RESUMEN
Here, we characterize the role of a π-helix in the molecular mechanisms underlying thermoadaptation in the glycoside hydrolase family 4 (GH4). The interspersed π-helix present in a subgroup is evolutionarily related to a conserved α-helix in other orthologs by a single residue insertion/deletion event. The insertional residue, Phe407, in a hyperthermophilic α-glucuronidase, makes specific interactions across the inter-subunit interface. In order to establish the sequence-structure-stability implications of the π-helix, the wild-type and the deletion variant (Δ407) were characterized. The variant showed a significant lowering of melting temperature and optimum temperature for the highest activity. Crystal structures of the proteins show a transformation of the π-helix to a continuous α-helix in the variant, identical to that in orthologs lacking this insertion. Thermodynamic parameters were determined from stability curves representing the temperature dependence of unfolding free energy. Though the proteins display maximum stabilities at similar temperatures, a higher melting temperature in the wild-type is achieved by a combination of higher enthalpy and lower heat capacity of unfolding. Comparisons of the structural changes, and the activity and thermodynamic profiles allow us to infer that specific non-covalent interactions, and the existence of residual structure in the unfolded state, are crucial determinants of its thermostability. These features permit the enzyme to balance the preservation of structure at a higher temperature with the thermodynamic stability required for optimum catalysis.
Asunto(s)
Bacillus subtilis/química , Proteínas Bacterianas/química , Glicósido Hidrolasas/química , Thermotoga maritima/química , Secuencia de Aminoácidos , Bacillus subtilis/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biocatálisis , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/enzimología , Escherichia coli/genética , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Calor , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Termodinámica , Thermotoga maritima/enzimologíaRESUMEN
Global and endothelial loss of PTP-PEST (also known as PTPN12) is associated with impaired cardiovascular development and embryonic lethality. Although hypoxia is implicated in vascular remodelling and angiogenesis, its effect on PTP-PEST remains unexplored. Here we report that hypoxia (1% oxygen) increases protein levels and catalytic activity of PTP-PEST in primary endothelial cells. Immunoprecipitation followed by mass spectrometry revealed that α subunits of AMPK (α1 and α2, encoded by PRKAA1 and PRKAA2, respectively) interact with PTP-PEST under normoxia but not in hypoxia. Co-immunoprecipitation experiments confirmed this observation and determined that AMPK α subunits interact with the catalytic domain of PTP-PEST. Knockdown of PTP-PEST abrogated hypoxia-mediated tyrosine dephosphorylation and activation of AMPK (Thr172 phosphorylation). Absence of PTP-PEST also blocked hypoxia-induced autophagy (LC3 degradation and puncta formation), which was rescued by the AMPK activator metformin (500â µM). Because endothelial autophagy is a prerequisite for angiogenesis, knockdown of PTP-PEST also attenuated endothelial cell migration and capillary tube formation, with autophagy inducer rapamycin (200â nM) rescuing angiogenesis. In conclusion, this work identifies for the first time that PTP-PEST is a regulator of hypoxia-induced AMPK activation and endothelial autophagy to promote angiogenesis.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Proteína Tirosina Fosfatasa no Receptora Tipo 12 , Proteínas Quinasas Activadas por AMP/genética , Autofagia , Células Endoteliales/metabolismo , Humanos , Hipoxia , Fosforilación , Proteínas Tirosina FosfatasasRESUMEN
Glycosyl hydrolases belonging to the family 4 (GH4) use a unique redox-based NAD+-dependent reaction mechanism involving anionic intermediates and requires a divalent metal ion and reducing conditions for catalytic activity. These enzymes display wide specificity and selectivity for their substrates. However, the structural basis of substrate binding, recognition and specificity remains poorly studied. Here, we report the crystal structure of Thermotoga maritima TmAgu4B, a GH4 α-glucuronidase, in complex with Co2+ and citrate. Analysis of GH4 structures show that the metal ion is present in a conserved octahedral coordination with conserved side chain atoms, the ligand atoms and an invariant water molecule. The data provides the first structural evidence for a metal-activated hydroxide ion that acts as the general base to deprotonate the C3-hydroxyl group of the glycone, a rate-limiting step in the mechanism. Furthermore, the citrate binding mode in the active site is analogous to a bound glucuronide substrate and provides insights into the mode of substrate interaction with the metal ion, the active site residues and, the structural basis of substrate recognition in a GH4 α-glucuronidase.