Faculty Opinions recommendation of Structures of the Catalytic Domain of Bacterial Primase DnaG in Complexes with DNA Provide Insight into Key Priming Events.

2018 ◽  
Author(s):  
Luca Pellegrini
Keyword(s):  
Catalytic Domain ◽  
Insight Into

scholarly journals Structure and function of factor XI

Blood ◽  
2010 ◽  
Vol 115 (13) ◽  
pp. 2569-2577 ◽  
Author(s):  
Jonas Emsley ◽  
Paul A. McEwan ◽  
David Gailani
Keyword(s):  
Catalytic Domain ◽  
Structural Features ◽  
Factor Ix ◽  
Missense Mutations ◽  
Factor Xi ◽  
Disk Structure ◽  
Ligand Binding Sites ◽  
And Function ◽  
Insight Into

AbstractFactor XI (FXI) is the zymogen of an enzyme (FXIa) that contributes to hemostasis by activating factor IX. Although bleeding associated with FXI deficiency is relatively mild, there has been resurgence of interest in FXI because of studies indicating it makes contributions to thrombosis and other processes associated with dysregulated coagulation. FXI is an unusual dimeric protease, with structural features that distinguish it from vitamin K–dependent coagulation proteases. The recent availability of crystal structures for zymogen FXI and the FXIa catalytic domain have enhanced our understanding of structure-function relationships for this molecule. FXI contains 4 “apple domains” that form a disk structure with extensive interfaces at the base of the catalytic domain. The characterization of the apple disk structure, and its relationship to the catalytic domain, have provided new insight into the mechanism of FXI activation, the interaction of FXIa with the substrate factor IX, and the binding of FXI to platelets. Analyses of missense mutations associated with FXI deficiency have provided additional clues to localization of ligand-binding sites on the protein surface. Together, these data will facilitate efforts to understand the physiology and pathology of this unusual protease, and development of therapeutics to treat thrombotic disorders.

scholarly journals Structural insight into industrially relevant glucoamylases: flexible positions of starch-binding domains

2018 ◽  
Vol 74 (5) ◽  
pp. 463-470 ◽  
Author(s):  
Christian Roth ◽  
Olga V. Moroz ◽  
Antonio Ariza ◽  
Lars K. Skov ◽  
Keiichi Ayabe ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Relative Concentration ◽  
Spatial Arrangement ◽  
Carbohydrate Binding ◽  
Polymeric Substrate ◽  
Binding Domains ◽  
Catalytic Mechanisms ◽  
The Individual ◽  
First Time ◽  
Insight Into

Glucoamylases are one of the most important classes of enzymes in the industrial degradation of starch biomass. They consist of a catalytic domain and a carbohydrate-binding domain (CBM), with the latter being important for the interaction with the polymeric substrate. Whereas the catalytic mechanisms and structures of the individual domains are well known, the spatial arrangement of the domains with respect to each other and its influence on activity are not fully understood. Here, the structures of three industrially used fungal glucoamylases, two of which are full length, have been crystallized and determined. It is shown for the first time that the relative orientation between the CBM and the catalytic domain is flexible, as they can adopt different orientations independently of ligand binding, suggesting a role as an anchor to increase the contact time and the relative concentration of substrate near the active site. The flexibility in the orientations of the two domains presented a considerable challenge for the crystallization of the enzymes.

scholarly journals The structure of the colorectal cancer-associated enzyme GalNAc-T12 reveals how nonconserved residues dictate its function

2019 ◽  
Vol 116 (41) ◽  
pp. 20404-20410 ◽  
Author(s):  
Amy J. Fernandez ◽  
Earnest James Paul Daniel ◽  
Sai Pooja Mahajan ◽  
Jeffrey J. Gray ◽  
Thomas A. Gerken ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Molecular Basis ◽  
Catalytic Domain ◽  
Binding Pocket ◽  
Substrate Selectivity ◽  
Peptide Substrate ◽  
X Ray ◽  
Biochemical Studies ◽  
Cancer Mutations ◽  
Insight Into

Polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts) initiate mucin type O-glycosylation by catalyzing the transfer of N-acetylgalactosamine (GalNAc) to Ser or Thr on a protein substrate. Inactive and partially active variants of the isoenzyme GalNAc-T12 are present in subsets of patients with colorectal cancer, and several of these variants alter nonconserved residues with unknown functions. While previous biochemical studies have demonstrated that GalNAc-T12 selects for peptide and glycopeptide substrates through unique interactions with its catalytic and lectin domains, the molecular basis for this distinct substrate selectivity remains elusive. Here we examine the molecular basis of the activity and substrate selectivity of GalNAc-T12. The X-ray crystal structure of GalNAc-T12 in complex with a di-glycosylated peptide substrate reveals how a nonconserved GalNAc binding pocket in the GalNAc-T12 catalytic domain dictates its unique substrate selectivity. In addition, the structure provides insight into how colorectal cancer mutations disrupt the activity of GalNAc-T12 and illustrates how the rules dictating GalNAc-T12 function are distinct from those for other GalNAc-Ts.

scholarly journals Molecular assemblies of the catalytic domain of SOS with KRas and oncogenic mutants

2021 ◽  
Vol 118 (12) ◽  
pp. e2022403118
Author(s):  
Zahra Moghadamchargari ◽  
Mehdi Shirzadeh ◽  
Chang Liu ◽  
Samantha Schrecke ◽  
Charles Packianathan ◽  
...  
Keyword(s):  
Ion Mobility ◽  
Catalytic Domain ◽  
Therapeutic Interventions ◽  
Nucleotide Exchange ◽  
Molecular Assemblies ◽  
Oncogenic Ras ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Son Of Sevenless ◽  
Insight Into

Ras is regulated by a specific guanine nucleotide exchange factor Son of Sevenless (SOS), which facilitates the exchange of inactive, GDP-bound Ras with GTP. The catalytic activity of SOS is also allosterically modulated by an active Ras (Ras–GTP). However, it remains poorly understood how oncogenic Ras mutants interact with SOS and modulate its activity. Here, native ion mobility–mass spectrometry is employed to monitor the assembly of the catalytic domain of SOS (SOScat) with KRas and three cancer-associated mutants (G12C, G13D, and Q61H), leading to the discovery of different molecular assemblies and distinct conformers of SOScat engaging KRas. We also find KRasG13D exhibits high affinity for SOScat and is a potent allosteric modulator of its activity. A structure of the KRasG13D•SOScat complex was determined using cryogenic electron microscopy providing insight into the enhanced affinity of the mutant protein. In addition, we find that KRasG13D–GTP can allosterically increase the nucleotide exchange rate of KRas at the active site more than twofold compared to KRas–GTP. Furthermore, small-molecule Ras•SOS disruptors fail to dissociate KRasG13D•SOScat complexes, underscoring the need for more potent disruptors. Taken together, a better understanding of the interaction between oncogenic Ras mutants and SOS will provide avenues for improved therapeutic interventions.

Connexin-43 Mediates Fcγ Receptor-Induced Calcium Signaling by Acting as an NAD/Cyclic ADP-Ribose Transporter.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3853-3853
Author(s):  
Eun-Kyung Song ◽  
Young-Rae Lee ◽  
Hong-Nu Yu ◽  
So-Young Rah ◽  
Uh-Hyun Kim ◽  
...  
Keyword(s):  
Calcium Signaling ◽  
Connexin 43 ◽  
Calcium Release ◽  
Catalytic Domain ◽  
Fcγ Receptor ◽  
Murine Macrophages ◽  
Cyclic Adp Ribose ◽  
Intracellular Target ◽  
Intracellular Action ◽  
Insight Into

Abstract Cyclic ADP-ribose (cADPR) is produced from NAD by CD38, a leukocyte receptor and ectoenzyme with its catalytic domain positioned outside of cells, and mobilizes Ca2+ from ryanodine sensitive Ca2+ stores. It has long been questioned how NAD approach CD38 and extracellularly generated cADPR reaches its intracellular target Ca2+ stores. Here we address this question by identifying connexin 43 hemichannels (Cx43) as a transporter of cyclic ADP-ribose as well as NAD in Fcg receptor (FcgR) triggering-induced calcium signaling. FcgR clustering triggers a Ca2+ transient via three sequential steps, Cx43-mediated NAD export, CD38-mediated cADPR production and subsequent Cx43-mediated cADPR import in murine macrophages. FcgR clustering induces Cx43 phosphorylation, thereby opening Cx43 and transporting NAD and cADPR. After cADPR-mediated Ca2+ transient, Cx43 is immediately dephosphorylated and closed, blocking further cADPR transport. This finding is the first evidence showing the involvement of Cx43 in cADPR-mediated Ca2+ release induced by physiological ligand where the apparent topological paradox of extracellular cADPR synthesis and its intracellular action exist. Our results provide new insight into the mechanism of intracellular calcium release triggered by the extracellularly generated calcium mobilizer cADPR, which is involved in (FcgR)-mediated calcium signaling in murine macrophages.

scholarly journals Structure of human endo-α-1,2-mannosidase (MANEA), an antiviral host-glycosylation target

2020 ◽  
Vol 117 (47) ◽  
pp. 29595-29601
Author(s):  
Łukasz F. Sobala ◽  
Pearl Z. Fernandes ◽  
Zalihe Hakki ◽  
Andrew J. Thompson ◽  
Jonathon D. Howe ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Antiviral Agents ◽  
Structural Features ◽  
Glycoside Hydrolases ◽  
Host Protein ◽  
Cellular Models ◽  
Mechanistic Basis ◽  
And Function ◽  
Insight Into ◽  
Mammalian Protein

Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the soleendo-acting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation.

scholarly journals Identification of GH15 Family Thermophilic Archaeal Trehalases That Function within a Narrow Acidic-pH Range

2015 ◽  
Vol 81 (15) ◽  
pp. 4920-4931 ◽  
Author(s):  
Masayoshi Sakaguchi ◽  
Satoru Shimodaira ◽  
Shin-nosuke Ishida ◽  
Miko Amemiya ◽  
Shotaro Honda ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Glycoside Hydrolase Family ◽  
Acidic Ph ◽  
Content Type ◽  
Domain Structures ◽  
Cazy Database ◽  
Ph Range ◽  
And Function ◽  
Reduced Activity ◽  
Insight Into

ABSTRACTTwo glucoamylase-like genes,TVN1315andTa0286, from the archaeaThermoplasma volcaniumandT. acidophilum, respectively, were expressed inEscherichia coli. The gene products, TVN1315 and Ta0286, were identified as archaeal trehalases. These trehalases belong to the CAZy database family GH15, although they have putative (α/α)6barrel catalytic domain structures similar to those of GH37 and GH65 family trehalases from other organisms. These newly identified trehalases function within a narrow range of acidic pH values (pH 3.2 to 4.0) and at high temperatures (50 to 60°C), and these enzymes displayKmvalues for trehalose higher than those observed for typical trehalases. These enzymes were inhibited by validamycin A; however, the inhibition constants (Ki) were higher than those of other trehalases. Three TVN1315 mutants, corresponding to E408Q, E571Q, and E408Q/E571Q mutations, showed reduced activity, suggesting that these two glutamic acid residues are involved in trehalase catalysis in a manner similar to that of glucoamylase. To date, TVN1315 and Ta0286 are the first archaeal trehalases to be identified, and this is the first report of the heterologous expression of GH15 family trehalases. The identification of these trehalases could extend our understanding of the relationships between the structure and function of GH15 family enzymes as well as glycoside hydrolase family enzymes; additionally, these enzymes provide insight into archaeal trehalose metabolism.

scholarly journals Isolation of a novel receptor tyrosine kinase cDNA expressed by developing erythroid progenitors

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1335-1343
Author(s):  
K Yee ◽  
TR Bishop ◽  
C Mather ◽  
LI Zon
Keyword(s):  
Receptor Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Catalytic Domain ◽  
Receptor Tyrosine Kinases ◽  
Erythroid Progenitors ◽  
Amino Acid Similarity ◽  
Hematopoietic Development ◽  
Cos Cells ◽  
Insight Into ◽  
Hematopoietic Cell Lines

Activation of distinct receptor tyrosine kinases (RTK), such as the products of the c-fms and c-kit proto-oncogenes, profoundly affects hematopoietic development. We have isolated a novel RTK cDNA, called met-related kinase (MRK), which is expressed on early erythroid progenitors. MRK is also expressed in many hematopoietic cell lines, and is not lineage restricted. Several regions within the catalytic domain of MRK have amino acid similarity to the c-met proto-oncogene and v-sea oncogene. Specific polyclonal anti-MRK antisera detects an 84- Kd polypeptide in COS cells transfected with an expression vector containing the MRK cDNA. Further studies of this novel RTK will provide potential insight into its role during hematopoiesis.

scholarly journals A point-mutation in the C-domain of CMP-sialic acid synthetase leads to lethality of medaka due to protein insolubility

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Di Wu ◽  
Hiromu Arakawa ◽  
Akiko Fujita ◽  
Hisashi Hashimoto ◽  
Masahiko Hibi ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Amino Acid ◽  
Catalytic Domain ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Ventricular Contraction ◽  
Synonymous Mutations ◽  
Animal Survival ◽  
The Stability ◽  
Insight Into

AbstractVertebrate CMP-sialic acid synthetase (CSS), which catalyzes the synthesis of CMP-sialic acid (CMP-Sia), consists of a 28 kDa-N-domain and a 20 kDa-C-domain. The N-domain is known to be a catalytic domain; however, the significance of the C-domain still remains unknown. To elucidate the function of the C-domain at the organism level, we screened the medaka TILLING library and obtained medaka with non-synonymous mutations (t911a), or single amino acid substitutions of CSS, L304Q, in the C-domain. Prominently, most L304Q medaka was lethal within 19 days post-fertilization (dpf). L304Q young fry displayed free Sia accumulation, and impairment of sialylation, up to 8 dpf. At 8 dpf, a marked abnormality in ventricular contraction and skeletal myogenesis was observed. To gain insight into the mechanism of L304Q-induced abnormalities, L304Q was biochemically characterized. Although bacterially expressed soluble L304Q and WT showed the similar Vmax/Km values, very few soluble L304Q was detected when expressed in CHO cells in sharp contrast to the WT. Additionally, the thermostability of various mutations of L304 greatly decreased, except for WT and L304I. These results suggest that L304 is important for the stability of CSS, and that an appropriate level of expression of soluble CSS is significant for animal survival.

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