scholarly journals Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme)

2005 ◽  
Vol 392 (1) ◽  
pp. 39-45 ◽  
Author(s):  
Hui-Chih Hung ◽  
Meng-Wei Kuo ◽  
Gu-Gang Chang ◽  
Guang-Yaw Liu
Keyword(s):  
Malate Dehydrogenase ◽  
Conformational Changes ◽  
Malic Enzyme ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Activation Mechanism ◽  
Amino Acid Residues ◽  
Allosteric Site ◽  
Charged Amino Acids

Human mitochondrial NAD(P)+-dependent malate dehydrogenase (decarboxylating) (malic enzyme) can be specifically and allosterically activated by fumarate. X-ray crystal structures have revealed conformational changes in the enzyme in the absence and in the presence of fumarate. Previous studies have indicated that fumarate is bound to the allosteric pocket via Arg67 and Arg91. Mutation of these residues almost abolishes the activating effect of fumarate. However, these amino acid residues are conserved in some enzymes that are not activated by fumarate, suggesting that there may be additional factors controlling the activation mechanism. In the present study, we tried to delineate the detailed molecular mechanism of activation of the enzyme by fumarate. Site-directed mutagenesis was used to replace Asp102, which is one of the charged amino acids in the fumarate binding pocket and is not conserved in other decarboxylating malate dehydrogenases. In order to explore the charge effect of this residue, Asp102 was replaced by alanine, glutamate or lysine. Our experimental data clearly indicate the importance of Asp102 for activation by fumarate. Mutation of Asp102 to Ala or Lys significantly attenuated the activating effect of fumarate on the enzyme. Kinetic parameters indicate that the effect of fumarate was mainly to decrease the Km values for malate, Mg2+ and NAD+, but it did not notably elevate kcat. The apparent substrate Km values were reduced by increasing concentrations of fumarate. Furthermore, the greatest effect of fumarate activation was apparent at low malate, Mg2+ or NAD+ concentrations. The Kact values were reduced with increasing concentrations of malate, Mg2+ and NAD+. The Asp102 mutants, however, are much less sensitive to regulation by fumarate. Mutation of Asp102 leads to the desensitization of the co-operative effect between fumarate and substrates of the enzyme.

scholarly journals Characterization of the ExoU activation mechanism using EPR and integrative modeling

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Maxx H. Tessmer ◽  
Samuel A. DeCero ◽  
Diego del Alamo ◽  
Molly O. Riegert ◽  
Jens Meiler ◽  
...  
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
Membrane Lipids ◽  
Binding Pocket ◽  
Activation Mechanism ◽  
Amino Acid Residues ◽  
Distance Distributions ◽  
Associated Proteins ◽  
Site Directed Spin Labeling ◽  
Double Electron Electron Resonance

AbstractExoU, a type III secreted phospholipase effector of Pseudomonas aeruginosa, serves as a prototype to model large, dynamic, membrane-associated proteins. ExoU is synergistically activated by interactions with membrane lipids and ubiquitin. To dissect the activation mechanism, structural homology was used to identify an unstructured loop of approximately 20 residues in the ExoU amino acid sequence. Mutational analyses indicate the importance of specific loop amino acid residues in mediating catalytic activity. Engineered disulfide cross-links show that loop movement is required for activation. Site directed spin labeling EPR and DEER (double electron–electron resonance) studies of apo and holo states demonstrate local conformational changes at specific sites within the loop and a conformational shift of the loop during activation. These data are consistent with the formation of a substrate-binding pocket providing access to the catalytic site. DEER distance distributions were used as constraints in RosettaDEER to construct ensemble models of the loop in both apo and holo states, significantly extending the range for modeling a conformationally dynamic loop.

Block of Human Heart hH1 Sodium Channels by the Enantiomers of Bupivacaine

2000 ◽  
Vol 93 (4) ◽  
pp. 1022-1033 ◽  
Author(s):  
Carla Nau ◽  
Sho-Ya Wang ◽  
Gary R. Strichartz ◽  
Ging Kuo Wang
Keyword(s):  
Human Heart ◽  
Point Mutations ◽  
Cell Voltage ◽  
Site Directed Mutagenesis ◽  
Na Channel ◽  
Amino Acid Residues ◽  
Na Channels ◽  
State Dependent ◽  
Positively Charged

Background S(-)-bupivacaine reportedly exhibits lower cardiotoxicity but similar local anesthetic potency compared with R(+)-bupivacaine. The bupivacaine binding site in human heart (hH1) Na+ channels has not been studied to date. The authors investigated the interaction of bupivacaine enantiomers with hH1 Na+ channels, assessed the contribution of putatively relevant residues to binding, and compared the intrinsic affinities to another isoform, the rat skeletal muscle (mu1) Na+ channel. Methods Human heart and mu1 Na+ channel alpha subunits were transiently expressed in HEK293t cells and investigated during whole cell voltage-clamp conditions. Using site-directed mutagenesis, the authors created point mutations at positions hH1-F1760, hH1-N1765, hH1-Y1767, and hH1-N406 by introducing the positively charged lysine (K) or the negatively charged aspartic acid (D) and studied their influence on state-dependent block by bupivacaine enantiomers. Results Inactivated hH1 Na+ channels displayed a weak stereoselectivity with a stereopotency ratio (+/-) of 1.5. In mutations hH1-F1760K and hH1-N1765K, bupivacaine affinity of inactivated channels was reduced by approximately 20- to 40-fold, in mutation hH1-N406K by approximately sevenfold, and in mutations hH1-Y1767K and hH1-Y1767D by approximately twofold to threefold. Changes in recovery of inactivated mutant channels from block paralleled those of inactivated channel affinity. Inactivated hH1 Na+ channels exhibited a slightly higher intrinsic affinity than mu1 Na+ channels. Conclusions Differences in bupivacaine stereoselectivity and intrinsic affinity between hH1 and mu1 Na+ channels are small and most likely of minor clinical relevance. Amino acid residues in positions hH1-F1760, hH1-N1765, and hH1-N406 may contribute to binding of bupivacaine enantiomers in hH1 Na+ channels, whereas the role of hH1-Y1767 remains unclear.

scholarly journals Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO

2019 ◽  
Vol 20 (6) ◽  
pp. 1444 ◽  
Author(s):  
Soria Iatmanen-Harbi ◽  
lucile Senicourt ◽  
Vassilios Papadopoulos ◽  
Olivier Lequin ◽  
Jean-Jacques Lacapere
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Changes ◽  
Protoporphyrin Ix ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Translocator Protein ◽  
Binding Affinities ◽  
Ligand Selectivity ◽  
High Affinity

The optimization of translocator protein (TSPO) ligands for Positron Emission Tomography as well as for the modulation of neurosteroids is a critical necessity for the development of TSPO-based diagnostics and therapeutics of neuropsychiatrics and neurodegenerative disorders. Structural hints on the interaction site and ligand binding mechanism are essential for the development of efficient TSPO ligands. Recently published atomic structures of recombinant mammalian and bacterial TSPO1, bound with either the high-affinity drug ligand PK 11195 or protoporphyrin IX, have revealed the membrane protein topology and the ligand binding pocket. The ligand is surrounded by amino acids from the five transmembrane helices as well as the cytosolic loops. However, the precise mechanism of ligand binding remains unknown. Previous biochemical studies had suggested that ligand selectivity and binding was governed by these loops. We performed site-directed mutagenesis to further test this hypothesis and measured the binding affinities. We show that aromatic residues (Y34 and F100) from the cytosolic loops contribute to PK 11195 access to its binding site. Limited proteolytic digestion, circular dichroism and solution two-dimensional (2-D) NMR using selective amino acid labelling provide information on the intramolecular flexibility and conformational changes in the TSPO structure upon PK 11195 binding. We also discuss the differences in the PK 11195 binding affinities and the primary structure between TSPO (TSPO1) and its paralogous gene product TSPO2.

scholarly journals Efficient Subgroup C Avian Sarcoma and Leukosis Virus Receptor Activity Requires the IgV Domain of the Tvc Receptor and Proper Display on the Cell Membrane

2008 ◽  
Vol 82 (22) ◽  
pp. 11419-11428 ◽  
Author(s):  
Audelia Munguia ◽  
Mark J. Federspiel
Keyword(s):  
Amino Acid ◽  
Cell Membrane ◽  
Conformational Changes ◽  
Aromatic Amino Acid ◽  
Receptor Activity ◽  
Site Directed Mutagenesis ◽  
Receptor Interaction ◽  
Common Mechanism ◽  
Amino Acid Residues ◽  
Avian Sarcoma

ABSTRACT We recently identified and cloned the receptor for subgroup C avian sarcoma and leukosis viruses [ASLV(C)], i.e., Tvc, a protein most closely related to mammalian butyrophilins, which are members of the immunoglobulin protein family. The extracellular domain of Tvc contains two immunoglobulin-like domains, IgV and IgC, which presumably each contain a disulfide bond important for native function of the protein. In this study, we have begun to identify the functional determinants of Tvc responsible for ASLV(C) receptor activity. We found that the IgV domain of the Tvc receptor is responsible for interacting with the glycoprotein of ASLV(C). Additional experiments demonstrated that a domain was necessary as a spacer between the IgV domain and the membrane-spanning domain for efficient Tvc receptor activity, most likely to orient the IgV domain a proper distance from the cell membrane. The effects on ASLV(C) glycoprotein binding and infection efficiency were also studied by site-directed mutagenesis of the cysteine residues of Tvc as well as conserved amino acid residues of the IgV Tvc domain compared to other IgV domains. In this initial analysis of Tvc determinants important for interacting with ASLV(C) glycoproteins, at least two aromatic amino acid residues in the IgV domain of Tvc, Trp-48 and Tyr-105, were identified as critical for efficient ASLV(C) infection. Interestingly, one or more aromatic amino acid residues have been identified as critical determinants in the other ASLV(A-E) receptors for a proper interaction with ASLV glycoproteins. This suggests that the ASLV glycoproteins may share a common mechanism of receptor interaction with an aromatic residue(s) on the receptor critical for triggering conformational changes in SU that initiate the fusion process required for efficient virus infection.

scholarly journals Role of Hydrophobic Residues in the Central Ectodomain of gp41 in Maintaining the Association between Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Subunits gp120 and gp41

2004 ◽  
Vol 78 (9) ◽  
pp. 4921-4926 ◽  
Author(s):  
Joanne York ◽  
Jack H. Nunberg
Keyword(s):  
Human Immunodeficiency Virus ◽  
Conformational Changes ◽  
Envelope Glycoprotein ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Virus Envelope ◽  
Hydrophobic Residues ◽  
Immunodeficiency Virus ◽  
Mediate Cell ◽  
The Stability

ABSTRACT The interaction between the gp120 and gp41 subunits of the human immunodeficiency virus envelope glycoprotein serves to stabilize the virion form of the complex and to transmit receptor-induced conformational changes in gp120 to trigger the membrane fusion activity of gp41. In this study, we used site-directed mutagenesis to identify amino acid residues in the central ectodomain of gp41 that contribute to the stability of the gp120-gp41 association. We identified alanine mutations at six positions, including four tryptophan residues, which result in mutant envelope glycoprotein complexes that fail to retain gp120 on the cell surface. These envelope glycoproteins readily shed their gp120 and are unable to mediate cell-cell fusion. These findings suggest an important role for the conserved bulky hydrophobic residues in stabilizing the gp120-gp41 complex.

scholarly journals Isolation from Ochrobactrum anthropi of a Novel Class II 5-Enopyruvylshikimate-3-Phosphate Synthase with High Tolerance to Glyphosate

2010 ◽  
Vol 76 (17) ◽  
pp. 6001-6005 ◽  
Author(s):  
Yong-Sheng Tian ◽  
Ai-Sheng Xiong ◽  
Jing Xu ◽  
Wei Zhao ◽  
Feng Gao ◽  
...  
Keyword(s):  
Genomic Library ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Construction Process ◽  
Ochrobactrum Anthropi ◽  
Gene Encoding ◽  
Phosphate Synthase

ABSTRACT Applying the genomic library construction process and colony screening, a novel aro A gene encoding 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified, cloned, and overexpressed, and the enzyme was purified to homogeneity. Furthermore, site-directed mutagenesis was employed to assess the role of single amino acid residues in glyphosate resistance.

scholarly journals Epigenetic modification in the expression of p73 p73 - epigenetic target for anticancer therapy

2019 ◽  
Vol 13 (2) ◽  
Author(s):  
Faiza Naseer ◽  
Mohammad Saleem
Keyword(s):  
Conformational Changes ◽  
Posttranslational Modifications ◽  
Epigenetic Modification ◽  
Amino Acid Residues ◽  
P53 Family ◽  
After Effects ◽  
Cycle Arrest ◽  
C Terminus ◽  
Upstream Promoter

A p73 is a new member of p53 family of transcription factor, having two types. First is TAp73, transcriptionally active and expressed via upstream promoter as a tumor suppressor and vital apoptotic inductor, it also has a key role in cell cycle arrest/differentiation and Second is ΔNp73 that is transcriptionally inactive and expressed via downstream regulator as oncogenes. Both types are expressed in various isoforms, which originate from alternative splicing events at the C-terminus. Upon DNA damage, posttranslational modifications cause conformational changes in various amino acid residues via induction or inhibition of various proteins, which are present in the structural domains of p73. These modifications may cause up- or down-regulation of p73 expression levels, as well as alters the transcriptional activity and/or stability of the protein. In this review, we have made an effort to assemble all existing data regarding the role of p73, its modification and after effects in cancer.

Glutamic acid-65 is an essential residue for catalysis in Proteus mirabilis glutathione S-transferase B1-1

2002 ◽  
Vol 363 (1) ◽  
pp. 189-193 ◽  
Author(s):  
Nerino ALLOCATI ◽  
Michele MASULLI ◽  
Enrico CASALONE ◽  
Silvia SANTUCCI ◽  
Bartolo FAVALORO ◽  
...  
Keyword(s):  
Proteus Mirabilis ◽  
Active Site ◽  
Structural Integrity ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Cd Spectra ◽  
Glutathione S Transferase ◽  
Terminal Amino

The functional role of three conserved amino acid residues in Proteus mirabilis glutathione S-transferase B1-1 (PmGST B1-1) has been investigated by site-directed mutagenesis. Crystallographic analyses indicated that Glu65, Ser103 and Glu104 are in hydrogen-bonding distance of the N-terminal amino group of the γ-glutamyl moiety of the co-substrate, GSH. Glu65 was mutated to either aspartic acid or leucine, and Ser103 and Glu104 were both mutated to alanine. Glu65 mutants (Glu65→Asp and Glu65→Leu) lost all enzyme activity, and a drastic decrease in catalytic efficiency was observed for Ser103→Ala and Glu104→Ala mutants toward both 1-chloro-2,4-dinitrobenzene and GSH. On the other hand, all mutants displayed similar intrinsic fluorescence, CD spectra and thermal stability, indicating that the mutations did not affect the structural integrity of the enzyme. Taken together, these results indicate that Ser103 and Glu104 are significantly involved in the interaction with GSH at the active site of PmGST B1-1, whereas Glu65 is crucial for catalysis.

scholarly journals Analysis of Binding Sites for the New Small-Molecule CCR5 Antagonist TAK-220 on Human CCR5

2005 ◽  
Vol 49 (11) ◽  
pp. 4708-4715 ◽  
Author(s):  
Masao Nishikawa ◽  
Katsunori Takashima ◽  
Toshiya Nishi ◽  
Rika A. Furuta ◽  
Naoyuki Kanzaki ◽  
...  
Keyword(s):  
Amino Acid ◽  
Small Molecule ◽  
Conformational Changes ◽  
Binding Pocket ◽  
Inhibitory Effect ◽  
Ccr5 Antagonist ◽  
Amino Acid Residues ◽  
Ccr5 Antagonists ◽  
Human Ccr5 ◽  
Hiv 1

ABSTRACT G protein-coupled receptor CCR5 is the main coreceptor for macrophage-tropic human immunodeficiency virus type 1 (HIV-1), and various small-molecule CCR5 antagonists are being developed to treat HIV-1 infection. It has been reported that such CCR5 antagonists, including TAK-779, bind to a putative binding pocket formed by transmembrane domains (TMs) 1, 2, 3 and 7 of CCR5, indicating the importance of the conformational changes of the TMs during virus entry. In this report, using a single-round infection assay with human CCR5 and its substitution mutants, we demonstrated that a new CCR5 antagonist, TAK-220, shares the putative interacting amino acid residues Asn252 and Leu255 in TM6 with TAK-779 but also requires the distinct residues Gly163 and Ile198 in TMs 4 and 5, respectively, for its inhibitory effect. We suggested that, together with molecular models of the interactions between the drugs and CCR5, the inhibitory activity of TAK-220 could involve direct interactions with amino acid residues in TMs 4, 5, and 6 in addition to those in the previously postulated binding pocket. The possible interaction of drugs with additional regions of the CCR5 molecule would help to develop a new small-molecule CCR5 antagonist.

scholarly journals Mu-class glutathione transferase from Xenopus laevis: molecular cloning, expression and site-directed mutagenesis

2002 ◽  
Vol 365 (3) ◽  
pp. 685-691 ◽  
Author(s):  
Antonella De LUCA ◽  
Bartolo FAVALORO ◽  
Stefania ANGELUCCI ◽  
Paolo SACCHETTA ◽  
Carmine Di ILIO
Keyword(s):  
Xenopus Laevis ◽  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Structural Instability ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Calculated Molecular Mass

A cDNA encoding a Mu-class glutathione transferase (XlGSTM1-1) has been isolated from a Xenopus laevis liver library, and its nucleotide sequence has been determined. XlGSTM1-1 is composed of 219 amino acid residues with a calculated molecular mass of 25359Da. Unlike many mammalian Mu-class GSTs, XlGSTM1-1 has a narrow spectrum of substrate specificity and it is also less effective in conjugating 1-chloro-2,4-dinitrobenzene. A notable structural feature of XlGSTM1-1 is the presence of the Cys-139 residue in place of the Glu-139, as well as the absence of the Cys-114 residue, present in other Mu-class GSTs, which is replaced by Ala. Site-directed mutagenesis experiments indicate that Cys-139 is not involved in the catalytic mechanism of XlGSTM1-1 but may be in part responsible for its structural instability, and experiments in vivo confirmed the role of this residue in stability. Evidence indicating that Arg-107 is essential for the 1-chloro-2,4-dinitrobenzene conjugation capacity of XlGSTM1-1 is also presented.

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