Conformational resolution of nucleotide cycling and effector interactions for multiple small GTPases determined in parallel
Notice bibliographique
Résumé
Small GTPases alternatively bind GDP/GTP guanine nucleotides to gate signaling pathways that direct most cellular processes. Numerous GTPases are implicated in oncogenesis, particularly the three RAS isoforms HRAS, KRAS, and NRAS and the RHO family GTPase RAC1. Signaling networks comprising small GTPases are highly connected, and there is some evidence of direct biochemical cross-talk between their functional G-domains. The activation potential of a given GTPase is contingent on a codependent interaction with the nucleotide and a Mg2+ ion, which bind to individual variants with distinct affinities coordinated by residues in the GTPase nucleotide-binding pocket. Here, we utilized a selective-labeling strategy coupled with real-time NMR spectroscopy to monitor nucleotide exchange, GTP hydrolysis, and effector interactions of multiple small GTPases in a single complex system. We provide insight into nucleotide preference and the role of Mg2+ in activating both WT and oncogenic mutant enzymes. Multiplexing revealed guanine nucleotide exchange factor (GEF), GTPase-activating protein (GAP), and effector-binding specificities in mixtures of GTPases and resolved that the three related RAS isoforms are biochemically equivalent. This work establishes that direct quantitation of the nucleotide-bound conformation is required to accurately determine an activation potential for any given GTPase, as small GTPases such as RAS-like proto-oncogene A (RALA) or the G12C mutant of KRAS display fast exchange kinetics but have a high affinity for GDP. Furthermore, we propose that the G-domains of small GTPases behave autonomously in solution and that nucleotide cycling proceeds independently of protein concentration but is highly impacted by Mg2+ abundance. Small GTPases alternatively bind GDP/GTP guanine nucleotides to gate signaling pathways that direct most cellular processes. Numerous GTPases are implicated in oncogenesis, particularly the three RAS isoforms HRAS, KRAS, and NRAS and the RHO family GTPase RAC1. Signaling networks comprising small GTPases are highly connected, and there is some evidence of direct biochemical cross-talk between their functional G-domains. The activation potential of a given GTPase is contingent on a codependent interaction with the nucleotide and a Mg2+ ion, which bind to individual variants with distinct affinities coordinated by residues in the GTPase nucleotide-binding pocket. Here, we utilized a selective-labeling strategy coupled with real-time NMR spectroscopy to monitor nucleotide exchange, GTP hydrolysis, and effector interactions of multiple small GTPases in a single complex system. We provide insight into nucleotide preference and the role of Mg2+ in activating both WT and oncogenic mutant enzymes. Multiplexing revealed guanine nucleotide exchange factor (GEF), GTPase-activating protein (GAP), and effector-binding specificities in mixtures of GTPases and resolved that the three related RAS isoforms are biochemically equivalent. This work establishes that direct quantitation of the nucleotide-bound conformation is required to accurately determine an activation potential for any given GTPase, as small GTPases such as RAS-like proto-oncogene A (RALA) or the G12C mutant of KRAS display fast exchange kinetics but have a high affinity for GDP. Furthermore, we propose that the G-domains of small GTPases behave autonomously in solution and that nucleotide cycling proceeds independently of protein concentration but is highly impacted by Mg2+ abundance.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule tête enseignante, pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».