1dk5: Difference between revisions

Revision as of 13:17, 21 February 2008

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 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

Page seeded by OCA on Thu Feb 21 12:17:26 2008

Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

OCA

@@ Line 1: / Line 1: @@
-[[Image:1dk5.jpg|left|200px]]<br /><applet load="1dk5" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1dk5.jpg|left|200px]]<br /><applet load="1dk5" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1dk5, resolution 2.8&Aring;" />
 '''CRYSTAL STRUCTURE OF ANNEXIN 24(CA32) FROM CAPSICUM ANNUUM'''<br />
 ==Overview==
-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.
+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 Structure==
-DK5 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Capsicum_annuum Capsicum annuum] with SO4 as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1DK5 OCA].
+DK5 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Capsicum_annuum Capsicum annuum] with <scene name='pdbligand=SO4:'>SO4</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1DK5 OCA].
 ==Reference==
@@ Line 24: / Line 24: @@
 [[Category: plant annexin]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Sun Nov 25 03:13:50 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:17:26 2008''