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@ARTICLE{Dioletis:139937,
author = {Dioletis, Evangelos and Dingley, Andrew and Driscoll, Paul
C},
title = {{S}tructural and functional characterization of the
recombinant death domain from death-associated protein
kinase.},
journal = {PLoS one},
volume = {8},
number = {7},
issn = {1932-6203},
address = {Lawrence, Kan.},
publisher = {PLoS},
reportid = {FZJ-2013-05904},
pages = {e70095},
year = {2013},
abstract = {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 α-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.},
cin = {ICS-6},
ddc = {500},
cid = {I:(DE-Juel1)ICS-6-20110106},
pnm = {452 - Structural Biology (POF2-452)},
pid = {G:(DE-HGF)POF2-452},
typ = {PUB:(DE-HGF)16},
pubmed = {pmid:23922916},
pmc = {pmc:PMC3726526},
UT = {WOS:000323369700132},
doi = {10.1371/journal.pone.0070095},
url = {https://juser.fz-juelich.de/record/139937},
}