Endo-β-D-1,4-mannanase fromChrysonilia sitophiladisplays a novel loop arrangement for substrate selectivity

2012 ◽  
Vol 68 (11) ◽  
pp. 1468-1478 ◽  
Author(s):  
Ana Maria D. Gonçalves ◽  
Catarina S. Silva ◽  
Tânia I. Madeira ◽  
Ricardo Coelho ◽  
Daniele de Sanctis ◽  
...  
Keyword(s):  
Substrate Selectivity ◽  
Relevant Feature ◽  
Structural Homology ◽  
Wild Type ◽  
Binding Region ◽  
Active Centre ◽  
Chrysonilia Sitophila ◽  
Binding Cleft ◽  
Binding Groove ◽  
Surface Loops

The crystal structure of wild-type endo-β-D-1,4-mannanase (EC 3.2.1.78) from the ascomyceteChrysonilia sitophila(CsMan5) has been solved at 1.40 Å resolution. The enzyme isolated directly from the source shows mixed activity as both an endo-glucanase and an endo-mannanase. CsMan5 adopts the (β/α)8-barrel fold that is well conserved within the GH5 family and has highest sequence and structural homology to the GH5 endo-mannanases. Superimposition with proteins of this family shows a unique structural arrangement of three surface loops of CsMan5 that stretch over the active centre, promoting an altered topography of the binding cleft. The most relevant feature results from the repositioning of a long loop at the extremity of the binding cleft, resulting in a shortened glycone-binding region with two subsites. The other two extended loops flanking the binding groove produce a narrower cleft compared with the wide architecture observed in GH5 homologues. Two aglycone subsites (+1 and +2) are identified and a nonconserved tryptophan (Trp271) at the +1 subsite may offer steric hindrance. Taken together, these findings suggest that the discrimination of mannan substrates is achieved through modified loop length and structure.

Localization of a Vitronectin Binding Region of Plasminogen Activator Inhibitor-1

1995 ◽  
Vol 73 (05) ◽  
pp. 829-834 ◽  
Author(s):  
Jaya Padmanabhan ◽  
David C Sane
Keyword(s):  
Binding Site ◽  
Plasminogen Activator ◽  
Inhibitory Activity ◽  
Plasminogen Activator Inhibitor ◽  
Structural Homology ◽  
Binding Region ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

SummaryThe PAI-1 binding site for VN was studied using two independent methods. PAI-1 was cleaved by Staph V8 protease, producing 8 fragments, only 2 of which bound to [125I]-VN. These fragments were predicted to overlap between residues 91-130. Since PAI-2 has structural homology to PAI-1, but does not bind to vitronectin, chimeras of PAI-1 and PAI-2 were constructed. Four chimeras, containing PAI-1 residues 1-70,1-105,1-114, and 1-167 were constructed and expressed in vitro. PAI-1, PAI-2, and all of the chimeras retained inhibitory activity for t-PA, but only the chimera containing PAI-1 residues 1-167 formed a complex with VN. Together, these results predict that the VN binding site of PAI-1 is between residues 115-130.

scholarly journals Direct Interaction between the Subunit RAP30 of Transcription Factor IIF (TFIIF) and RNA Polymerase Subunit 5, Which Contributes to the Association between TFIIF and RNA Polymerase II

2001 ◽  
Vol 276 (15) ◽  
pp. 12266-12273 ◽  
Author(s):  
Wenxiang Wei ◽  
Dorjbal Dorjsuren ◽  
Yong Lin ◽  
Weiping Qin ◽  
Takahiro Nomura ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Middle Part ◽  
Wild Type ◽  
Binding Region ◽  
Pol Ii ◽  
Transcription Factor Iif ◽  
B Virus

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30in vitrousing purified recombinant proteins andin vivoin COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47–120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101–170) and the N-terminus (aa 1–100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding inin vitroandin vivoassays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

Analysis of the essential and excision repair functions of the RAD3 gene of Saccharomyces cerevisiae by mutagenesis

1986 ◽  
Vol 6 (4) ◽  
pp. 1218-1227
Author(s):  
L Naumovski ◽  
E C Friedberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Excision Repair ◽  
Random Mutagenesis ◽  
Nucleotide Binding ◽  
Yeast Cells ◽  
Wild Type ◽  
Binding Region ◽  
Site Specific ◽  
Essential Function

The RAD3 gene of Saccharomyces cerevisiae, which is involved in excision repair of DNA and is essential for cell viability, was mutagenized by site-specific and random mutagenesis. Site-specific mutagenesis was targeted to two regions near the 5' and 3' ends of the coding region, selected on the basis of amino acid sequence homology with known nucleotide binding and with known specific DNA-binding proteins, respectively. Two mutations in the putative nucleotide-binding region and one in the putative DNA-binding region inactivate the excision repair function of the gene, but not the essential function. A gene encoding two tandem mutations in the putative DNA-binding region is defective in both excision repair and essential functions of RAD3. Seven plasmids were isolated following random mutagenesis with hydroxylamine. Mutations in six of these plasmids were identified by gap repair of mutant plasmids from the chromosome of strains with previously mapped rad3 mutations, followed by DNA sequencing. Three of these contain missense mutations which inactivate only the excision repair function. The other three carry nonsense mutations which inactivate both the excision repair and essential functions. Collectively our results indicate that the RAD3 excision repair function is more sensitive to inactivation than is the essential function. Overexpression of wild-type Rad3 protein and a number of rad3 mutant proteins did not affect the UV resistance of wild-type yeast cells. However, overexpression of Rad3-2 protein rendered wild-type cells partially UV sensitive, indicating that excess Rad3-2 protein is dominant to the wild-type form. These and other results suggest that Rad3-2 protein retains its affinity for damaged DNA or other substrates, but is not catalytically active in excision repair.

scholarly journals Specificity and mutagenesis bias of the mycobacterial alternative mismatch repair analyzed by mutation accumulation studies

2020 ◽  
Vol 6 (7) ◽  
pp. eaay4453
Author(s):  
A. Castañeda-García ◽  
I. Martín-Blecua ◽  
E. Cebrián-Sastre ◽  
A. Chiner-Oms ◽  
M. Torres-Puente ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Mycobacterium Smegmatis ◽  
Genome Stability ◽  
Structural Homology ◽  
Spontaneous Mutations ◽  
Wild Type ◽  
Key Factor ◽  
Maintain Genome Stability ◽  
Mmr Proteins

The postreplicative mismatch repair (MMR) is an almost ubiquitous DNA repair essential for maintaining genome stability. It has been suggested that Mycobacteria have an alternative MMR in which NucS, an endonuclease with no structural homology to the canonical MMR proteins (MutS/MutL), is the key factor. Here, we analyze the spontaneous mutations accumulated in a neutral manner over thousands of generations by Mycobacterium smegmatis and its MMR-deficient derivative (ΔnucS). The base pair substitution rates per genome per generation are 0.004 and 0.165 for wild type and ΔnucS, respectively. By comparing the activity of different bacterial MMR pathways, we demonstrate that both MutS/L- and NucS-based systems display similar specificity and mutagenesis bias, revealing a functional evolutionary convergence. However, NucS is not able to repair indels in vivo. Our results provide an unparalleled view of how this mycobacterial system works in vivo to maintain genome stability and how it may affect Mycobacterium evolution.

scholarly journals Inhibition of Metalloprotease Botulinum Serotype A from a Pseudo-peptide Binding Mode to a Small Molecule That Is Active in Primary Neurons

2006 ◽  
Vol 282 (7) ◽  
pp. 5004-5014 ◽  
Author(s):  
James C. Burnett ◽  
Gordon Ruthel ◽  
Christian M. Stegmann ◽  
Rekha G. Panchal ◽  
Tam L. Nguyen ◽  
...  
Keyword(s):  
Small Molecule ◽  
Light Chain ◽  
Binding Mode ◽  
Research Strategy ◽  
Titration Calorimetry ◽  
Serotype A ◽  
Therapeutic Development ◽  
Botulinum Neurotoxin Serotype A ◽  
Binding Cleft ◽  
Surface Loops

An efficient research strategy integrating empirically guided, structure-based modeling and chemoinformatics was used to discover potent small molecule inhibitors of the botulinum neurotoxin serotype A light chain. First, a modeled binding mode for inhibitor 2-mercapto-3-phenylpropionyl-RATKML (Ki = 330 nm) was generated, and required the use of a molecular dynamic conformer of the enzyme displaying the reorientation of surface loops bordering the substrate binding cleft. These flexible loops are conformationally variable in x-ray crystal structures, and the model predicted that they were pivotal for providing complementary binding surfaces and solvent shielding for the pseudo-peptide. The docked conformation of 2-mercapto-3-phenylpropionyl-RATKML was then used to refine our pharmacophore for botulinum serotype A light chain inhibition. Data base search queries derived from the pharmacophore were employed to mine small molecule (non-peptidic) inhibitors from the National Cancer Institute's Open Repository. Four of the inhibitors possess Ki values ranging from 3.0 to 10.0 μm. Of these, NSC 240898 is a promising lead for therapeutic development, as it readily enters neurons, exhibits no neuronal toxicity, and elicits dose-dependent protection of synaptosomal-associated protein (of 25 kDa) in a primary culture of embryonic chicken neurons. Isothermal titration calorimetry showed that the interaction between NSC 240898 and the botulinum A light chain is largely entropy-driven, and occurs with a 1:1 stoichiometry and a dissociation constant of 4.6 μm.

Structure of the Mutant E92K of [2Fe–2S] Ferredoxin I from Spinacia oleracea at 1.7 Å Resolution

1998 ◽  
Vol 54 (6) ◽  
pp. 1353-1358 ◽  
Author(s):  
Claudia Binda ◽  
Alessandro Coda ◽  
Alessandro Aliverti ◽  
Giuliana Zanetti ◽  
Andrea Mattevi
Keyword(s):  
Spinacia Oleracea ◽  
Crystal Packing ◽  
Side Chain ◽  
Molecular Replacement ◽  
Amino Acid Residues ◽  
Hydrogen Bond Network ◽  
Wild Type ◽  
Active Centre ◽  
Bond Network ◽  
Ferredoxin I

Ferredoxin I (Fd I) from Spinacia oleracea is composed of 97 amino-acid residues and a [2Fe–2S] cluster. The crystal structure of the E92K mutant of Fd I was solved by molecular replacement and refined to an R factor of 19.6% for 11755 reflections at 1.7 Å resolution. The overall structure and the active centre of spinach Fd is highly conserved with respect to ferredoxins of known structure. The E92K mutation appears to disturb a hydrogen-bond network which stabilizes the loop bearing the [2Fe–2S] cluster. This observation provides a rationale for the reduced electron-transfer efficiency displayed by the E92K mutant. Inspection of the crystal packing reveals that the side chain of Lys92 is engaged in an intermolecular interaction with Asp26 of a symmetry-related molecule. This feature may explain why only the mutant E92K and not wild-type Fd I could be successfully crystallized.

Histidine-regulated activity of M-ficolin

2008 ◽  
Vol 417 (2) ◽  
pp. 485-491 ◽  
Author(s):  
Michikazu Tanio ◽  
Toshiyuki Kohno
Keyword(s):  
Conformational Equilibrium ◽  
Innate Immune ◽  
High Avidity ◽  
Wild Type ◽  
Binding Region ◽  
Functional Studies ◽  
Ph Dependency ◽  
Brevibacillus Choshinensis ◽  
Trimer Formation ◽  
Recognition Molecule

Human M-ficolin is a pathogen-associated molecular recognition molecule in the innate immune system, and it binds to some sugars, such as GlcNAc (N-acetylglucosamine), on pathogen surfaces. From previous structural and functional studies of the FD1 (M-ficolin fibrinogen-like domain), we proposed that the ligand-binding region of FD1 exists in a conformational equilibrium between active and non-active states depending on three groups with a pKa of 6.2, which are probably histidine residues, and suggested that the 2-state conformational equilibrium as well as the trimer formation contributes to the discrimination mechanism between self and non-self of FD1 [Tanio, M., Kondo, S., Sugio, S. and Kohno, T. (2007) J. Biol. Chem. 282, 3889–3895]. To investigate the origins of the pH dependency, mutational analyses were performed on FD1 expressed by Brevibacillus choshinensis. The GlcNAc binding study of a series of single histidine mutants of FD1 demonstrated that His251, His284 and His297 are required for the activity, and thus we concluded that the three histidines are the origins of the pH dependency of FD1. Monomeric mutants of FD1 show weaker affinity for the ligand than the trimeric wild-type, indicating that trimer formation confers high avidity for the ligand. In addition, analyses of the GlcNAc association and dissociation of FD1 provided evidence that FD1 always exchanges between the active and non-active states with the pH-dependent populations in solution. The biological roles of the histidine-regulated conformational equilibrium of M-ficolin are discussed in terms of the self and non-self discrimination mechanism.

scholarly journals Studies of SpoIIAB mutant proteins elucidate the mechanisms that regulate the developmental transcription factor σF in Bacillus subtilis

2004 ◽  
Vol 384 (1) ◽  
pp. 169-178 ◽  
Author(s):  
Jwu-Ching SHU ◽  
Joanna CLARKSON ◽  
Michael D. YUDKIN
Keyword(s):  
Conformational Change ◽  
Sigma Factor ◽  
Reaction Scheme ◽  
Wild Type ◽  
Binding Region ◽  
Mutant Proteins ◽  
Binding Partner ◽  
Binding Partners ◽  
Crucial Feature ◽  
Mutant Cells

σF, the first compartment-specific sigma factor of sporulation, is regulated by an anti-sigma factor, SpoIIAB (AB) and its antagonist SpoIIAA (AA). AB can bind to σF in the presence of ATP or to AA in the presence of ADP; in addition, AB can phosphorylate AA. The ability of AB to switch between its two binding partners regulates σF. Early in sporulation, AA activates σF by releasing it from its complex with AB. We have previously proposed a reaction scheme for the phosphorylation of AA by AB which accounts for AA's regulatory role. A crucial feature of this scheme is a conformational change in AB that accompanies its switch in binding partner. In the present study, we have studied three AB mutants, all of which have amino-acid replacements in the nucleotide-binding region; AB-E104K (Glu104→Lys) and AB-T49K (Thr49→Lys) fail to activate σF, and AB-R105A (Arg105→Ala) activates it prematurely. We used techniques of enzymology, surface plasmon resonance and fluorescence spectroscopy to analyse the defects in each mutant. AB-E104K was deficient in binding to AA, AB-T49K was deficient in binding to ADP and AB-R105A bound ADP exceptionally strongly. Although the release of σF from all three mutant proteins was impaired, and all three failed to undergo the wild-type conformational change when switching binding partners, the phenotypes of the mutant cells were best accounted for by the properties of the respective AB species in forming complexes with AA and ADP. The behaviour of the mutants enables us to propose convincing mechanisms for the regulation of σF in wild-type bacteria.

scholarly journals Phosphatidylinositol-4,5 Bisphosphate Produced by PIP5KIγ Regulates Gelsolin, Actin Assembly, and Adhesion Strength of N-Cadherin Junctions

2007 ◽  
Vol 18 (8) ◽  
pp. 3026-3038 ◽  
Author(s):  
T. Y. El Sayegh ◽  
P. D. Arora ◽  
K. Ling ◽  
C. Laschinger ◽  
P. A. Janmey ◽  
...  
Keyword(s):  
Adhesion Strength ◽  
Intercellular Adhesion ◽  
Actin Binding ◽  
Type I ◽  
Actin Assembly ◽  
Wild Type ◽  
Binding Region ◽  
A Cell ◽  
Intercellular Adhesions ◽  
Phosphatidylinositol 4

Phosphoinositides regulate several actin-binding proteins but their role at intercellular adhesions has not been defined. We found that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) was generated at sites of N-cadherin–mediated intercellular adhesion and was a critical regulator of intercellular adhesion strength. Immunostaining for PI(4,5)P2 or transfection with GFP-PH-PLCδ showed that PI(4,5)P2 was enriched at sites of N-cadherin adhesions and this enrichment required activated Rac1. Isoform-specific immunostaining for type I phosphatidylinositol 4-phosphate 5 kinase (PIP5KI) showed that PIP5KIγ was spatially associated with N-cadherin–Fc beads. Association of PIP5KIγ with N-cadherin adhesions was in part dependent on the activation of RhoA. Transfection with catalytically inactive PIP5KIγ blocked the enrichment of PI(4,5)P2 around beads. Catalytically inactive PIP5KIγ or a cell-permeant peptide that mimics and competes for the PI(4,5)P2-binding region of the actin-binding protein gelsolin inhibited incorporation of actin monomers in response to N-cadherin ligation and reduced intercellular adhesion strength by more than twofold. Gelsolin null fibroblasts transfected with a gelsolin severing mutant containing an intact PI(4,5)P2 binding region, demonstrated intercellular adhesion strength similar to wild-type transfected controls. We conclude that PIP5KIγ-mediated generation of PI(4,5)P2 at sites of N-cadherin contacts regulates intercellular adhesion strength, an effect due in part to PI(4,5)P2-mediated regulation of gelsolin.

scholarly journals Induction versus progression of brain tumor development: differential functions for the pRB- and p53-targeting domains of simian virus 40 T antigen.

1994 ◽  
Vol 14 (4) ◽  
pp. 2686-2698 ◽  
Author(s):  
M T Sáenz Robles ◽  
H Symonds ◽  
J Chen ◽  
T Van Dyke
Keyword(s):  
Tumor Progression ◽  
Choroid Plexus ◽  
Simian Virus 40 ◽  
Cell Types ◽  
Simian Virus ◽  
T Antigen ◽  
Cellular Proteins ◽  
Wild Type ◽  
Binding Region ◽  
Amino Terminal

The ability of simian virus 40-encoded large T antigen to disrupt the growth control of a variety of cell types is related to its ability to interfere with certain cellular proteins, such as p53 and the retinoblastoma susceptibility gene product (pRB). We have used wild-type and mutant forms of T antigen in transgenic mice to dissect the roles of pRB, p53, and other cellular proteins in tumorigenesis of different cell types. In this study, using a cell-specific promoter to target expression specifically to brain epithelium (the choroid plexus) and to B and T lymphoid cells, we characterize the tumorigenic capacity of a T-antigen fragment that comprises only the amino-terminal 121 residues. This fragment (dl1137) retains the ability to interact with pRB and p107 but lacks the p53-binding domain. While loss of the p53-binding region results in loss of the capacity to induce lymphoid abnormalities, dl1137 retains the ability to induce choroid plexus tumors that are histologically indistinguishable from those induced by wild-type T antigen. Tumors induced by dl1137 develop much more slowly, however, reaching an end point at around 8 months of age rather than at 1 to 2 months. Analysis of tumor progression indicates that tumor induction by dl1137 does not require secondary genetic or epigenetic events. Rather, the tumor growth rate is significantly slowed, indicating that the T-antigen C-terminal region contributes to tumor progression in this cell type. In contrast, the pRB-binding region appears essential for tumorigenesis as mutation of residue 107, known to disrupt pRB and p107 binding to wild-type T antigen, abolishes the ability of the dl1137 protein to induce growth abnormalities in the brain.

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