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Titolo:
Mutation of highly conserved arginine residues disrupts the structure and function of annexin V
Autore:
Campos, B; Wang, ST; Retzinger, GS; Kaetzel, MA; Seaton, BA; Karin, NJ; Johnson, JD; Dedman, JR;
Indirizzi:
Univ Cincinnati, Coll Med, Dept Mol & Cellular Physiol, Cincinnati, OH 45267 USA Univ Cincinnati Cincinnati OH USA 45267 Physiol, Cincinnati, OH 45267 USA Univ Cincinnati, Coll Med, Dept Pathol & Lab Med, Cincinnati, OH 45267 USAUniv Cincinnati Cincinnati OH USA 45267 Lab Med, Cincinnati, OH 45267 USA Ohio State Univ, Dept Med Biochem, Columbus, OH 43210 USA Ohio State UnivColumbus OH USA 43210 Med Biochem, Columbus, OH 43210 USA Boston Univ, Sch Med, Dept Physiol, Struct Biol Grp, Boston, MA 02118 USA Boston Univ Boston MA USA 02118 ol, Struct Biol Grp, Boston, MA 02118 USA Univ Texas, Sch Med, Dept Integrat Biol, Houston, TX USA Univ Texas Houston TX USA , Sch Med, Dept Integrat Biol, Houston, TX USA
Titolo Testata:
ARCHIVES OF MEDICAL RESEARCH
fascicolo: 5, volume: 30, anno: 1999,
pagine: 360 - 367
SICI:
0188-4409(199909/10)30:5<360:MOHCAR>2.0.ZU;2-8
Fonte:
ISI
Lingua:
ENG
Soggetto:
LIPOCORTIN FAMILY; BINDING PROTEINS; MEMBRANE-BINDING; EXPRESSION; MUTAGENESIS; CRYSTAL; SITE;
Keywords:
annexins; calcium-binding proteins; mutagenesis; structure;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
36
Recensione:
Indirizzi per estratti:
Indirizzo: Dedman, JR Univ Cincinnati, Coll Med, Dept Mol & Cellular Physiol, Cincinnati, OH 45267 USA Univ Cincinnati Cincinnati OH USA 45267 ncinnati, OH 45267 USA
Citazione:
B. Campos et al., "Mutation of highly conserved arginine residues disrupts the structure and function of annexin V", ARCH MED R, 30(5), 1999, pp. 360-367

Abstract

Background. Annexins are a family of structurally related proteins that bind to phospholipid membranes in a Ca2+-dependent manner. Annexins are characterized by highly conserved canonical domains of approximately 70 amino acids. Annexin V contains four such domains. Each of these domains has a highly conserved arginine (R). Methods. To evaluate the role of the conserved arginines in the molecular structure of annexin V, negatively charged amino acids were substituted forarginines at positions R43, R115, R199, and R274 using site-directed mutagenesis,Results. Mutants R199D and R274E were rapidly degraded when expressed in bacteria, and were not further characterized. R43E exhibited an electrophoretic mobility similar to the wild-type protein, while R115E migrated significantly in a slower fashion, suggesting a less compact conformation. R43E and R115E exhibited much greater susceptibility to proteolytic digestion thanthe wild type. While Ca2+-dependence for phospholipid binding was similar in both mutants (half-maximal 50-80 mu M Ca2+), R43E and R115E exhibited a 6- and 2-fold decrease in phospholipid affinity, respectively. Consistent with the different phospholipid affinities of the annexins, a phospholipid-dependent clotting reaction, the activated partial thromboplastin time (aPTT), was significantly prolonged by the wild-type protein and mutants R115E and R115A. The aPTT was unaffected by R43E. Conclusions. Our data suggest that mutation of these highly conserved arginine residues in each of the four canonical domains of annexin have differential effects on the phospholipid binding, tertiary structure, and proteolytic susceptibility of annexin V. The site I mutation, R43E, produced a large decrease in phospholipid affinity associated with an increase in proteolytic susceptibility. The site II mutation, R115E, produced a small change inphospholipid binding but a significant modification of electrophoretic mobility. Our data suggest that highly conserved arginine residues are required to stabilize the tertiary structure of annexin V by establishing hydrogenbonds and ionic bridges. (C) 1999 IMSS, Published by Elsevier Science Inc.

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Documento generato il 28/09/20 alle ore 22:09:13