Differential contributions of Glu96, Asp102 and Asp111 to coagulation Factor V/Va metal ion binding and subunit stability

2009 ◽  
Vol 422 (2) ◽  
pp. 257-264 ◽  
Author(s):  
Jina Song ◽  
Kimberley Talbot ◽  
Jeffrey Hewitt ◽  
Ross T. A. MacGillivray ◽  
Edward L. G. Pryzdial
Keyword(s):  
Metal Ion ◽  
Coagulation Factor ◽  
Normal Metal ◽  
Ion Binding ◽  
Factor V ◽  
Loss Of Function ◽  
Metal Ion Binding ◽  
Subunit Dissociation ◽  
Subunit Interface ◽  
Metal Ion Interactions

Blood coagulation FV (Factor V) is activated by thrombin-mediated excision of the B domain, resulting in a non-covalent heterodimer, FVa (activated FV). Previous studies implicated Glu96, Asp102 and Asp111 in the essential Ca2+-dependent FVa subunit interaction. In the present study, FV E96A, D102A and D111A were purified and evaluated for function, subunit dissociation and metal ion binding. Chromogenic and clotting assays in the presence of procoagulant vesicles showed that each variant was inhibited (∼20–40%). D111A was further inhibited (>90%) after cleavage by thrombin. Comparable function was observed on activated platelets. D111A inhibition correlated to spontaneous subunit dissociation and severely impaired Ca2+ binding. The Cu2+ interaction was also inhibited, suggesting interdependent Ca2+ and Cu2+ binding to FV. The parental FV (FV-810; wild-type human FV missing residues 811–1491) used here is fully active without proteolysis because the B domain is truncated. Therefore, a FVa-like functional configuration exists for intact D111A independent of normal metal ion interactions. Unlike D111A, the thrombin-mediated FVa derived from E96A and D102A had only moderately enhanced subunit dissociation upon chelation and had normal metal ion binding. For FV-810-, E96A- and D102A-derived FVa, loss of function after chelation significantly preceded subunit dissociation. This study defines the highly conserved segment spanning Glu96–Asp111 in FV as multifunctional. Of the three amino acids evaluated, Asp111 is essential and probably functions through direct and indirect effects on Ca2+ and Cu2+ interactions. Glu96 and Asp102 individually influence FV/FVa by more subtle effects, possibly at the metal ion-dependent subunit interface.

scholarly journals Structure ofD-tagatose 3-epimerase-like protein fromMethanocaldococcus jannaschii

2014 ◽  
Vol 70 (7) ◽  
pp. 890-895 ◽  
Author(s):  
Keiko Uechi ◽  
Goro Takata ◽  
Kazunari Yoneda ◽  
Toshihisa Ohshima ◽  
Haruhiko Sakuraba
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Open Reading Frame ◽  
Ion Binding ◽  
Asymmetric Unit ◽  
Metal Ion Binding ◽  
Pseudomonas Cichorii ◽  
Reading Frame ◽  
Subunit Interface ◽  
Binding Cleft

The crystal structure of a D-tagatose 3-epimerase-like protein (MJ1311p) encoded by a hypothetical open reading frame, MJ1311, in the genome of the hyperthermophilic archaeonMethanocaldococcus jannaschiiwas determined at a resolution of 2.64 Å. The asymmetric unit contained two homologous subunits, and the dimer was generated by twofold symmetry. The overall fold of the subunit proved to be similar to those of the D-tagatose 3-epimerase fromPseudomonas cichoriiand the D-psicose 3-epimerases fromAgrobacterium tumefaciensandClostridium cellulolyticum. However, the situation at the subunit–subunit interface differed substantially from that in D-tagatose 3-epimerase family enzymes. In MJ1311p, Glu125, Leu126 and Trp127 from one subunit were found to be located over the metal-ion-binding site of the other subunit and appeared to contribute to the active site, narrowing the substrate-binding cleft. Moreover, the nine residues comprising a trinuclear zinc centre in endonuclease IV were found to be strictly conserved in MJ1311p, although a distinct groove involved in DNA binding was not present. These findings indicate that the active-site architecture of MJ1311p is quite unique and is substantially different from those of D-tagatose 3-epimerase family enzymes and endonuclease IV.

Photophysical Characterisation, Metal Ion Binding and Enantiomeric Recognition of Chiral Ligands Containing Phenazine Fluorophore

2004 ◽  
Vol 69 (4) ◽  
pp. 885-896 ◽  
Author(s):  
Luisa Stella Dolci ◽  
Péter Huszthy ◽  
Erika Samu ◽  
Marco Montalti ◽  
Luca Prodi ◽  
...  
Keyword(s):  
Metal Ion ◽  
Photophysical Properties ◽  
Ion Binding ◽  
Metal Ion Binding ◽  
Ammonium Ions ◽  
Enantiomerically Pure ◽  
Binding Process ◽  
High Affinity ◽  
Enantiomeric Recognition ◽  
Chiral Species

Enantiomerically pure dimethyl- and diisobutyl-substituted phenazino-18-crown-6 ligands bind metal and ammonium ions and also primary aralkylammonium perchlorates in acetonitrile with high affinity, causing pronounced changes in their luminescence properties. In addition, they show enantioselectivity towards chiral primary aralkylammonium perchlorates. The possibility to monitor the binding process by photoluminescence spectroscopy can gain ground for the design of very efficient enantioselective chemosensors for chiral species. The observed changes in the photophysical properties are also an important tool for understanding the interactions present in the adduct.

scholarly journals Snapshots of a Non-Canonical RdRP in Action

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1260
Author(s):  
Diego S. Ferrero ◽  
Michela Falqui ◽  
Nuria Verdaguer
Keyword(s):  
Metal Ion ◽  
Rna Viruses ◽  
Ion Binding ◽  
Genome Replication ◽  
Metal Ion Binding ◽  
Insect Virus ◽  
X Ray ◽  
Template Strand ◽  
Nucleotide Incorporation ◽  
Elongation Process

RNA viruses typically encode their own RNA-dependent RNA polymerase (RdRP) to ensure genome replication and transcription. The closed “right hand” architecture of RdRPs encircles seven conserved structural motifs (A to G) that regulate the polymerization activity. The four palm motifs, arranged in the sequential order A to D, are common to all known template dependent polynucleotide polymerases, with motifs A and C containing the catalytic aspartic acid residues. Exceptions to this design have been reported in members of the Permutotetraviridae and Birnaviridae families of positive single stranded (+ss) and double-stranded (ds) RNA viruses, respectively. In these enzymes, motif C is located upstream of motif A, displaying a permuted C–A–B–D connectivity. Here we study the details of the replication elongation process in the non-canonical RdRP of the Thosea asigna virus (TaV), an insect virus from the Permutatetraviridae family. We report the X-ray structures of three replicative complexes of the TaV polymerase obtained with an RNA template-primer in the absence and in the presence of incoming rNTPs. The structures captured different replication events and allowed to define the critical interactions involved in: (i) the positioning of the acceptor base of the template strand, (ii) the positioning of the 3’-OH group of the primer nucleotide during RNA replication and (iii) the recognition and positioning of the incoming nucleotide. Structural comparisons unveiled a closure of the active site on the RNA template-primer binding, before rNTP entry. This conformational rearrangement that also includes the repositioning of the motif A aspartate for the catalytic reaction to take place is maintained on rNTP and metal ion binding and after nucleotide incorporation, before translocation.

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