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Published on: September 28, 2018
Structural and functional characterization of the recombinant death domain from death-associated protein kinase
Evangelos Dioletis1, Andrew J Dingley, Paul C Driscoll
1Research Department of Structural & Molecular Biology, University College London, London, United Kingdom.
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.
Insights
The death-associated protein kinase death domain (DAPk-DD) lacks a stable fold but still binds ERK2. This suggests its function may not require a classical structure, challenging current structural biology assignments.
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- Death-associated protein kinase (DAPk) is a key regulator in cell death signaling pathways.
- DAPk possesses a unique multi-domain structure, including a C-terminal death domain (DD) that positively regulates its activity.
- Understanding the DAPk death domain's structure and function is crucial for elucidating cell death mechanisms.
Purpose of the Study:
- To investigate the structural properties of the recombinant death domain of DAPk (DAPk-DD).
- To determine the interaction between DAPk-DD and extracellular-regulated kinase-2 (ERK2).
- To clarify the structural basis for DAPk-DD's interaction with ERK2.
Main Methods:
- Recombinant DAPk-DD was expressed as a fusion protein with the B1 domain of streptococcal protein G.
- Circular dichroism (CD) spectroscopy, NMR spectroscopy, and fluorescence assays were used to assess protein structure.
- In vitro binding assays measured the interaction kinetics between DAPk-DD and ERK2.
Main Results:
- Recombinant DAPk-DD exhibited low regular secondary structure and lacked a stable globular fold.
- Despite its disordered state, DAPk-DD bound to ERK2 with a 1:1 stoichiometry and a K d in the low micromolar range.
- Only the full-length DAPk-DD, not a partial 'D-motif', effectively bound ERK2.
- ERK2 binding did not induce significant secondary structure changes in DAPk-DD.
Conclusions:
- The DAPk death domain, in isolation, does not adopt a canonical, stable fold.
- The lack of a defined structure does not preclude DAPk-DD's ability to bind its partner ERK2.
- The findings suggest that DAPk-DD's function may rely on interactions independent of a stable globular conformation, potentially requiring cellular context or partners for proper folding.
