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Structural and functional characterization of recombinant mouse annexin A11: influence of calcium binding.

ABSTRACT: Annexin A11 is one of the 12 vertebrate subfamilies in the annexin superfamily of calcium/phospholipid-binding proteins, distinguishable by long, non-homologous N-termini rich in proline, glycine and tyrosine residues. As there is negligible structural information concerning this annexin subfamily apart from primary sequence data, we have cloned, expressed and purified recombinant mouse annexin A11 to investigate its structural and functional properties. CD spectroscopy reveals two main secondary-structure contributions, alpha-helix and random coil (approx. 30% each), corresponding mainly to the annexin C-terminal tetrad and the N-terminus respectively. On calcium binding, an increase in alpha-helix and a decrease in random coil are detected. Fluorescence spectroscopy reveals that its only tryptophan residue, located at the N-terminus, is completely exposed to the solvent; calcium binding promotes a change in tertiary structure, which does not affect this tryptophan residue but involves the movement of approximately four tyrosine residues to a more hydrophobic environment. These calcium-induced structural changes produce a significant thermal stabilization, with an increase of approx. 14 degrees C in the melting temperature. Annexin A11 binds to acidic phospholipids and to phosphatidylethanolamine in the presence of calcium; weaker calcium-independent binding to phosphatidylserine, phosphatidic acid and phosphatidylethanolamine was also observed. The calcium-dependent binding to phosphatidylserine is accompanied by an increase in alpha-helix and a decrease in random-coil contents, with translocation of the tryptophan residue towards a more hydrophobic environment. This protein induces vesicle aggregation but requires non-physiological calcium concentrations in vitro. A three-dimensional model, consistent with these data, was generated to conceptualize annexin A11 structure-function relationships.

SUBMITTER: Lecona E

PROVIDER: S-EPMC1223495 | biostudies-other | 2003 Jul

REPOSITORIES: biostudies-other

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Project description:BackgroundAmong the EF-Hand calcium-binding proteins the subgroup of S100 proteins constitute a large family with numerous and diverse functions in calcium-mediated signaling. The evolutionary origin of this family is still uncertain and most studies have examined mammalian family members.ResultsWe have performed an extensive search in several teleost genomes to establish the s100 gene family in fish. We report that the teleost S100 repertoire comprises fourteen different subfamilies which show remarkable similarity across six divergent teleost species. Individual species feature distinctive subsets of thirteen to fourteen genes that result from local gene duplications and gene losses. Eight of the fourteen S100 subfamilies are unique for teleosts, while six are shared with mammalian species and three of those even with cartilaginous fish. Several S100 family members are found in jawless fish already, but none of them are clear orthologs of cartilaginous or bony fish s100 genes. All teleost s100 genes show the expected structural features and are subject to strong negative selection. Many aspects of the genomic arrangement and location of mammalian s100 genes are retained in the teleost s100 gene family, including a completely conserved intron/exon border between the two EF hands. Zebrafish s100 genes exhibit highly specific and characteristic expression patterns, showing both redundancy and divergence in their cellular expression. In larval tissue expression is often restricted to specific cell types like keratinocytes, hair cells, ionocytes and olfactory receptor neurons as demonstrated by in situ hybridization.ConclusionThe origin of the S100 family predates at least the segregation of jawed from jawless fish and some extant family members predate the divergence of bony from cartilaginous fish. Despite a complex pattern of gene gains and losses the total repertoire size is remarkably constant between species. On the expression level the teleost S100 proteins can serve as precise markers for several different cell types. At least some of their functions may be related to those of their counterparts in mammals. Accordingly, our findings provide an excellent basis for future studies of the functions and interaction partners of s100 genes and finally their role in diseases, using the zebrafish as a model organism.

Project description:Death-associated protein kinase (DAPk) is a calcium/calmodulin-regulated Ser/Thr-protein kinase that functions at an important point of integration for cell death signaling pathways. DAPk has a structurally unique multi-domain architecture, including a C-terminally positioned death domain (DD) that is a positive regulator of DAPk activity. In this study, recombinant DAPk-DD was observed to aggregate readily and could not be prepared in sufficient yield for structural analysis. However, DAPk-DD could be obtained as a soluble protein in the form of a translational fusion protein with the B1 domain of streptococcal protein G. In contrast to other DDs that adopt the canonical six amphipathic ?-helices arranged in a compact fold, the DAPk-DD was found to possess surprisingly low regular secondary structure content and an absence of a stable globular fold, as determined by circular dichroism (CD), NMR spectroscopy and a temperature-dependent fluorescence assay. Furthermore, we measured the in vitro interaction between extracellular-regulated kinase-2 (ERK2) and various recombinant DAPk-DD constructs. Despite the low level of structural order, the recombinant DAPk-DD retained the ability to interact with ERK2 in a 1?1 ratio with a K d in the low micromolar range. Only the full-length DAPk-DD could bind ERK2, indicating that the apparent 'D-motif' located in the putative sixth helix of DAPk-DD is not sufficient for ERK2 recognition. CD analysis revealed that binding of DAPk-DD to ERK2 is not accompanied by a significant change in secondary structure. Taken together our data argue that the DAPk-DD, when expressed in isolation, does not adopt a classical DD fold, yet in this state retains the capacity to interact with at least one of its binding partners. The lack of a stable globular structure for the DAPk-DD may reflect either that its folding would be supported by interactions absent in our experimental set-up, or a limitation in the structural bioinformatics assignment of the three-dimensional structure.

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Structural and functional characterization of recombinant mouse annexin A11: influence of calcium binding.

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