1DK5

CRYSTAL STRUCTURE OF ANNEXIN 24(CA32) FROM CAPSICUM ANNUUM

Summary for 1DK5

• Entry DOI: 10.2210/pdb1dk5/pdb
• Descriptor: ANNEXIN 24(CA32), SULFATE ION (3 entities in total)
• Functional Keywords: plant annexin, capsicum annuum, bell pepper, calcium binding protein, metal binding protein
• Biological source: Capsicum annuum
• Total number of polymer chains: 2
• Total formula weight: 77051.72

Authors

Hofmann, A.,Proust, J.,Dorowski, A.,Schantz, R.,Huber, R. (deposition date: 1999-12-06, release date: 2000-12-06, Last modification date: 2024-02-07)

Primary citation

Hofmann, A.,Proust, J.,Dorowski, A.,Schantz, R.,Huber, R.
Annexin 24 from Capsicum annuum. X-ray structure and biochemical characterization.
J.Biol.Chem., 275:8072-8082, 2000

PubMed Abstract: 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.

PubMed: 10713128
DOI: 10.1074/jbc.275.11.8072

Experimental method

X-RAY DIFFRACTION (2.8 Å)