ABSTRACT
The tumour suppressor protein RASSF1A is phosphorylated by Aurora A kinase, thereby impairing its tumour suppressor function. Consequently, inhibiting the interaction between Aurora A and RASSF1A may be used for anti-tumour therapy. We used recombinant variants of RASSF1A to map the sites of interaction with Aurora A. The phosphorylation kinetics of three truncated RASSF1A variants has been analysed. Compared to the RASSF1A form lacking the 120 residue long N-terminal part, the Km value of the phosphorylation is increased from 10 to 45 µM upon additional deletion of the C-terminal SARAH domain. On the other hand, deletion of the flexible loop (Δ177-197) that precedes the phosphorylation site/s (T202/S203) results in a reduction of the kcat value from about 40 to 7 min-1. Direct physical interaction between the isolated SARAH domain and Aurora A was revealed by SPR. These data demonstrate that the SARAH domain of RASSF1A is involved in the binding to Aurora A kinase. Structural modelling confirms that a novel complex is feasible between the SARAH domain and the kinase domain of Aurora A. In addition, a regulatory role of the loop in the catalytic phosphorylation reaction has been demonstrated both experimentally and by structural modelling.
Subject(s)
Aurora Kinase A/metabolism , Protein Interaction Domains and Motifs , Receptors, Opioid, kappa/metabolism , Aurora Kinase A/chemistry , Aurora Kinase A/genetics , Binding Sites , Chromatography, Gel , Models, Molecular , Mutation , Phosphorylation , Protein Multimerization , Receptors, Opioid, kappa/chemistry , Receptors, Opioid, kappa/genetics , Surface Plasmon ResonanceABSTRACT
Coupling of structural flexibility and biological function is an essential feature of proteins. The role of relative domain movements in enzyme function has been evidenced in many cases. However, the way of communication between protein domains and its manifestation in their movements as well as in the biological function are rarely delineated. In this review we summarize comprehensive studies with a typical hinge-bending two-domain enzyme, 3-phosphoglycerate kinase. A possible mechanism is proposed by which the two substrates that bind to different domains trigger the operation of the molecular hinges, located in the interdomain region. Various crystal structures of the enzyme have been determined with different relative domain positions, suggesting that domain closure brings the two substrates together for the catalysis. Substrate-caused conformational changes in the binary and the ternary complexes have been tested with the solubilized enzyme using physical methods, such as differential scanning calorimetry, small angle X-ray scattering and infrared spectroscopy. The results indicated the existence of strong cooperativity between the two domains and that the presence of both substrates is necessary for the domain closure. Comparison of the atomic contacts in the structures has led to selection of conserved side-chains, which may be involved in the domain movement. On this basis a hypothesis was put forward about the molecular mechanism of interdomain co-operation. Enzyme kinetic, ligand binding and small angle X-ray scattering studies with various site-directed mutants have confirmed this hypothesis. Namely, a special H-bonding network (a double molecular switch) seems to be responsible for operation of the main molecular hinge at the beta-strand L under the concerted action of both substrates.
