Bora phosphorylation substitutes in trans for T-loop phosphorylation in Aurora A to promote mitotic entry.

Polo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry.

Protein global fold determination using site-directed spin and isotope labeling.

We describe a simple experimental approach for the rapid determination of protein global folds. This strategy utilizes site-directed spin labeling (SDSL) in combination with isotope enrichment to determine long-range distance restraints between amide protons and the unpaired electron of a nitroxide spin label using the paramagnetic effect on relaxation rates. The precision and accuracy of calculating a protein global fold from only paramagnetic effects have been demonstrated on barnase, a well-characterized protein. Two monocysteine derivatives of barnase, (H102C) and (H102A/Q15C), were 15N enriched, and the paramagnetic nitroxide spin label, MTSSL, attached to the single Cys residue of each. Measurement of amide 1H longitudinal relaxation times, in both the oxidized and reduced states, allowed the determination of the paramagnetic contribution to the relaxation processes. Correlation times were obtained from the frequency dependence of these relaxation processes at 800, 600, and 500 MHz. Distances in the range of 8 to 35 A were calculated from the magnitude of the paramagnetic contribution to the relaxation processes and individual amide 1H correlation times. Distance restraints from the nitroxide spin to amide protons were used as restraints in structure calculations. Using nitroxide to amide 1H distances as long-range restraints and known secondary structure restraints, barnase global folds were calculated having backbone RMSDs <3 A from the crystal structure. This approach makes it possible to rapidly obtain the overall topology of a protein using a limited number of paramagnetic distance restraints.

Solution structures of the C-terminal domain of cardiac troponin C free and bound to the N-terminal domain of cardiac troponin I.

The N-terminal domain of cardiac troponin I (cTnI) comprising residues 33-80 and lacking the cardiac-specific amino terminus forms a stable binary complex with the C-terminal domain of cardiac troponin C (cTnC) comprising residues 81-161. We have utilized heteronuclear multidimensional NMR to assign the backbone and side-chain resonances of Ca2+-saturated cTnC(81-161) both free and bound to cTnI(33-80). No significant differences were observed between secondary structural elements determined for free and cTnI(33-80)-bound cTnC(81-161). We have determined solution structures of Ca2+-saturated cTnC(81-161) free and bound to cTnI(33-80). While the tertiary structure of cTnC(81-161) is qualitatively similar to that observed free in solution, the binding of cTnI(33-80) results mainly in an opening of the structure and movement of the loop region between helices F and G. Together, these movements provide the binding site for the N-terminal domain of cTnI. The putative binding site for cTnI(33-80) was determined by mapping amide proton and nitrogen chemical shift changes, induced by the binding of cTnI(33-80), onto the C-terminal cTnC structure. The binding interface for cTnI(33-80), as suggested from chemical shift changes, involves predominantly hydrophobic interactions located in the expanded hydrophobic pocket. The largest chemical shift changes were observed in the loop region connecting helices F and G. Inspection of available TnC sequences reveals that these residues are highly conserved, suggesting a common binding motif for the Ca2+/Mg2+-dependent interaction site in the TnC/TnI complex.

NMR analysis of cardiac troponin C-troponin I complexes: effects of phosphorylation.

Phosphorylation of the cardiac specific amino-terminus of troponin I has been demonstrated to reduce the Ca2+ affinity of the cardiac troponin C regulatory site. Recombinant N-terminal cardiac troponin I proteins, cardiac troponin I(33-80), cardiac troponin I(1-80), cardiac troponin I(1-80)DD and cardiac troponin I(1-80)pp, phosphorylated by protein kinase A, were used to form stable binary complexes with recombinant cardiac troponin C. Cardiac troponin I(1-80)DD, having phosphorylated Ser residues mutated to Asp, provided a stable mimetic of the phosphorylated state. In all complexes, the N-terminal domain of cardiac troponin I primarily makes contact with the C-terminal domain of cardiac troponin C. The nonphosphorylated cardiac specific amino-terminus, cardiac troponin I(1-80), was found to make additional interactions with the N-terminal domain of cardiac troponin C.

Effects of troponin I phosphorylation on conformational exchange in the regulatory domain of cardiac troponin C.

Conformational exchange has been demonstrated within the regulatory domain of calcium-saturated cardiac troponin C when bound to the NH2-terminal domain of cardiac troponin I-(1-80), and cardiac troponin I-(1-80)DD, having serine residues 23 and 24 mutated to aspartate to mimic the phosphorylated form of the protein. Binding of cardiac troponin I-(1-80) decreases conformational exchange for residues 29, 32, and 34. Comparison of average transverse cross correlation rates show that both the NH2- and COOH-terminal domains of cardiac troponin C tumble with similar correlation times when bound to cardiac troponin I-(1-80). In contrast, the NH2- and COOH-terminal domains in free cardiac troponin C and cardiac troponin C bound cardiac troponin I-(1-80)DD tumble independently. These results suggest that the nonphosphorylated cardiac specific NH2 terminus of cardiac troponin I interacts with the NH2-terminal domain of cardiac troponin C.