RESUMO
The serine/threonine mammalian sterile 20-like kinase (MST1) is involved in promotion of caspase-dependent and independent apoptosis. Phosphorylation and oligomerization are required for its activation. The oligomerization domain, denoted as SARAH domain, forms an antiparallel coiled coil dimer, and it is important for both MST1 autophosphorylation and interactions with other proteins like the Rassf proteins containing also a SARAH domain. Here we show that the monomeric state of SARAH is thermodynamically unstable and that homodimerization is coupled with folding. Moreover, the influence of the inhibitory domain on SARAH stability and affinity is addressed. By investigating the thermal denaturation using differential scanning calorimetry and circular dichroism, we have found that the SARAH domain dissociates and unfolds cooperatively, without a stable intermediate monomeric state. Combining the data with information from isothermal titration calorimetry, a low thermodynamic stability of the monomeric species is obtained. Thus, it is proposed that the transition from MST1 SARAH homodimer to some specific heterodimer implies a non-native monomer intermediate. The inhibitory domain is found to be highly flexible and intrinsically unfolded, not only in isolation but also in the dimeric state of the inhibitory-SARAH construct. The existence of two caspase recognition motifs within the inhibitory domain suggests that its structural flexibility might be important for activation of MST1 during apoptosis. Moreover, the inhibitory domain increases the thermodynamic stability of the SARAH dimer and the homodimer affinity, while having almost no effect on the SARAH domain in the monomeric state. These results emphasize the importance of flexibility and binding-induced folding for specificity, affinity, and the capacity to switch from one state to another.
Assuntos
Dimerização , Fator de Crescimento de Hepatócito/química , Dobramento de Proteína , Proteínas Proto-Oncogênicas/química , Algoritmos , Calorimetria , Varredura Diferencial de Calorimetria , Caspases/metabolismo , Dicroísmo Circular , Reagentes de Ligações Cruzadas/química , Estabilidade Enzimática , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Modelos Moleculares , Nefelometria e Turbidimetria , Concentração Osmolar , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Desnaturação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
Human guanylate binding protein 1 (hGBP1) belongs to the dynamin superfamily of large GTPases (LGs). In the course of GTP hydrolysis, the protein undergoes structural changes leading to self-assembly of the protein, which is a characteristic property of all family members. For self-assembly, the protein employs two distinct interaction sites, one of which is located within the LG domain of the protein located at the N-terminus, and the second is located in the C-terminal alpha-helical domain. Here, we identify intramolecular contacts between the LG domain and the helical part of hGBP1, which relay nucleotide-dependent structural changes from the N-terminus to the C-terminus and thereby mediate tetramer formation of the protein through a second contact site at the C-terminus. Furthermore, we demonstrate the impact of this intramolecular communication on the enzymatic activity of hGBP1 and on its cellular localization.
