scholarly journals Conformation and concerted dynamics of the integrin-binding site and the C-terminal region of echistatin revealed by homonuclear NMR

2005 ◽  
Vol 387 (1) ◽  
pp. 57-66 ◽  
Author(s):  
Daniel MONLEÓN ◽  
Vicent ESTEVE ◽  
Helena KOVACS ◽  
Juan J. CALVETE ◽  
Bernardo CELDA
Keyword(s):  
Amino Acids ◽  
Terminal Region ◽  
Full Length ◽  
Binding Motif ◽  
Recognition Sequence ◽  
Functional Regions ◽  
Nuclear Overhauser ◽  
Nuclear Overhauser Effects ◽  
Backbone Atoms ◽  
Binding Loop

Echistatin is a potent antagonist of the integrins αvβ3, α5β1 and αIIbβ3. Its full inhibitory activity depends on an RGD (Arg-Gly-Asp) motif expressed at the tip of the integrin-binding loop and on its C-terminal tail. Previous NMR structures of echistatin showed a poorly defined integrin-recognition sequence and an incomplete C-terminal tail, which left the molecular basis of the functional synergy between the RGD loop and the C-terminal region unresolved. We report a high-resolution structure of echistatin and an analysis of its internal motions by off-resonance ROESY (rotating-frame Overhauser enhancement spectroscopy). The full-length C-terminal polypeptide is visible as a β-hairpin running parallel to the RGD loop and exposing at the tip residues Pro43, His44 and Lys45. The side chains of the amino acids of the RGD motif have well-defined conformations. The integrin-binding loop displays an overall movement with maximal amplitude of 30°. Internal angular motions in the 100–300 ps timescale indicate increased flexibility for the backbone atoms at the base of the integrin-recognition loop. In addition, backbone atoms of the amino acids Ala23 (flanking the R24GD26 tripeptide) and Asp26 of the integrin-binding motif showed increased angular mobility, suggesting the existence of major and minor hinge effects at the base and the tip, respectively, of the RGD loop. A strong network of NOEs (nuclear Overhauser effects) between residues of the RGD loop and the C-terminal tail indicate concerted motions between these two functional regions. A full-length echistatin–αvβ3 docking model suggests that echistatin's C-terminal amino acids may contact αv-subunit residues and provides new insights to delineate structure–function correlations.

scholarly journals Human xylosyltransferase I and N-terminal truncated forms: functional characterization of the core enzyme

2006 ◽  
Vol 394 (1) ◽  
pp. 163-171 ◽  
Author(s):  
Sandra Müller ◽  
Jennifer Disse ◽  
Manuela Schöttler ◽  
Sylvia Schön ◽  
Christian Prante ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Binding Capacity ◽  
Functional Characterization ◽  
Terminal Region ◽  
Binding Motif ◽  
Heparin Binding ◽  
Loss Of Function ◽  
The Impact

Human XT-I (xylosyltransferase I; EC 2.4.2.26) initiates the biosynthesis of the glycosaminoglycan linkage region and is a diagnostic marker of an enhanced proteoglycan biosynthesis. In the present study, we have investigated mutant enzymes of human XT-I and assessed the impact of the N-terminal region on the enzymatic activity. Soluble mutant enzymes of human XT-I with deletions at the N-terminal domain were expressed in insect cells and analysed for catalytic activity. As many as 260 amino acids could be truncated at the N-terminal region of the enzyme without affecting its catalytic activity. However, truncation of 266, 272 and 273 amino acids resulted in a 70, 90 and >98% loss in catalytic activity. Interestingly, deletion of the single 12 amino acid motif G261KEAISALSRAK272 leads to a loss-of-function XT-I mutant. This is in agreement with our findings analysing the importance of the Cys residues where we have shown that C276A mutation resulted in a nearly inactive XT-I enzyme. Moreover, we investigated the location of the heparin-binding site of human XT-I using the truncated mutants. Heparin binding was observed to be slightly altered in mutants lacking 289 or 568 amino acids, but deletion of the potential heparin-binding motif P721KKVFKI727 did not lead to a loss of heparin binding capacity. The effect of heparin or UDP on the XT-I activity of all mutants was not significantly different from that of the wild-type. Our study demonstrates that over 80% of the nucleotide sequence of the XT-I-cDNA is necessary for expressing a recombinant enzyme with full catalytic activity.

scholarly journals Norovirus VPg Binds RNA through a Conserved N-Terminal K/R Basic Patch

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1282
Author(s):  
Alice M. McSweeney ◽  
Vivienne L. Young ◽  
Vernon K. Ward
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Terminal Region ◽  
Binding Potential ◽  
Binding Motif ◽  
Murine Norovirus ◽  
Functional Protein ◽  
Mobility Shift ◽  
Cycle Arrest ◽  
Electrophoretic Mobility Shift Assays

The viral protein genome-linked (VPg) of noroviruses is a multi-functional protein that participates in essential roles during the viral replication cycle. Predictive analyses indicate that murine norovirus (MNV) VPg contains a disordered N-terminal region with RNA binding potential. VPg proteins were expressed with an N-terminal spidroin fusion protein in insect cells and the interaction with RNA investigated by electrophoretic mobility shift assays (EMSA) against a series of RNA probes (pentaprobes) representing all possible five nucleotide combinations. MNV VPg and human norovirus (HuNV) VPg proteins were directly bound to RNA in a non-specific manner. To identify amino acids involved in binding to RNA, all basic (K/R) residues in the first 12 amino acids of MNV VPg were mutated to alanine. Removal of the K/R amino acids eliminated RNA binding and is consistent with a K/R basic patch RNA binding motif within the disordered N-terminal region of norovirus VPgs. Finally, we show that mutation of the K/R basic patch required for RNA binding eliminates the ability of MNV VPg to induce a G0/G1 cell cycle arrest.

scholarly journals The arachidonate-activatable, NADPH oxidase-associated H+ channel is contained within the multi-membrane-spanning N-terminal region of gp91-phox

1997 ◽  
Vol 325 (3) ◽  
pp. 701-705 ◽  
Author(s):  
Lydia M. HENDERSON ◽  
Stephen THOMAS ◽  
George BANTING ◽  
J. Brian CHAPPELL
Keyword(s):  
Amino Acids ◽  
Nadph Oxidase ◽  
Cell Line ◽  
Proton Motive Force ◽  
Transmembrane Domains ◽  
Terminal Region ◽  
Full Length ◽  
Protein Component ◽  
Cho Cell ◽  
Membrane Spanning

The generation of superoxide by the NADPH oxidase of neutrophils is accompanied by the efflux of H+ ions through a H+ channel. gp91-phox, a protein component of the oxidase, has been shown previously to function as a H+ channel [Henderson, Banting and Chappell (1995) J. Biol. Chem. 270, 5909–5916]. We have constructed a CHO cell line (CHO-N) that expresses an N-terminal fragment of gp91-phox containing the predicted multiple transmembrane domains of the protein. These cells exhibit H+ fluxes in response to an imposed proton motive force and in the presence of arachidonate (to open the channel). The H+ fluxes were indistinguishable from those observed in cells expressing full-length gp91-phox. Therefore the N-terminal 230 amino acids of gp91-phox contain all that is required to function as the NADPH oxidase-associated H+ channel.

scholarly journals NMR solution conformation of heparin-derived tetrasaccharide

1996 ◽  
Vol 318 (1) ◽  
pp. 93-102 ◽  
Author(s):  
Dmitri MIKHAILOV ◽  
Kevin H. MAYO ◽  
Ioncho R. VLAHOV ◽  
Toshihiko TOIDA ◽  
Azra PERVIN ◽  
...  
Keyword(s):  
Chair Conformation ◽  
Coupling Constants ◽  
Solution Conformation ◽  
1H And 13C Nmr ◽  
Small Deviations ◽  
J Coupling Constants ◽  
Nuclear Overhauser ◽  
Nuclear Overhauser Effects ◽  
Dynamics Simulations ◽  
Ring Conformations

The solution conformation of the homogeneous, heparin-derived tetrasaccharide ΔUA2S(1 → 4)-α-d-GlcNpS6S(1 → 4)-α-l-IdoAp2S(1 → 4)-α-d-GlcNpS6S (residues A, B, C and D respectively, where IdoA is iduronic acid) has been investigated by using 1H- and 13C-NMR. Ring conformations have been defined by J-coupling constants and inter-proton nuclear Overhauser effects (NOEs), and the orientation of one ring with respect to the other has been defined by inter-ring NOEs. NOE-based conformational modelling has been done by using the iterative relaxation matrix approach (IRMA), restrained molecular dynamics simulations and energy minimization to refine structures and to distinguish between minor structural differences and equilibria between various ring forms. Both glucosamine residues B and D are in the 4C1 chair conformation. The 6-O-sulphate group is oriented in the gauche–trans configuration in the D ring, whereas in the B ring the gauche–gauche rotomer predominates. Uronate (A) and iduronate (C) residues are mostly represented by 1H2 and 2S0 twisted boat forms, respectively, with small deviations in expected coupling constants and NOEs suggesting minor contributions from other A and C ring conformations.

Receptor and Molecular Mechanism of AGGF1 Signaling in Endothelial Cell Functions and Angiogenesis

2021 ◽  
Author(s):  
Jingjing Wang ◽  
Huixin Peng ◽  
Ayse Anil Timur ◽  
Vinay Pasupuleti ◽  
Yufeng Yao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Endothelial Cell ◽  
Neutralizing Antibodies ◽  
Molecular Mechanisms ◽  
Capillary Tube ◽  
Therapeutic Angiogenesis ◽  
Angiogenic Factor ◽  
Single Amino Acid ◽  
Binding Motif ◽  
Cell Functions

Objective: Angiogenic factor AGGF1 (angiogenic factor and G-patch and FHA [Forkhead-associated] domain 1) promotes angiogenesis as potently as VEGFA (vascular endothelial growth factor A) and regulates endothelial cell (EC) proliferation, migration, specification of multipotent hemangioblasts and venous ECs, hematopoiesis, and vascular development and causes vascular disease Klippel-Trenaunay syndrome when mutated. However, the receptor for AGGF1 and the underlying molecular mechanisms remain to be defined. Approach and Results: Using functional blocking studies with neutralizing antibodies, we identified α5β1 as the receptor for AGGF1 on ECs. AGGF1 interacts with α5β1 and activates FAK (focal adhesion kinase), Src, and AKT. Functional analysis of 12 serial N-terminal deletions and 13 C-terminal deletions by every 50 amino acids mapped the angiogenic domain of AGGF1 to a domain between amino acids 604-613 (FQRDDAPAS). The angiogenic domain is required for EC adhesion and migration, capillary tube formation, and AKT activation. The deletion of the angiogenic domain eliminated the effects of AGGF1 on therapeutic angiogenesis and increased blood flow in a mouse model for peripheral artery disease. A 40-mer or 15-mer peptide containing the angiogenic domain blocks AGGF1 function, however, a 15-mer peptide containing a single amino acid mutation from −RDD- to −RGD- (a classical RGD integrin-binding motif) failed to block AGGF1 function. Conclusions: We have identified integrin α5β1 as an EC receptor for AGGF1 and a novel AGGF1-mediated signaling pathway of α5β1-FAK-Src-AKT for angiogenesis. Our results identify an FQRDDAPAS angiogenic domain of AGGF1 crucial for its interaction with α5β1 and signaling.

scholarly journals The Capsid (ORF2) Protein of Hepatitis E Virus in Feces Is C-Terminally Truncated

Pathogens ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 24
Author(s):  
Takashi Nishiyama ◽  
Koji Umezawa ◽  
Kentaro Yamada ◽  
Masaharu Takahashi ◽  
Satoshi Kunita ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Culture ◽  
Structure Prediction ◽  
Hepatitis E Virus ◽  
Culture Media ◽  
Molecular Size ◽  
Hepatitis E ◽  
Terminal Region ◽  
Translation Product ◽  
Orf2 Protein

The hepatitis E virus (HEV) is a causative agent of hepatitis E. HEV virions in circulating blood and culture media are quasi-enveloped, while those in feces are nonenveloped. The capsid (ORF2) protein associated with an enveloped HEV virion is reported to comprise the translation product of leucine 14/methionine 16 to 660 (C-terminal end). However, the nature of the ORF2 protein associated with fecal HEV remains unclear. In the present study, we compared the molecular size of the ORF2 protein among fecal HEV, cell-culture-generated HEV (HEVcc), and detergent-treated protease-digested HEVcc. The ORF2 proteins associated with fecal HEV were C-terminally truncated and showed the same size as those of the detergent-treated protease-digested HEVcc virions (60 kDa), in contrast to those of the HEVcc (68 kDa). The structure prediction of the ORF2 protein (in line with previous studies) demonstrated that the C-terminal region (54 amino acids) of an ORF2 protein is in flux, suggesting that proteases target this region. The nonenveloped nondigested HEV structure prediction indicates that the C-terminal region of the ORF2 protein moves to the surface of the virion and is unnecessary for HEV infection. Our findings clarify the maturation of nonenveloped HEV and will be useful for studies on the HEV lifecycle.

Structure assignment of some 3,4′-diquinolinyl sulfides studied by Nuclear Overhauser Effects, X-ray analysis and reactions with sodium methoxide

2010 ◽  
Vol 109 (10) ◽  
pp. 509-514 ◽  
Author(s):  
Stanislaw Boryczka ◽  
Andrzej Maślankiewicz ◽  
Miroslaw Wyszomirski ◽  
Teresa Borowiak ◽  
Maciej Kubicki
Keyword(s):  
Sodium Methoxide ◽  
X Ray ◽  
Nuclear Overhauser ◽  
Nuclear Overhauser Effects ◽  
Structure Assignment
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