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1.
J Struct Biol ; 213(1): 107691, 2021 03.
Article in English | MEDLINE | ID: mdl-33387654

ABSTRACT

GTP Cyclohydrolase I (GCH1) catalyses the conversion of guanosine triphosphate (GTP) to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). BH4 functions as co-factor in neurotransmitter biosynthesis. BH4 homeostasis is a promising target to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). Dependent on the relative cellular concentrations of effector ligands, BH4 and phenylalanine, GFRP binds GCH1 to form inhibited or activated complexes, respectively. We determined high-resolution structures of the ligand-free and -bound human GFRP and GCH1-GFRP complexes by X-ray crystallography. Highly similar binding modes of the substrate analogue 7-deaza-GTP to active and inhibited GCH1-GFRP complexes confirm a novel, dissociation rate-controlled mechanism of non-competitive inhibition to be at work. Further, analysis of all structures shows that upon binding of the effector molecules, the conformations of GCH1 or GFRP are altered and form highly complementary surfaces triggering a picomolar interaction of GFRP and GCH1 with extremely slow koff values, while GCH1-GFRP complexes rapidly disintegrate in absence of BH4 or phenylalanine. Finally, comparing behavior of full-length and N-terminally truncated GCH1 we conclude that the disordered GCH1 N-terminus does not have impact on complex formation and enzymatic activity. In summary, this comprehensive and methodologically diverse study helps to provide a better understanding of the regulation of GCH1 by GFRP and could thus stimulate research on GCH1 modulating drugs.


Subject(s)
GTP Cyclohydrolase/chemistry , GTP Cyclohydrolase/metabolism , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Biophysics/methods , Crystallography, X-Ray/methods , Feedback , Humans , Phenylalanine/chemistry , Phenylalanine/metabolism
2.
Proc Natl Acad Sci U S A ; 117(50): 31838-31849, 2020 12 15.
Article in English | MEDLINE | ID: mdl-33229582

ABSTRACT

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1-GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.


Subject(s)
GTP Cyclohydrolase/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Allosteric Regulation , Allosteric Site/genetics , Biopterins/analogs & derivatives , Biopterins/metabolism , Cryoelectron Microscopy , Crystallography, X-Ray , GTP Cyclohydrolase/genetics , GTP Cyclohydrolase/ultrastructure , Mutagenesis, Site-Directed , Phenylalanine/metabolism , Protein Structure, Quaternary
3.
Article in English | MEDLINE | ID: mdl-23722832

ABSTRACT

Keap1 is a substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex and plays an important role in the cellular response to oxidative stress. It binds Nrf2 with its Kelch domain and thus triggers the ubiquitinylation and degradation of Nrf2. Oxidative stress prevents the degradation of Nrf2 and leads to the activation of cytoprotective genes. Therefore, Keap1 is an attractive drug target in inflammatory diseases. The support of a medicinal chemistry effort by structural research requires a robust crystallization system in which the crystals are preferably suited for performing soaking experiments. This facilitates the generation of protein-ligand complexes in a routine and high-throughput manner. The structure of human Keap1 has been described previously. In this crystal form, however, the binding site for Nrf2 was blocked by a crystal contact. This interaction was analysed and mutations were introduced to disrupt this crystal contact. One double mutation (E540A/E542A) crystallized in a new crystal form in which the binding site for Nrf2 was not blocked and was accessible to small-molecule ligands. The crystal structures of the apo form of the mutated Keap1 Kelch domain (1.98 Å resolution) and of the complex with an Nrf2-derived peptide obtained by soaking (2.20 Å resolution) are reported.


Subject(s)
Carrier Proteins/chemistry , Chemical Engineering/methods , Intracellular Signaling Peptides and Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Crystallization , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Kelch-Like ECH-Associated Protein 1 , Ligands , NF-E2-Related Factor 2/chemistry , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Protein Structure, Secondary/genetics , Protein Structure, Tertiary/genetics
4.
Biochemistry ; 52(20): 3523-31, 2013 May 21.
Article in English | MEDLINE | ID: mdl-23621087

ABSTRACT

Glucokinase (GK) plays a major role in the regulation of blood glucose homeostasis in both the liver and the pancreas. In the liver, GK is controlled by the GK regulatory protein (GKRP). GKRP in turn is activated by fructose 6-phosphate (F6P) and inactivated by fructose 1-phosphate (F1P). Disrupting the GK-GKRP complex increases the activity of GK in the cytosol and is considered an attractive concept for the regulation of blood glucose. We have determined the crystal structure of GKRP in its inactive F1P-bound form. The binding site for F1P is located deeply buried at a domain interface, and H-D exchange experiments confirmed that F1P and F6P compete for this site. The structure of the inactive GKRP-F1P complex provides a starting point for understanding the mechanism of fructose phosphate-dependent GK regulation at an atomic level.


Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Binding Sites , Crystallography, X-Ray , Fructosephosphates/chemistry , Fructosephosphates/metabolism , Humans , Models, Molecular , Protein Conformation
5.
J Mol Biol ; 381(1): 150-9, 2008 Aug 01.
Article in English | MEDLINE | ID: mdl-18586267

ABSTRACT

The crystal structure of the full mosquitocidal toxin from Bacillus sphaericus (MTX(holo)) has been determined at 2.5 A resolution by the molecular replacement method. The resulting structure revealed essentially the complete chain consisting of four ricin B-type domains curling around the catalytic domain in a hedgehog-like assembly. As the structure was virtually identical in three different crystal packings, it is probably not affected by packing contacts. The structure of MTX(holo) explains earlier autoinhibition data. An analysis of published complexes comprising ricin B-type lectin domains and sugar molecules shows that the general construction principle applies to all four lectin domains of MTX(holo), indicating 12 putative sugar-binding sites. These sites are sequence-related to those of the cytotoxin pierisin from cabbage butterfly, which are known to bind glycolipids. It seems therefore likely that MTX(holo) also binds glycolipids. The seven contact interfaces between the five domains are predominantly polar and not stronger than common crystal contacts so that in an appropriate environment, the multidomain structure would likely uncurl into a string of single domains. The structure of the isolated catalytic domain plus an extended linker was established earlier in three crystal packings, two of which showed a peculiar association around a 7-fold axis. The catalytic domain of the reported MTX(holo) closely resembles all three published structures, except one with an appreciable deviation of the 40 N-terminal residues. A comparison of all structures suggests a possible scenario for the translocation of the toxin into the cytosol.


Subject(s)
Culicidae , Glycosides/chemistry , Glycosides/metabolism , Insecticides/chemistry , Insecticides/metabolism , Triterpenes/chemistry , Triterpenes/metabolism , Amino Acid Sequence , Animals , Bacillus/enzymology , Bacillus/genetics , Binding Sites , Carbohydrates/chemistry , Catalytic Domain , Crystallography, X-Ray , Glycosides/genetics , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Folding , Protein Structure, Tertiary , Sequence Alignment
6.
J Mol Biol ; 357(4): 1226-36, 2006 Apr 07.
Article in English | MEDLINE | ID: mdl-16483607

ABSTRACT

The catalytic domain of a mosquitocidal toxin prolonged by a C-terminal 44 residue linker connecting to four ricin B-like domains was crystallized. Three crystal structures were established at resolutions between 2.5A and 3.0A using multi-wavelength and single-wavelength anomalous X-ray diffraction as well as molecular replacement phasing techniques. The chainfold of the toxin fragment corresponds to those of ADP-ribosylating enzymes. At pH 4.3 the fragment is associated in a C(7)-symmetric heptamer in agreement with an aggregate of similar size observed by size-exclusion chromatography. In two distinct crystal forms, the heptamers formed nearly spherical, D(7)-symmetric tetradecamers. Another crystal form obtained at pH 6.3 contained a recurring C(2)-symmetric tetramer, which, however, was not stable in solution. On the basis of the common chainfold and NAD(+)-binding site of all ADP-ribosyl transferases, the NAD(+)-binding site of the toxin was assigned at a high confidence level. In all three crystal forms the NAD(+) site was occupied by part of the 44 residue linker, explaining the known inhibitory effect of this polypeptide region. The structure showed that the cleavage site for toxin activation is in a highly mobile loop that is exposed in the monomer. Since it contains the inhibitory linker as a crucial part of the association contact, the observed heptamer is inactive. Moreover, the heptamer cannot be activated by proteolysis because the activation loop is at the ring center and not accessible for proteases. Therefore the heptamer, or possibly the tetradecamer, seems to represent an inactive storage form of the toxin.


Subject(s)
Aedes , Bacillus/chemistry , Bacterial Proteins/chemistry , Bacterial Toxins/chemistry , Insecticides/chemistry , Peptides/chemistry , Amino Acid Sequence , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Toxins/genetics , Bacterial Toxins/metabolism , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Insecticides/metabolism , Models, Molecular , Molecular Sequence Data , NAD/metabolism , Peptides/genetics , Peptides/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Structure-Activity Relationship
7.
J Biol Chem ; 280(45): 37833-8, 2005 Nov 11.
Article in English | MEDLINE | ID: mdl-16157585

ABSTRACT

The large cytotoxins of Clostridia species glycosylate and thereby inactivate small GTPases of the Rho family. Clostridium difficile toxins A and B and Clostridium sordellii lethal toxin use UDP-glucose as the donor for glucosylation of Rho/Ras GTPases. In contrast, alpha-toxin from Clostridium novyi N-acetylglucosaminylates Rho GTPases by using UDP-N-acetylglucosamine as a donor substrate. Based on the crystal structure of C. difficile toxin B, we studied the sugar donor specificity of the toxins by site-directed mutagenesis. The changing of Ile-383 and Gln-385 in toxin B to serine and alanine, respectively, largely increased the acceptance of UDP-N-acetylglucosamine as a sugar donor for modification of RhoA. The K(m) value was reduced from 960 to 26 mum for the double mutant. Accordingly, the potential of the double mutant of toxin B to hydrolyze UDP-N-acetylglucosamine was higher than that for UDP-glucose. The changing of Ile-383 and Gln-385 in the lethal toxin of C. sordellii allowed modification of Ras in the presence of UDP-N-acetyl-glucosamine and reduced the acceptance of UDP-glucose as a donor for glycosylation. Vice versa, the changing of the equivalent residues in C. novyi alpha-toxin from Ser-385 and Ala-387 to isoleucine and glutamine, respectively, reversed the donor specificity of the toxin from UDP-N-acetylglucosamine to UDP-glucose. These data demonstrate that two amino acid residues are crucial for the co-substrate specificity of clostridial glycosylating toxins.


Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Toxins/genetics , Bacterial Toxins/metabolism , Mutagenesis, Site-Directed , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Toxins/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Kinetics , Mutation , Protein Binding , Protein Conformation , Sequence Homology, Amino Acid , Substrate Specificity , Uridine Diphosphate Glucose/chemistry , Uridine Diphosphate Glucose/metabolism , Uridine Diphosphate N-Acetylglucosamine/chemistry , Uridine Diphosphate N-Acetylglucosamine/metabolism
8.
J Mol Biol ; 351(5): 973-81, 2005 Sep 02.
Article in English | MEDLINE | ID: mdl-16054646

ABSTRACT

Toxin B is a member of the family of large clostridial cytotoxins which are of great medical importance. Its catalytic fragment was crystallized in the presence of UDP-glucose and Mn2+. The structure was determined at 2.2 A resolution, showing that toxin B belongs to the glycosyltransferase type A family. However, toxin B contains as many as 309 residues in addition to the common chainfold, which most likely contribute to the target specificity. A superposition with other glycosyltransferases shows the expected positions of the acceptor oxygen atom during glucosyl transfer and indicates further that the reaction proceeds probably along a single-displacement pathway. The C1'' donor carbon atom position is defined by the bound UDP and glucose. It assigns the surface area of toxin B that forms the interface to the target protein during the modifying reaction. A docking attempt brought the known acceptor atom, Thr37 O(gamma1) of the switch I region of the RhoA:GDP target structure, near the expected position. The relative orientation of the two proteins was consistent with both being attached to a membrane. Sequence comparisons between toxin B variants revealed that the highest exchange rate occurs around the active center at the putative docking interface, presumably due to a continuous hit-and-evasion struggle between Clostridia and their eukaryotic hosts.


Subject(s)
Bacterial Proteins/genetics , Bacterial Toxins/genetics , Manganese/chemistry , Uridine Diphosphate Glucose/chemistry , Binding Sites , Carbon/chemistry , Catalysis , Clostridioides difficile/metabolism , Crystallography, X-Ray , Escherichia coli/metabolism , Glucose/chemistry , Glutathione Transferase/metabolism , Glycosyltransferases/metabolism , Hydrolysis , Ligands , Models, Molecular , Molecular Conformation , Mutation , Protein Binding , Protein Conformation , Protein Isoforms , Stereoisomerism , Uridine Diphosphate/chemistry
9.
Chem Biol ; 11(1): 121-6, 2004 Jan.
Article in English | MEDLINE | ID: mdl-15113001

ABSTRACT

The membrane protein squalene-hopene cyclase was cocrystallized with 2-azasqualene and analyzed by X-ray diffraction to 2.13 A resolution. The conformation of this close analog was clearly established, and it agreed with the common textbook presentation. The bound squalene undergoes only small conformational changes during the formation of rings A through D, thus requiring no intermediate. However, ring E formation is hindered by an entropic barrier, which may explain its absence in the steroids. The structure analysis revealed a mobile region between the active center cavity and the membrane, which may melt, opening a passage for squalene and hopene.


Subject(s)
Intramolecular Transferases/chemistry , Membrane Proteins/chemistry , Squalene/chemistry , Triterpenes/chemical synthesis , Bacillaceae/enzymology , Binding Sites , Catalysis , Cell Membrane/metabolism , Crystallography, X-Ray , Cyclization , Intramolecular Transferases/analysis , Intramolecular Transferases/metabolism , Membrane Proteins/analysis , Membrane Proteins/metabolism , Models, Molecular , Protein Conformation , Squalene/analogs & derivatives
10.
J Med Chem ; 46(11): 2083-92, 2003 May 22.
Article in English | MEDLINE | ID: mdl-12747780

ABSTRACT

The binding structures of 11 human oxidosqualene cyclase inhibitors designed as cholesterol-lowering agents were determined for the squalene-hopene cyclase from Alicyclobacillus acidocaldarius, which is the only structurally known homologue of the human enzyme. The complexes were produced by cocrystallization, and the structures were elucidated by X-ray diffraction analyses. All inhibitors were bound in the large active center cavity. The detailed binding structures are presented and discussed in the light of the IC50 values of these 11 as well as 17 other inhibitors. They provide a consistent picture for the inhibition of the bacterial enzyme and can be used to adjust and improve homology models of the human enzyme. The detailed active center structures of the two enzymes are too different to show an IC50 correlation.


Subject(s)
Anticholesteremic Agents/chemistry , Enzyme Inhibitors/chemistry , Intramolecular Transferases/antagonists & inhibitors , Amines/chemistry , Anticholesteremic Agents/pharmacology , Bacillaceae/chemistry , Benzene Derivatives/chemistry , Benzophenones/chemistry , Benzophenones/pharmacology , Binding Sites , Crystallography, X-Ray , Cyclopropanes/chemistry , Enzyme Inhibitors/pharmacology , Humans , In Vitro Techniques , Intramolecular Transferases/chemistry , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Models, Molecular , Protein Binding , Structure-Activity Relationship
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