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Balance of Conformational States Affect the Intrinsic Hydrolysis of NRas When Compared to Other Ras Isoforms
Author(s) -
Reid Derion F,
Mattos Carla
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.764.14
Subject(s) - hras , gtpase , neuroblastoma ras viral oncogene homolog , guanine nucleotide exchange factor , gtp' , gene isoform , kras , signal transduction , guanosine , chemistry , guanosine diphosphate , small gtpase , biochemistry , microbiology and biotechnology , gtp binding protein regulators , g protein , biology , guanosine triphosphate , mutation , enzyme , gene
Ras GTPases are small proteins that are involved in various signal transduction pathways that regulate cellular proliferation, survival, migration, and apoptosis. Although the first crystal structure of Ras was determined more than two decades ago, biochemical and structural differences in the G‐domain of the three isoforms, H‐, K‐ and N‐Ras is only now beginning to emerge. Each isoform functions as a molecular switch where guanine nucleotide exchange factors (GEFs) facilitate the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) turning its signal on. Ras signaling is turned off via intrinsic hydrolysis or through the aid of GTPase activating proteins (GAP). Ras mutants are involved in 20% of human cancers, yet there are no inhibitors that target these proteins effectively. Most of the effort was originally focused on HRas as the classical representative of all three isoforms and more recently on KRas, the most frequently mutated in cancers. Thus, the structural biology and biochemistry of NRas have been largely overlooked. We have determined hydrolysis rate constants for NRas and determined the crystal structure of wild‐type NRas bound to GppNHp (GNP), a non‐hydrolyzable analogue of GTP, for comparison with H‐Ras and K‐Ras. Although each of the three isoforms follow a one‐step mechanism of hydrolysis, K‐ and NRas have lower rates than HRas, with structural features that promote disorder in switch II near the active site. N‐Ras mutants at G12 and Q61 are commonly found in melanoma tumors. These conserved residues are found in the phosphate‐binding loop (G12) and switch II (Q61) that help make up the active site of NRas. The structures of NRas mutants reveal distinct mechanisms through which they affect catalysis, consistent with lower hydrolysis rate constants found for G12D, G12S, Q61H, and Q61K compared to wild‐type NRas. Biochemical and structural understanding of NRas mutants will help further influence the development of novel therapeutic targets of NRas mutant melanoma. Support or Funding Information This research was funded by NSF Grant Number MCB‐1517295.