Probing the role of loop 2 in Ras function with unnatural amino acids.

Abstract

The YDPT sequence motif (residues 32-35) in loop 2 (residues 32-40) of Ha-Ras p21 protein is conserved in the Ras protein family. X-ray crystal structures have revealed significant conformational differences in this region between the GTP-and GDP-bound forms. Moreover, mutations in this region block neoplastic transformation and prevent interaction with GTPase-activating protein (GAP), suggesting that this region may contribute to the effector function of Ras. To better understand the structural features required for GAP interaction and GTPase activity, the expanded repertoire of unnatural amino acid mutagenesis has been used to investigate the roles of the key residues, Pro-34, . A Pro-34--methanoproline mutant, in which residue 34 is locked in the trans conformation, was found to retain high levels of intrinsic and GAP-activated GTPase activity, making unlikely conformational isomerization at this position. Deletion of a single methyl group from Be (Ile-36 --norvaline) abolished GAP activation of Ras, revealing a remarkable specificity in this protein-protein interaction. Finally, replacement of Thr-35 with diastereomeric allo-threonine led to inactivation of Ras, demonstrating the importance of the orientation of this critical residue in Ras function.Mammalian proteins encoded by the ras genes function as molecular switches in the signaling events associated with cell growth and differentiation (1-4). The chemical basis for the regulation involves cycling of the protein between the inactive (off) GDP-bound state and the active (on) GTPbound state. The low intrinsic GTPase activity ofcellular Ras protein is increased by interaction with the cytosolic protein GTPase-activating protein (GAP), a possible effector or negative regulator of Ras. In addition, GDP-GTP exchange factors (5-8), which have recently been identified as possible positive regulators, enhance the dissociation ofGDP from the protein and facilitate rebinding of GTP. Point mutations that result in a modest decrease in the intrinsic GTPase activity of Ras or GAP (9-12)-stimulated GTPase activity are associated with a large number of human cancers.The YDPT sequence motif (residues 32-35 in loop 2 ofRas) is conserved throughout the Ras protein family. X-ray crystal structures (13-20) of the GDP-, guanosine 5'-[,8,y-methyleneltriphosphate-, and guanosine 5'-[P,lyimido]triphosphate-bound forms of Ras have shown that the loop 2 region undergoes significant conformational changes upon hydrolysis ofGTP. The 3-hydroxyl group ofThr-35, which associates with Mg2+ in the GTP-Ras complex, moves out toward solvent in the GDP complex. In addition, the side chain of Ile-36, which is solvent-exposed in the GTP complex, is positioned closer to the protein surface in the GDP complex. This flexibility has been proposed to be a functionally important feature of Ras and perhaps in the GTPase family in general. in this region that block neoplastic transformation also prevent interaction ofRas with GAP. WeThe publication costs of this article were defrayed in p...

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