scholarly journals Functional characterization of the non-catalytic ectodomains of the nucleotide pyrophosphatase/phosphodiesterase NPP1

2003 ◽  
Vol 371 (2) ◽  
pp. 321-330 ◽  
Author(s):  
Rik GIJSBERS ◽  
Hugo CEULEMANS ◽  
Mathieu BOLLEN
Keyword(s):  
Catalytic Activity ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Functional Characterization ◽  
Structure Stability ◽  
Subunit Structure ◽  
Terminal Domain ◽  
Nucleotide Pyrophosphatase ◽  
Domain Truncation ◽  
And Function

The ubiquitous nucleotide pyrophosphatases/phosphodiesterases NPP1–3 consist of a short intracellular N-terminal domain, a single transmembrane domain and a large extracellular part, comprising two somatomedin-B-like domains, a catalytic domain and a poorly defined C-terminal domain. We show here that the C-terminal domain of NPP1–3 is structurally related to a family of DNA/RNA non-specific endonucleases. However, none of the residues that are essential for catalysis by the endonucleases are conserved in NPP1–NPP3, suggesting that the nuclease-like domain of NPP1–3 does not represent a second catalytic domain. Truncation analysis revealed that the nuclease-like domain of NPP1 is required for protein stability, for the targeting of NPP1 to the plasma membrane and for the expression of catalytic activity. We also demonstrate that 16 conserved cysteines in the somatomedin-B-like domains of NPP1, in concert with two flanking cysteines, mediate the dimerization of NPP1. The K173Q polymorphism of NPP1, which maps to the second somatomedin-B-like domain and has been associated with the aetiology of insulin resistance, did not affect the dimerization or catalytic activity of NPP1, and did not endow NPP1 with an affinity for the insulin receptor. Our data suggest that the non-catalytic ectodomains contribute to the subunit structure, stability and function of NPP1–3.

scholarly journals Structure of Mycobacterium tuberculosis Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases

2021 ◽  
Author(s):  
Ved Mehta ◽  
Basavraj Khanppnavar ◽  
Dina Schuster ◽  
Irene Vercellino ◽  
Angela Kosturanova ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Catalytic Activity ◽  
Adenylyl Cyclase ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Vital Role ◽  
Cytosolic Side ◽  
Tm Domain ◽  
And Function

AbstractMycobacterium tuberculosis adenylyl cyclase (AC) Cya is an evolutionary ancestor of the mammalian membrane ACs and a model system for studies of their structure and function. Although the vital role of ACs in cellular signaling is well established, the function of their transmembrane (TM) regions remains unknown. Here we describe the cryo-EM structure of Cya bound to a stabilizing nanobody at 3.6 Å resolution. The TM helices 1-5 form a structurally conserved domain that facilitates the assembly of the helical and catalytic domains. The TM region contains discrete pockets accessible from the extracellular and cytosolic side of the membrane. Neutralization of the negatively charged extracellular pocket Ex1 destabilizes the cytosolic helical domain and reduces the catalytic activity of the enzyme. The TM domain acts as a functional component of Cya, guiding the assembly of the catalytic domain and providing the means for direct regulation of catalytic activity in response to extracellular ligands.One-Sentence SummaryCryo-EM structure of M. tuberculosis adenylyl cyclase Cya provides clues to the role of its transmembrane domain

scholarly journals Identification of three critical acidic residues of poly(ADP-ribose) glycohydrolase involved in catalysis: determining the PARG catalytic domain

2005 ◽  
Vol 388 (2) ◽  
pp. 493-500 ◽  
Author(s):  
Chandra N. PATEL ◽  
David W. KOH ◽  
Myron K. JACOBSON ◽  
Marcos A. OLIVEIRA
Keyword(s):  
Catalytic Activity ◽  
Catalytic Domain ◽  
Functional Characterization ◽  
Sequence Alignments ◽  
Inhibitor Binding ◽  
Conserved Sequence ◽  
Binding Residue ◽  
Acidic Residues ◽  
Hydrolysis Of

PARG [poly(ADP-ribose) glycohydrolase] catalyses the hydrolysis of α(1″→2′) or α(1‴→2″) O-glycosidic linkages of ADP-ribose polymers to produce free ADP-ribose. We investigated possible mechanistic similarities between PARG and glycosidases, which also cleave O-glycosidic linkages. Glycosidases typically utilize two acidic residues for catalysis, thus we targeted acidic residues within a conserved region of bovine PARG that has been shown to contain an inhibitor-binding site. The targeted glutamate and aspartate residues were changed to asparagine in order to minimize structural alterations. Mutants were purified and assayed for catalytic activity, as well as binding, to an immobilized PARG inhibitor to determine ability to recognize substrate. Our investigation revealed residues essential for PARG catalytic activity. Two adjacent glutamic acid residues are found in the conserved sequence Gln755-Glu-Glu757, and a third residue found in the conserved sequence Val737-Asp-Phe-Ala-Asn741. Our functional characterization of PARG residues, along with recent identification of an inhibitor-binding residue Tyr796 and a glycine-rich region Gly745-Gly-Gly747 important for PARG function, allowed us to define a PARG ‘signature sequence’ [vDFA-X3-GGg-X6–8-vQEEIRF-X3-PE-X14-E-X12-YTGYa], which we used to identify putative PARG sequences across a range of organisms. Sequence alignments, along with our mapping of PARG functional residues, suggest the presence of a conserved catalytic domain of approx. 185 residues which spans residues 610–795 in bovine PARG.

scholarly journals Crystal structure of JHP933 from Helicobacter pylori J99

2014 ◽  
Vol 70 (a1) ◽  
pp. C823-C823
Author(s):  
Sang Jae Lee ◽  
Ji Young Yoon ◽  
Bong-Jin Lee ◽  
Se Won Suh
Keyword(s):  
Crystal Structure ◽  
Helicobacter Pylori ◽  
Peptic Ulcer ◽  
Functional Characterization ◽  
Domain Architecture ◽  
Structural Similarity ◽  
Terminal Domain ◽  
Plasticity Region ◽  
H Pylori ◽  
And Function

Helicobacter pylori infection is the main cause of chronic gastritis, gastric mucosal atrophy, peptic ulcer, and some forms of gastric cancer. There has been considerable interest in strain-specific genes found outside of the cag pathogenicity island, especially genes in the plasticity regions of H. pylori. In H. pylori strain J99, the plasticity region contains 48 genes ranging from jhp0914 to jhp0961. Because little is known about many of these genes in the plasticity region, further studies are necessary to elucidate their roles in H. pylori-associated pathogenesis. The JHP933 protein, encoded by the jhp0933 gene in the plasticity region of H. pylori J99, is one of the prevalently expressed proteins in some gastritis and peptic ulcer patients. However, its structure and function remain unknown. Here, we have determined the crystal structure of JHP933, revealing the first two-domain architecture of DUF1814 family. The N-terminal domain has the nucleotidyltransferase fold and the C-terminal domain is a helix bundle. Structural similarity of JHP933 to known nucleotidyltransferases is very remote, suggesting that it may function as a novel nucleotidyltransferase. It is expected that this study will facilitate functional characterization of JHP933 to obtain an insight into its role in pathogenesis by the H. pylori plasticity region.

scholarly journals Plasma membrane-targeted ras GTPase-activating protein is a potent suppressor of p21ras function.

1993 ◽  
Vol 13 (4) ◽  
pp. 2420-2431 ◽  
Author(s):  
D C Huang ◽  
C J Marshall ◽  
J F Hancock
Keyword(s):  
Plasma Membrane ◽  
Catalytic Activity ◽  
Catalytic Domain ◽  
Cell Transformation ◽  
Negative Regulator ◽  
Membrane Localization ◽  
Plasma Membrane Localization ◽  
Terminal Domain ◽  
3T3 Fibroblasts ◽  
C Terminus

Although p21ras is localized to the plasma membrane, proteins it interacts with, such as the GTPase-activating proteins (GAPs) ras GAP and neurofibromin (NF1), are not, suggesting that one function of p21ras GTP may be to target such proteins to the plasma membrane. To investigate the effects of targeting ras GAP to the plasma membrane, ras C-terminal motifs sufficient for plasma membrane localization of p21ras were cloned onto the C terminus of ras GAP. Plasma membrane-targeted ras GAP is growth inhibitory to NIH 3T3 fibroblasts and COS cells. This growth inhibition correlates with GAP catalytic activity, since the plasma membrane-targeted C-terminal catalytic domain or the GAP-related domain of neurofibromin is inhibitory, whereas the similarly targeted N-terminal domain is not. Moreover, the inhibition is abrogated by the inactivating mutation L902I, which abolishes ras GAP catalytic activity. Coexpression of oncogenic mutant ras rescues cell viability, but the majority of rescued colonies are phenotypically untransformed. Furthermore, in focus assays, targeted ras GAP suppresses transformation by oncogenic mutant ras, and in reversion assays, targeted ras GAP can revert cells transformed by oncogenic mutant ras. Neither the targeted or nontargeted N-terminal domain nor the L902I mutant of ras GAP has any transforming activity. These data demonstrate that ras GAP can function as a negative regulator of ras and that plasma membrane localization potentiates this activity. However, if ras GAP is involved in the effector functions of p21ras, it can only be part of the effector complex for cell transformation.

scholarly journals Assessment of Structural Units Deletions in the Archaeal Oligosaccharyltransferase AglB

2020 ◽  
Author(s):  
Conrado Pedebos ◽  
Hugo Verli
Keyword(s):  
Structural Integrity ◽  
Transmembrane Domain ◽  
Functional Characterization ◽  
Wild Type Enzyme ◽  
Structural Units ◽  
Terminal Domain ◽  
Glycan Chain ◽  
The Stability ◽  
Dynamics Simulations ◽  
Binding Interfaces

AbstractOligosaccharyltransferases (OSTs) are enzymes that catalyze the transfer of a glycan chain to an acceptor protein. Their structure is composed by a transmembrane domain and a periplasmic / C-terminal domain, which can be divided into structural units. The Archaeoglobus fulgidus OST, AfAglB, has unique structural units with unknown functions. Here, we evaluate the stability role proposed for AfAglB units by employing molecular modelling and molecular dynamics simulations, to examine the effect of single and double deletions in the enzyme structure. Our results show a strong effect on the dynamics of the C-terminal domain for the mutated systems with increased fluctuations near the deleted areas. Conformational profile and stability are deeply affected, mainly in the double unit deletion, modifying the enzyme behavior and binding interfaces. Coordination at the catalytic site was not disrupted, indicating that the mutated enzymes could retain activity at some level. Hotspots of variation were identified and rationalized with previous data. Our data shows that structural units may provide stabilization interactions, contributing for integrity of the wild-type enzyme at high temperatures. By correlating our findings to structural units mutagenesis experimental data available, it was observed that structural units deletion can interfere with OSTs stability and dynamics but it is not directly related to catalysis. Instead, they may influence the OST structural integrity, and, potentially, thermostability. This work offers a basis for future experiments involving OSTs structural and functional characterization, as well as for protein engineering.

scholarly journals Role of the N-terminal transmembrane domain in the endo-lysosomal targeting and function of the human ABCB6 protein

2015 ◽  
Vol 467 (1) ◽  
pp. 127-139 ◽  
Author(s):  
Katalin Kiss ◽  
Nora Kucsma ◽  
Anna Brozik ◽  
Gabor E. Tusnady ◽  
Ptissam Bergam ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Intracellular Localization ◽  
Atp Binding ◽  
Multivesicular Bodies ◽  
Molecular Dissection ◽  
Terminal Domain ◽  
Lysosomal Targeting ◽  
And Function

The intracellular localization of ATP-binding cassette, sub family B (ABCB) 6 is a matter of debate. We show that ABCB6 is internalized from the plasma membrane to multivesicular bodies and lysosomes. Molecular dissection of the ABCB6 protein reveals a role of its N-terminal domain in targeting.

Plasma membrane-targeted ras GTPase-activating protein is a potent suppressor of p21ras function

1993 ◽  
Vol 13 (4) ◽  
pp. 2420-2431
Author(s):  
D C Huang ◽  
C J Marshall ◽  
J F Hancock
Keyword(s):  
Plasma Membrane ◽  
Catalytic Activity ◽  
Catalytic Domain ◽  
Cell Transformation ◽  
Negative Regulator ◽  
Membrane Localization ◽  
Plasma Membrane Localization ◽  
Terminal Domain ◽  
3T3 Fibroblasts ◽  
C Terminus

Although p21ras is localized to the plasma membrane, proteins it interacts with, such as the GTPase-activating proteins (GAPs) ras GAP and neurofibromin (NF1), are not, suggesting that one function of p21ras GTP may be to target such proteins to the plasma membrane. To investigate the effects of targeting ras GAP to the plasma membrane, ras C-terminal motifs sufficient for plasma membrane localization of p21ras were cloned onto the C terminus of ras GAP. Plasma membrane-targeted ras GAP is growth inhibitory to NIH 3T3 fibroblasts and COS cells. This growth inhibition correlates with GAP catalytic activity, since the plasma membrane-targeted C-terminal catalytic domain or the GAP-related domain of neurofibromin is inhibitory, whereas the similarly targeted N-terminal domain is not. Moreover, the inhibition is abrogated by the inactivating mutation L902I, which abolishes ras GAP catalytic activity. Coexpression of oncogenic mutant ras rescues cell viability, but the majority of rescued colonies are phenotypically untransformed. Furthermore, in focus assays, targeted ras GAP suppresses transformation by oncogenic mutant ras, and in reversion assays, targeted ras GAP can revert cells transformed by oncogenic mutant ras. Neither the targeted or nontargeted N-terminal domain nor the L902I mutant of ras GAP has any transforming activity. These data demonstrate that ras GAP can function as a negative regulator of ras and that plasma membrane localization potentiates this activity. However, if ras GAP is involved in the effector functions of p21ras, it can only be part of the effector complex for cell transformation.

scholarly journals Structural and functional analysis of tomato sterol C22 desaturase

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Laura Gutiérrez-García ◽  
Montserrat Arró ◽  
Teresa Altabella ◽  
Albert Ferrer ◽  
Albert Boronat
Keyword(s):  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Current Knowledge ◽  
Complex Mixture ◽  
Structural Features ◽  
Structure And Function ◽  
Cell Fractionation ◽  
Globular Domain ◽  
And Function ◽  
Functional Components

Abstract Background Sterols are structural and functional components of eukaryotic cell membranes. Plants produce a complex mixture of sterols, among which β-sitosterol, stigmasterol, campesterol, and cholesterol in some Solanaceae, are the most abundant species. Many reports have shown that the stigmasterol to β-sitosterol ratio changes during plant development and in response to stresses, suggesting that it may play a role in the regulation of these processes. In tomato (Solanum lycopersicum), changes in the stigmasterol to β-sitosterol ratio correlate with the induction of the only gene encoding sterol C22-desaturase (C22DES), the enzyme specifically involved in the conversion of β-sitosterol to stigmasterol. However, despite the biological interest of this enzyme, there is still a lack of knowledge about several relevant aspects related to its structure and function. Results In this study we report the subcellular localization of tomato C22DES in the endoplasmic reticulum (ER) based on confocal fluorescence microscopy and cell fractionation analyses. Modeling studies have also revealed that C22DES consists of two well-differentiated domains: a single N-terminal transmembrane-helix domain (TMH) anchored in the ER-membrane and a globular (or catalytic) domain that is oriented towards the cytosol. Although TMH is sufficient for the targeting and retention of the enzyme in the ER, the globular domain may also interact and be retained in the ER in the absence of the N-terminal transmembrane domain. The observation that a truncated version of C22DES lacking the TMH is enzymatically inactive revealed that the N-terminal membrane domain is essential for enzyme activity. The in silico analysis of the TMH region of plant C22DES revealed several structural features that could be involved in substrate recognition and binding. Conclusions Overall, this study contributes to expand the current knowledge on the structure and function of plant C22DES and to unveil novel aspects related to plant sterol metabolism.

DdNek2, the first non-vertebrate homologue of human Nek2, is involved in the formation of microtubule-organizing centers

2002 ◽  
Vol 115 (9) ◽  
pp. 1919-1929
Author(s):  
Ralph Gräf
Keyword(s):  
Catalytic Activity ◽  
Leucine Zipper ◽  
Catalytic Domain ◽  
Amino Acid Identity ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Microtubule Organizing Centers ◽  
Terminal Domain ◽  
C Terminus ◽  
Early Mitosis

Dictyostelium Nek2 (DdNek2) is the first structural and functional non-vertebrate homologue of human Nek2, a NIMA-related serine/threonine kinase required for centrosome splitting in early mitosis. DdNek2 shares 43% overall amino-acid identity with its human counterpart and 54% identity within the catalytic domain. Both proteins can be subdivided in an N-terminal catalytic domain, a leucine zipper and a C-terminal domain. Kinase assays with bacterially expressed DdNek2 and C-terminal deletion mutants revealed that catalytic activity requires the presence of the leucine zipper and that autophosphorylation occurs at the C-terminus. Microscopic analyses with DdNek2 antibodies and expression of a GFP-DdNek2 fusion protein in Dictyostelium showed that DdNek2 is a permanent centrosomal resident and suggested that it is a component of the centrosomal core. The GFP-DdNek2-overexpressing mutants frequently exhibit supernumerary microtubule-organizing centers (MTOCs). This phenotype did not require catalytic activity because it was also observed in cells expressing inactive GFP-K33R. However, it was shown to be caused by overexpression of the C-terminal domain since it also occurred in GFP-mutants expressing only the C-terminus or a leucine zipper/C-terminus construct but not in those mutants expressing only the catalytic domain or a catalytic domain/leucine zipper construct. These results suggest that DdNek2 is involved in the formation of MTOCs. Furthermore, the localization of the GFP-fusion proteins revealed two independent centrosomal targeting domains of DdNek2, one within the catalytic or leucine zipper domain and one in the C-terminal domain.

scholarly journals The Leishmania donovani histidine acid ecto-phosphatase LdMAcP: insight into its structure and function

2015 ◽  
Vol 467 (3) ◽  
pp. 473-486 ◽  
Author(s):  
Amalia Papadaki ◽  
Anastasia S. Politou ◽  
Despina Smirlis ◽  
Maria P. Kotini ◽  
Konstadina Kourou ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Mammalian Cells ◽  
Leishmania Donovani ◽  
Catalytic Domain ◽  
Functional Characterization ◽  
High Sequence Identity ◽  
Mouse Macrophages ◽  
Report Data ◽  
And Function

Acid ecto-phosphatase activity has been implicated in Leishmania donovani promastigote virulence. In the present study, we report data contributing to the molecular/structural and functional characterization of the L. donovani LdMAcP (L. donovani membrane acid phosphatase), member of the histidine acid phosphatase (HAcP) family. LdMAcP is membrane-anchored and shares high sequence identity with the major secreted L. donovani acid phosphatases (LdSAcPs). Sequence comparison of the LdMAcP orthologues in Leishmania sp. revealed strain polymorphism and species specificity for the L. donovani complex, responsible for visceral leishmaniasis (Khala azar), proposing thus a potential value of LdMAcP as an epidemiological or diagnostic tool. The extracellular orientation of the LdMAcP catalytic domain was confirmed in L. donovani promastigotes, wild-type (wt) and transgenic overexpressing a recombinant LdMAcP–mRFP1 (monomeric RFP1) chimera, as well as in transiently transfected mammalian cells expressing rLdMAcP–His. For the first time it is demonstrated in the present study that LdMAcP confers tartrate resistant acid ecto-phosphatase activity in live L. donovani promastigotes. The latter confirmed the long sought molecular identity of at least one enzyme contributing to this activity. Interestingly, the L. donovani rLdMAcP–mRFP1 promastigotes generated in this study, showed significantly higher infectivity and virulence indexes than control parasites in the infection of J774 mouse macrophages highlighting thereby a role for LdMAcP in the parasite's virulence.

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