An undecided coiled coil: the leucine zipper of Nek2 kinase exhibits atypical conformational exchange dynamics.

Leucine zippers are oligomerization domains used in a wide range of proteins. Their structure is based on a highly conserved heptad repeat sequence in which two key positions are occupied by leucines. The leucine zipper of the cell cycle-regulated Nek2 kinase is important for its dimerization and activation. However, the sequence of this leucine zipper is most unusual in that leucines occupy only one of the two hydrophobic positions. The other position, depending on the register of the heptad repeat, is occupied by either acidic or basic residues. Using NMR spectroscopy, we show that this leucine zipper exists in two conformations of almost equal population that exchange with a rate of 17 s(-1). We propose that the two conformations correspond to the two possible registers of the heptad repeat. This hypothesis is supported by a cysteine mutant that locks the protein in one of the two conformations. NMR spectra of this mutant showed the predicted 2-fold reduction of peaks in the (15)N HSQC spectrum and the complete removal of cross peaks in exchange spectra. It is possible that interconversion of these two conformations may be triggered by external signals in a manner similar to that proposed recently for the microtubule binding domain of dynein and the HAMP domain. As a result, the leucine zipper of Nek2 kinase is the first example where the frameshift of coiled-coil heptad repeats has been directly observed experimentally.

NEMO oligomerization and its ubiquitin-binding properties.

The IKK [IkappaB (inhibitory kappaB) kinase] complex is a key regulatory component of NF-kappaB (nuclear factor kappaB) activation and is responsible for mediating the degradation of IkappaB, thereby allowing nuclear translocation of NF-kappaB and transcription of target genes. NEMO (NF-kappaB essential modulator), the regulatory subunit of the IKK complex, plays a pivotal role in this process by integrating upstream signals, in particular the recognition of polyubiquitin chains, and relaying these to the activation of IKKalpha and IKKbeta, the catalytic subunits of the IKK complex. The oligomeric state of NEMO is controversial and the mechanism by which it regulates activation of the IKK complex is poorly understood. Using a combination of hydrodynamic techniques we now show that apo-NEMO is a highly elongated, dimeric protein that is in weak equilibrium with a tetrameric assembly. Interaction with peptides derived from IKKbeta disrupts formation of the tetrameric NEMO complex, indicating that interaction with IKKalpha and IKKbeta and tetramerization are mutually exclusive. Furthermore, we show that NEMO binds to linear di-ubiquitin with a stoichiometry of one molecule of di-ubiquitin per NEMO dimer. This stoichiometry is preserved in a construct comprising the second coiled-coil region and the leucine zipper and in one that essentially spans the full-length protein. However, our data show that at high di-ubiquitin concentrations a second weaker binding site becomes apparent, implying that two different NEMO-di-ubiquitin complexes are formed during the IKK activation process. We propose that the role of these two complexes is to provide a threshold for activation, thereby ensuring sufficient specificity during NF-kappaB signalling.

Structure and regulation of the human Nek2 centrosomal kinase.

The dimeric Ser/Thr kinase Nek2 regulates centrosome cohesion and separation through phosphorylation of structural components of the centrosome, and aberrant regulation of Nek2 activity can lead to aneuploid defects characteristic of cancer cells. Mutational analysis of autophosphorylation sites within the kinase domain identified by mass spectrometry shows a complex pattern of positive and negative regulatory effects on kinase activity that are correlated with effects on centrosomal splitting efficiency in vivo. The 2.2-A resolution x-ray structure of the Nek2 kinase domain in complex with a pyrrole-indolinone inhibitor reveals an inhibitory helical motif within the activation loop. This helix presents a steric barrier to formation of the active enzyme and generates a surface that may be exploitable in the design of specific inhibitors that selectively target the inactive state. Comparison of this "auto-inhibitory" conformation with similar arrangements in cyclin-dependent kinase 2 and epidermal growth factor receptor kinase suggests a role for dimerization-dependent allosteric regulation that combines with autophosphorylation and protein phosphatase 1c phosphatase activity to generate the precise spatial and temporal control required for Nek2 function in centrosomal maturation.

The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain.

Polo-like kinases (Plks) perform crucial functions in cell-cycle progression and multiple stages of mitosis. Plks are characterized by a C-terminal noncatalytic region containing two tandem Polo boxes, termed the Polo-box domain (PBD), which has recently been implicated in phosphodependent substrate targeting. We show that the PBDs of human, Xenopus, and yeast Plks all recognize similar phosphoserine/threonine-containing motifs. The 1.9 A X-ray structure of a human Plk1 PBD-phosphopeptide complex shows that the Polo boxes each comprise beta6alpha structures that associate to form a 12-stranded beta sandwich domain. The phosphopeptide binds along a conserved, positively charged cleft located at the edge of the Polo-box interface. Mutations that specifically disrupt phosphodependent interactions abolish cell-cycle-dependent localization and provide compelling phenotypic evidence that PBD-phospholigand binding is necessary for proper mitotic progression. In addition, phosphopeptide binding to the PBD stimulates kinase activity in full-length Plk1, suggesting a conformational switching mechanism for Plk regulation and a dual functionality for the PBD.