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Structures of N-terminally processed KRAS provide insight into the role of N-acetylation.

  • Author(s): Dharmaiah, Srisathiyanarayanan;
  • Tran, Timothy H;
  • Messing, Simon;
  • Agamasu, Constance;
  • Gillette, William K;
  • Yan, Wupeng;
  • Waybright, Timothy;
  • Alexander, Patrick;
  • Esposito, Dominic;
  • Nissley, Dwight V;
  • McCormick, Frank;
  • Stephen, Andrew G;
  • Simanshu, Dhirendra K
  • et al.
Abstract

Although post-translational modification of the C-terminus of RAS has been studied extensively, little is known about N-terminal processing. Mass spectrometric characterization of KRAS expressed in mammalian cells showed cleavage of the initiator methionine (iMet) and N-acetylation of the nascent N-terminus. Interestingly, structural studies on GDP- and GMPPNP-bound KRAS lacking the iMet and N-acetylation resulted in Mg2+-free structures of KRAS with flexible N-termini. In the Mg2+-free KRAS-GDP structure, the flexible N-terminus causes conformational changes in the interswitch region resulting in a fully open conformation of switch I. In the Mg2+-free KRAS-GMPPNP structure, the flexible N-terminus causes conformational changes around residue A59 resulting in the loss of Mg2+ and switch I in the inactive state 1 conformation. Structural studies on N-acetylated KRAS-GDP lacking the iMet revealed the presence of Mg2+ and a conformation of switch regions also observed in the structure of GDP-bound unprocessed KRAS with the iMet. In the absence of the iMet, the N-acetyl group interacts with the central beta-sheet and stabilizes the N-terminus and the switch regions. These results suggest there is crosstalk between the N-terminus and the Mg2+ binding site, and that N-acetylation plays an important role by stabilizing the N-terminus of RAS upon excision of the iMet.

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