Neutron Crystal Structure of RAS GTPase Puts in Question the Protonation State of the GTP γ-Phosphate*

By R. Knihtila, G. Holzapfel, K. Weiss, F. Meilleur, Carla Mattos. 2015

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Background: The GTP nucleotide is thought to be fully deprotonated when bound to RAS. Results: The neutron crystal structure of RAS bound to the GTP analogue GppNHp shows a protonated γ-phosphate. Conclusion: The active site of RAS significantly increases the pKa of the nucleotide. Significance: This work provides insight to the GTP hydrolysis mechanism, with implications to the superfamily of small GTPases. RAS GTPase is a prototype for nucleotide-binding proteins that function by cycling between GTP and GDP, with hydrogen atoms playing an important role in the GTP hydrolysis mechanism. It is one of the most well studied proteins in the superfamily of small GTPases, which has representatives in a wide range of cellular functions. These proteins share a GTP-binding pocket with highly conserved motifs that promote hydrolysis to GDP. The neutron crystal structure of RAS presented here strongly supports a protonated γ-phosphate at physiological pH. This counters the notion that the phosphate groups of GTP are fully deprotonated at the start of the hydrolysis reaction, which has colored the interpretation of experimental and computational data in studies of the hydrolysis mechanism. The neutron crystal structure presented here puts in question our understanding of the pre-catalytic state associated with the hydrolysis reaction central to the function of RAS and other GTPases.

Subfields: Biochemistry, Materials Chemistry, Molecular Biology

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Citations in the corpus

Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix (2019, background): “It is therefore possible to locate H (or D) atoms and to deduce their protonation states; this knowledge helps in understanding catalytic mechanisms and ligand binding (Yamaguchi et al., 2009; Bryan et al., 2013; Knihtila et al., 2015).”
Evaluation of models determined by neutron diffraction and proposed improvements to their validation and deposition (2018, background): “…al., 2003; Weber et al., 2013; Haupt et al., 2014; Casadei et al., 2014; Howard et al., 2016), neutron diffraction is able to provide valuable information for the understanding of catalytic mechanisms and ligand binding (Yamaguchi et al., 2009; Bryan et al., 2013; Knihtila et al., 2015).”