Design, expression, purification and crystallization of human 14-3-3ζ protein chimera with phosphopeptide from proapoptotic protein BAD.

14-3-3 protein isoforms regulate multiple processes in eukaryotes, including apoptosis and cell division. 14-3-3 proteins preferentially recognize phosphorylated unstructured motifs, justifying the protein-peptide binding approach to study 14-3-3/phosphotarget complexes. Tethering of human 14-3-3σ with partner phosphopeptides via a short linker has provided structural information equivalent to the use of synthetic phosphopeptides, simultaneously facilitating purification and crystallization. Nevertheless, the broader applicability to other 14-3-3 isoforms and phosphopeptides was unclear. Here, we designed a novel 14-3-3ζ chimera with a conserved phosphopeptide from BAD, whose complex with 14-3-3 is a gatekeeper of apoptosis regulation. The chimera could be bacterially expressed and purified without affinity tags. Co-expressed PKA efficiently phosphorylates BAD within the chimera and blocks its interaction with a known 14-3-3 phosphotarget, suggesting occupation of the 14-3-3 grooves by the tethered BAD phosphopeptide. Efficient crystallization of the engineered protein suggests suitability of the "chimeric" approach for studies of other relevant 14-3-3 complexes.

Bim, Bad and Bmf: intrinsically unstructured BH3-only proteins that undergo a localized conformational change upon binding to prosurvival Bcl-2 targets.

All BH3-only proteins, key initiators of programmed cell death, interact tightly with multiple binding partners and have sequences of low complexity, properties that are the hallmark of intrinsically unstructured proteins (IUPs). We show, using spectroscopic methods, that the BH3-only proteins Bim, Bad and Bmf are unstructured in the absence of binding partners. Detailed sequence analyses are consistent with this observation and suggest that most BH3-only proteins are unstructured. When Bim binds and inactivates prosurvival proteins, most residues remain disordered, only the BH3 element becomes structured, and the short alpha-helical molecular recognition element can be considered to behave as a 'bead on a string'. Coupled folding and binding is typical of many IUPs that have important signaling roles, such as BH3-only proteins, as the inherent structural plasticity favors interaction with multiple targets. This understanding offers promise for the development of BH3 mimetics, as multiple modes of binding are tolerated.