Összes szerző

Györffy Dániel

az alábbi absztraktok szerzői között szerepel:

Hajdú István
Allosteric mechanism in both the activity and regulation of Rho kinase 2

Aug 29 - kedd

09:30 – 09:45

Molekuláris biofizika

E04

Allosteric mechanism in both the activity and regulation of Rho kinase 2

István Hajdú1, Barbara M Végh1, Dániel Györffy1,2, Attila Baksa1, András Szilágyi1, Péter Závodszky1,2

1Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary

2Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, Budapest, Hungary

Rho-associated protein kinase 2 (ROCK2) is a membrane-anchored, long, flexible, multidomain, multifunctional protein. Our solution small-angle X-ray scattering (SAXS) study revealed that ROCK2 population is a dynamic mixture of folded and partially extended conformers. Binding of RhoA to the coiled-coil domain shifts the equilibrium towards the partially extended state. Enzyme activity measurements suggest that binding of natural protein substrates to the kinase domain breaks up the interaction between the N-terminal kinase and C-terminal regulatory domains. We observed, that besides RhoA, binding of bioactive lipids to the C-terminal lipid binding domains also play an important part in the activation of the enzyme.

ROCK2 phosphorylates both Amyloid-beta Precursor Protein (at Thr654) and the APP-cleaving enzyme BACE1 (at Ser498) regulating amyloidogenic processing. Using direct physical methods in combination with in silico approach, we found that BACE1 binds to ROCK2 with a low affinity (Kd=18 µM), while no binding of APP to ROCK2 alone could be detected. Strong association (Kd =3.5 nM) of APP to the weak ROCK2-BACE1 complex was observed, although no stable ternary complex was detected, i.e. BACE1 was displaced by APP. We constructed a sequential functional model: (1) BACE1 weakly binds to ROCK2 and induces an allosteric conformational change in ROCK2; (2) APP strongly binds to the ROCK2/BACE1 complex, and BACE1 is released (3) ROCK2 phosphorylates APP at Thr654 (leading to a longer stay in the early endosome during APP processing). Using mutational and fragmenting approach, we concluded that two binding sites are involved in the ROCK2/APP interaction: (1) The substrate binding groove, where the APP646-664 sequence containing Thr654 sits (2) and the allosteric binding site, where the APP665-695 sequence binds. These results open the way to attack the allosteric site to prevent APP phosphorylation without inhibiting the activity of ROCK2 towards its other substrates.