alkoxy transition state intermediate during the FAH catalyzed reaction, and reveals a Mg(2+) bound in the enzyme's active site. The analysis of FAH structures corresponding to different catalytic states reveals significant active site side-chain motions that may also be related to catalytic function. Thus, these results advance the understanding of an essential catabolic reaction associated with a fatal metabolic disease and provide insight into the structure-based development of FAH inhibitors.

Acetoacetates↗

PubMed2001

The crystal structure of yeast thiamin pyrophosphokinase.

BACKGROUND: Thiamin pyrophosphokinase (TPK) catalyzes the transfer of a pyrophosphate group from ATP to vitamin B1 (thiamin) to form the coenzyme thiamin pyrophosphate (TPP). Thus, TPK is important for the formation of a coenzyme required for central metabolic functions. TPK has no sequence homologs in the PDB and functions by an unknown mechanism. The TPK structure has been determined as a significant step toward elucidating its catalytic action. RESULTS: The crystal structure of Saccharomyces cerevisiae TPK complexed with thiamin has been determined at 1.8 A resolution. TPK is a homodimer, and each subunit consists of two domains. One domain resembles a Rossman fold with four alpha helices on each side of a 6 strand parallel beta sheet. The other domain has one 4 strand and one 6 strand antiparallel beta sheet, which form a flattened sandwich structure containing a jelly-roll topology. The active site is located in a cleft at the dimer interface and is formed from residues from domains of both subunits. The TPK dimer contains two compound active sites at the subunit interface. CONCLUSIONS: The structure of TPK with one substrate bound identifies the location of the thiamin binding site and probable catalytic residues. The structure also suggests a likely binding site for ATP. These findings are further supported by TPK sequence homologies. Although possessing no significant sequence homology with other pyrophospokinases, thiamin pyrophosphokinase may operate by a mechanism of pyrophosphoryl transfer similar to those described for pyrophosphokinases functioning in nucleotide biosynthesis.

Adenosine Triphosphate↗

PubMed2001

Structural basis of pheromone binding to mouse major urinary protein (MUP-I).

The mouse major urinary proteins are pheromone-binding proteins that function as carriers of volatile effectors of mouse physiology and behavior. Crystal structures of recombinant mouse major urinary protein-I (MUP-I) complexed with the synthetic pheromones, 2-sec-butyl-4,5-dihydrothiazole and 6-hydroxy-6-methyl-3-heptanone, have been determined at high resolution. The purification of MUP-I from mouse liver and a high-resolution structure of the natural isolate are also reported. These results show the binding of 6-hydroxy-6-methyl-3-heptanone to MUP-I, unambiguously define ligand orientations for two pheromones within the MUP-I binding site, and suggest how different chemical classes of pheromones can be accommodated within the MUP-I beta-barrel.

Animals↗

PubMed2000

Structural and functional analysis of mutations in alkaptonuria.

Alkaptonuria (AKU), the prototypic inborn error of metabolism, was the first human disease to be interpreted as a Mendelian trait by Garrod and Bateson at the beginning of last century. AKU results from impaired function of homogentisate dioxygenase (HGO), an enzyme required for the catabolism of phenylalanine and tyrosine. With the novel 7 AKU and 22 fungal mutations reported here, a total of 84 mutations impairing this enzyme have been found in the HGO gene from humans and model organisms. Forty-three of these mutations result in single amino acid substitutions. This mutational information is analysed here in the context of the HGO structure and function using kinetic assays performed using purified AKU mutant enzymes and the crystal structure of human HGO. HGO is a topologically complex structure which assembles as a functional hexamer arranged as a dimer of trimers. We show how the intricate pattern of intra- and inter-subunit interactions and the extensive surfaces required for subunit folding and association of this oligomeric enzyme can be inactivated at multiple levels by single-residue substitutions. This explains, in part, the predominance of missense mutations (67%) in AKU.

Alkaptonuria↗

PubMed2000

Crystal structure of human homogentisate dioxygenase.

Homogentisate dioxygenase (HGO) cleaves the aromatic ring during the metabolic degradation of Phe and Tyr. HGO deficiency causes alkaptonuria (AKU), the first human disease shown to be inherited as a recessive Mendelian trait. Crystal structures of apo-HGO and HGO containing an iron ion have been determined at 1.9 and 2.3 A resolution, respectively. The HGO protomer, which contains a 280-residue N-terminal domain and a 140-residue C-terminal domain, associates as a hexamer arranged as a dimer of trimers. The active site iron ion is coordinated near the interface between subunits in the HGO trimer by a Glu and two His side chains. HGO represents a new structural class of dioxygenases. The largest group of AKU associated missense mutations affect residues located in regions of contact between subunits.

Alkaptonuria↗

PubMed2000

Asymmetries in the nucleosome core particle at 2.5 A resolution.

The 2.5 A X-ray crystal structure of the nucleosome core particle presented here provides significant additions to the understanding of the nucleosome, the fundamental unit of chromatin structure. Extensions are made to the structure of the N-terminal histone tails and details are provided on hydration and ion binding. The structure is composed of twofold symmetric molecules, native chicken histone octamer cores and the DNA palindrome, which were expected to form a perfectly twofold symmetric nucleosome core particle. In fact, the result is asymmetric owing to the binding of the DNA to the protein surface and to the packing of the particles in the crystal lattice. An analysis is made of the asymmetries by comparisons both within the nucleosome core particle and to the structure of the histone octamer core of the nucleosome.

Amino Acid Sequence↗

PubMed1999

Crystal structure and mechanism of a carbon-carbon bond hydrolase.

BACKGROUND: Fumarylacetoacetate hydrolase (FAH) catalyzes the final step of tyrosine and phenylalanine catabolism, the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate, to yield fumarate and acetoacetate. FAH has no known sequence homologs and functions by an unknown mechanism. Carbon-carbon hydrolysis reactions are essential for the human metabolism of aromatic amino acids. FAH deficiency causes the fatal metabolic disease hereditary tyrosinemia type I. Carbon-carbon bond hydrolysis is also important in the microbial metabolism of aromatic compounds as part of the global carbon cycle. RESULTS: The FAH crystal structure has been determined by rapid, automated analysis of multiwavelength anomalous diffraction data. The FAH polypeptide folds into a 120-residue N-terminal domain and a 300-residue C-terminal domain. The C-terminal domain defines an unusual beta-strand topology and a novel 'mixed beta-sandwich roll' structure. The structure of FAH complexed with its physiological products was also determined. This structure reveals fumarate binding near the entrance to the active site and acetoacetate binding to an octahedrally coordinated calcium ion located in close proximity to a Glu-His dyad. CONCLUSIONS: FAH represents the first structure of a hydrolase that acts specifically on carbon-carbon bonds. FAH also defines a new class of metalloenzymes characterized by a unique alpha/beta fold. A mechanism involving a Glu-His-water catalytic triad is suggested based on structural observations, sequence conservation and mutational analysis. The histidine imidazole group is proposed to function as a general base. The Ca(2+) is proposed to function in binding substrate, activating the nucleophile and stabilizing a carbanion leaving group. An oxyanion hole formed from sidechains is proposed to stabilize a tetrahedral alkoxide transition state. The proton transferred to the carbanion leaving group is proposed to originate from a lysine sidechain. The results also reveal the molecular basis for mutations causing the hereditary tyrosinemia type 1.

Amino Acids↗

PubMed1999

Macromolecular crystal annealing: evaluation of techniques and variables.

Additional examples of successful application of macromolecular crystal annealing are presented. A qualitative evaluation of variables related to the annealing process was conducted using a variety of macromolecular crystals to determine in which cases parameters may be varied and in which cases the original macromolecular crystal annealing protocol is preferred. A hypothesis is presented relating the solvent content of the crystal to the specific protocol necessary for the successful application of annealing.

Animals↗

PubMed1998

Macromolecular crystal annealing: overcoming increased mosaicity associated with cryocrystallography.

Although cryogenic data collection has become the method of choice for macromolecular crystallography, the flash-cooling step can dramatically increase the mosaicity of some crystals. Macromolecular crystal annealing significantly reduces the mosaicity of flash-cooled crystals without affecting molecular structure. The process, which cycles a flash-cooled crystal to ambient temperature and back to cryogenic temperature, is simple, quick and requires no special equipment. The annealing process has been applied to crystals of several different macromolecules grown from different precipitants and using a variety of cryoprotectants. The protocol for macromolecular crystal annealing also has been applied to restore diffraction from flash-cooled crystals that were mishandled during transfer to or from cryogenic storage. These results will be discussed in relation to crystal mosaicity and effects of radiation damage in flash-cooled crystals.

Crystallization↗

PubMed1997

The crystal structure of the mouse glandular kallikrein-13 (prorenin converting enzyme).

A crystal structure of the serine protease, mouse glandular kallikrein 13 (mGK-13) has been determined at 2.6-A resolution. This enzyme, isolated from the mouse submandibular gland, is also known as prorenin-converting enzyme and cleaves submandibular gland Ren-2 prorenin to yield active renin. The mGK-13 structure is similar to other members of the mammalian serine protease family, having five conserved disulfide bonds and an active site located in the cleft between two beta-barrel domains. The mGK-13 structure reveals for the first time an ordered kallikrein loop conformation containing a short 3(10) helix. This loop is disordered in the related porcine pancreatic kallikrein and rat submandibular tonin structures. The kallikrein loop is in close spatial proximity to the active site and is also involved in a dimeric arrangement of mGK-13. The catalytic specificity of mGK-13 for Ren-2 prorenin was studied by modeling a prorenin-derived peptide into the active site of mGK-13. This model emphasizes two electronegative substrate specificity pockets on the mGK-13 surface, which could accommodate the dibasic P2 and P1 residues at the site of prorenin cleavage by mGK-13.

Animals↗

PubMed1995

Alteration of NH2-terminal residues of nerve growth factor affects activity and Trk binding without affecting stability or conformation.

The role of the NH2-terminal region of nerve growth factor (NGF) was studied with an NGF delta 9/13 deletion mutant, overexpressed in a baculovirus system, and mouse NGF truncated at Met-9 by cleavage with CNBr (des-(1-9)-NGF). Structural studies have been performed on the purified proteins, in addition to biological activity assessment, in order to determine effects of such modifications on global conformation and stability. The activity of NGF delta 9/13 was reduced below detectable levels, and the activity of the des-(1-9)-NGF form was decreased by at least a 50-fold in a PC12 bioassay. Competitive binding of NGF delta 9/13 to low affinity receptors on PC12 cells was not impaired; however, the mutant was not capable of competing for the cold chase-stable, high affinity binding of NGF to the cells. The binding of NGF delta 9/13 to Sf21 cells ectopically expressing the TrkA NGF receptor was also abolished. Thus, deletion of residues 9-13 significantly altered the binding affinity for the high affinity receptors on PC12 cells and for the TrkA receptor, but not for the low affinity receptor. Neither the secondary structure, determined by circular dichroism, nor the conformational stability determined by equilibrium denaturation of NGF delta 9/13 was altered as compared with wild type NGF. Slight conformational and stability perturbations of des-(1-9)-NGF were revealed by the same analysis; however, these changes were found to reflect the influence of the formic acid treatment, not the truncation of 9 residues. Our results support the conclusion that the NH2-terminal domain encompassing residues 1-9 and 9-13 is essential for maintaining the binding capability of NGF for high affinity TrkA receptors. Moreover, conformational and stability data show that the functional results of these modifications of the NH2-terminal region are directly due to receptor binding and not to secondary effects of improper folding or other indirect structural changes.

Animals↗

PubMed1994

Comparative equilibrium denaturation studies of the neurotrophins: nerve growth factor, brain-derived neurotrophic factor, neurotrophin 3, and neurotrophin 4/5.

The neurotrophins are a family of small dimeric proteins required for the development and survival of vertebrate neurons. Solvent denaturation studies were used to compare recombinant human nerve growth factor (hNGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4/5 (NT-4/5) to nerve growth factor isolated from mouse submaxillary glands (mNGF). Although greater than 50% sequence identity is conserved among this family, significant structural differences were revealed by the folding and unfolding of these proteins. Denaturation in guanidine hydrochloride and renaturation at pH 7 and 3.5 were monitored by fluorescence intensity, fluorescence polarization, and circular dichroism. The midpoint of equilibrium unfolding curves for all four neurotrophins was independent of the technique but was dependent on protein concentration, indicating that a two-state model involving native neurotrophin dimers and denatured neurotrophin monomers (N2 = 2D) describes the equilibrium between folded and unfolded neurotrophins. The conformational stabilities of the dimeric neurotrophins revealed that mNGF had the lowest conformational stability (19.3 kcal/mol); hNGF, NT-3, and NT-4/5 had intermediate stabilities, and BDNF had the highest stability (26.4 kcal/mol). Recovery of native spectroscopic characteristics upon removal of denaturant indicated that the unfolding process is reversible. Accordingly, unfolding and refolding curves were coincident for mNGF or NT-4/5 at pH 7 and 3.5 and for BDNF at pH 3.5. However, BDNF and NT-3 unfolding and refolding curves were not coincident at pH 7. The stability of the neurotrophins decreased as pH decreased, with compact monomeric intermediates (N2 = [2I] = 2D) becoming populated below pH 4. The differences in stability, pH dependence, and coincidence of refolding curves distinguish the homologous structures of the neurotrophins.

Animals↗

PubMed1994

Circular dichroism and crosslinking studies of the interaction between four neurotrophins and the extracellular domain of the low-affinity neurotrophin receptor.

Interactions between the purified recombinant receptor extracellular domain (RED) of the human low-affinity neurotrophin receptor (LANR) and recombinant human brain-derived neurotrophic factor, neurotrophin-3 (NT-3) and neuotrophin-4/5 have been studied by chemical crosslinking and circular dichroism. Conformational changes subsequent to binding have been shown by these procedures. First, relative affinities of the neurotrophins for RED were determined by binding competition assays in which radioiodinated nerve growth factor (NGF) from mouse submaxillary gland was crosslinked to RED in the presence of varying amounts of unlabeled neurotrophin competitors. RED bound each of the 3 recombinant human neurotrophins with affinities that were indistinguishable from authentic mouse NGF. These results are the first measurement of binding of the neurotrophin family to their common receptor using purified components. In order to study the effect of binding on the conformation of the proteins, CD measurements were made before and after mixing neurotrophins and RED, as had previously been done with NGF and RED (Timm DE, Vissavajjhala P, Ross AH, Neet KE, 1992, Protein Sci 1:1023-1031). Similar changes in CD spectra occurred upon combination of each of the neurotrophins and RED, with negative changes near 220-225 nm and positive changes near 190-200 nm; however, significant differences existed among the various neurotrophin-RED difference spectra. The NT-3/RED complex showed the largest spectral change and NGF the smallest. Thus, specific conformational changes in secondary structure of neurotrophin, RED, or both accompany the binding of each neurotrophin to the extracellular domain of the LANR.

Binding, Competitive↗

PubMed1994

Conformational stability of dimeric proteins: quantitative studies by equilibrium denaturation.

The conformational stability of dimeric globular proteins can be measured by equilibrium denaturation studies in solvents such as guanidine hydrochloride or urea. Many dimeric proteins denature with a 2-state equilibrium transition, whereas others have stable intermediates in the process. For those proteins showing a single transition of native dimer to denatured monomer, the conformational stabilities, delta Gu (H2O), range from 10 to 27 kcal/mol, which is significantly greater than the conformational stability found for monomeric proteins. The relative contribution of quaternary interactions to the overall stability of the dimer can be estimated by comparing delta Gu (H2O) from equilibrium denaturation studies to the free energy associated with simple dissociation in the absence of denaturant. In many cases the large stabilization energy of dimers is primarily due to the intersubunit interactions and thus gives a rationale for the formation of oligomers. The magnitude of the conformational stability is related to the size of the polypeptide in the subunit and depends upon the type of structure in the subunit interface. The practical use, interpretation, and utility of estimation of conformational stability of dimers by equilibrium denaturation methods are discussed.

Ligands↗

PubMed1993

Characterization of the lectin from the bulbs of Eranthis hyemalis (winter aconite) as an inhibitor of protein synthesis.

The lectin from Eranthis hyemalis has been previously characterized as consisting of two polypeptide chains covalently linked by disulfide bond(s) (Cammue, B. P., Peeters, B., and Peumans, W. J. (1985) Biochem. J. 227, 949-955). We have further characterized the biochemical properties of the lectin and demonstrated that it possesses the property of inhibition of protein synthesis using in vitro eukaryotic translation systems. The protein also possesses antiviral activity against the plant virus, alfalfa mosaic virus, and larvicidal activity against the southern corn rootworm, Diabrotica undecimpunctata howardii, a major insect pest of the maize plant. Both isoelectric focusing on gels and chromatofocusing indicated heterogeneity of the protein, with three species having isoelectric points in the range 4-5. The disulfide bond(s) can be rapidly reduced with beta-mercaptoethanol under native conditions. The reduced alkylated polypeptide chains remain associated under native conditions to form a species, EHL', that elutes at the same position as the native protein and has the same molecular weight by sedimentation equilibrium experiments. However, circular dichroism and fluorescence measurements indicated conformational differences between the species.

Amino Acid Sequence↗

PubMed1993

Generation and transit pathway of H+ is critical for inhibition of palmar sweating by iontophoresis in water.

Passing galvanic current across the skin (known as "tap water iontophoresis" or TWI) inhibits sweating; however, its mechanism of action is unclear. Using improved methods, we confirmed that anodal current has more of an inhibitory effect than cathodal current, water is superior to saline, and the inhibitory effect is a function of the amperage used. To address the importance of current flowing through the pores, a layer of silicone grease was placed on the skin to reduce the shunt pathway across the epidermis. With silicone, total skin conductance decreased 60% without the sweat pores being occluded, swelling of the stratum corneum and collapse of the poral lumen was prevented, and current-induced inhibition of sweating was enhanced, most likely because of an increase in current density in the pores. The pH of anodal water, but not of saline, dropped to 3, whereas that of cathodal water increased to 10 during passage of current through the skin. Acidified anodal water was superior to alkaline water. Sweat glands isolated from TWI-induced anhidrotic palmar skin responded to methacholine in vitro, but the sweat rate and pharmacological sensitivity were slightly lowered. Thus the strong acidity generated by hydrolysis of water in the anodal bath and the further accumulation of H+ in the sweat duct by anodal current may be responsible for TWI-induced inhibition of sweating due to an unknown lesion(s) in the duct or sweat pore. The secretory coil function may also be altered because of exposure to intense acidity during TWI. The importance of H+ movement into the sweat pore for inhibition of sweating could be further exploited to develop new strategies for the control of sweating.

Adolescent↗