1dk5
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CRYSTAL STRUCTURE OF ANNEXIN 24(CA32) FROM CAPSICUM ANNUUM
OverviewOverview
This work provides the first three-dimensional structure of a member of, the plant annexin family and correlates these findings with biochemical, properties of this protein. Annexin 24(Ca32) from Capsicum annuum was, purified as a native protein from bell pepper and was also prepared by, recombinant techniques. To overcome the problem of precipitation of the, recombinant wild-type protein in crystallization trials, two mutants were, designed. Whereas an N-terminal truncation mutant turned out to be an, unstable protein, the N-terminal His-tagged annexin 24(Ca32) was, crystallized, and the three-dimensional structure was determined by x-ray, diffraction at 2. 8 A resolution. The structure refined to an R-factor of, 0.216 adopts the typical annexin fold; the detailed structure, however, is, different from non-plant annexins, especially in domains I and III and in, the membrane binding loops on the convex side. Within the unit cell there, are two molecules per asymmetric unit, which differ in conformation of the, IAB-loop. Both conformers show Trp-35 on the surface. The loop-out, conformation is stabilized by tight interactions of this tryptophan with, residue side chains of a symmetry-related molecule and enforced by a bound, sulfate. Characterization of this plant annexin using biophysical methods, revealed calcium-dependent binding to phospholipid vesicles with, preference for phosphatidylcholine over phosphatidylserine and, magnesium-dependent phosphodiesterase activity in vitro as shown with, adenosine triphosphate as the substrate. A comparative unfolding study of, recombinant annexin 24(Ca32) wild type and of the His-tag fusion protein, indicates higher stability of the latter. The effect of this N-terminal, modification is also visible from CD spectra. Both proteins were subjected, to a FURA-2-based calcium influx assay, which gave high influx rates for, the wild-type but greatly reduced influx rates for the fusion protein. We, therefore conclude that the N-terminal domain is indeed a major regulatory, element modulating different annexin properties by allosteric mechanisms.
About this StructureAbout this Structure
1DK5 is a Single protein structure of sequence from Capsicum annuum with SO4 as ligand. Full crystallographic information is available from OCA.
ReferenceReference
Annexin 24 from Capsicum annuum. X-ray structure and biochemical characterization., Hofmann A, Proust J, Dorowski A, Schantz R, Huber R, J Biol Chem. 2000 Mar 17;275(11):8072-82. PMID:10713128
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