Annexin 24 from Capsicum annuum - X-ray structure and biochemical characterization

This work provides the first three-dimensional structure of a member of the plant annexin family and correlates these findings with biochemical proper ties of this protein. Annexin 24(Ca32) from Capsicum annuum was purified as a native protein fr om bell pepper and was also prepared by recombinant te chniques. To overcome the problem of precipitation of the recombinant wildt ype protein in crystallization trials, two mutants were designed. Whereas a n N-terminal truncation mutant turned out to be an unstable protein, the N- terminal His-tagged annexin 24(Ca32) was crystallized, and the three-dimens ional structure was determined by x-ray diffraction at 2.8 Angstrom resolut ion, The structure refined to an R-factor of 0.216 adopts the typical annex in fold; the detailed structure, however, is different from non-plant annex ins, especially in domains I and III and in the membrane binding loops on t he convex side. Within the unit cell there are two molecules per asymmetric unit, which differ in conformation of the LQB-loop. Both conformers show T rp-35 on the surface. The loop-out conformation is stabilized by tight inte ractions of this tryptophan with residue side chains of a symmetry-related molecule and enforced by a bound sulfate. Characterization of this plant an nexin using biophysical methods revealed calcium-dependent binding to phosp holipid vesicles with preference for phosphatidylcholine over phosphatidyls erine 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 protei n indicates higher stability of the latter. The effect of this N-terminal m odification is also visible from CD spectra. Both proteins were subjected t o 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 ther efore conclude that the N-terminal domain is indeed a major regulatory elem ent modulating different annexin properties by allosteric mechanisms.