# Peptides That Block RAS-p21 Protein-Induced Cell Transformation
### Structure of RAS-p21  
The X-ray crystal structures of wild-type and oncogenic forms of RAS-p21 have been determined (12,13). In the crystal structure of RAS-p21 bound to the activating non-hydrolyzable GTP analogue, the \(\Upsilon\)-phosphoroamidate form of GTP (GppNHp), there are distinct domains that interact with this activating ligand [12]. These include the P loop domain, containing residues 4-20 that contain the critical mutational sites including Gly 12 and Gly 13; the switch 1 domain, containing residues 32-47; an effector domain, of which has been shown to be involved in the interaction of RAS-p21 with critical downstream targets such as RAF discussed in the next section below; and the switch 2 domain, consisting of residues 55-76 and containing other critical mutational sites such as Gln 61 and Gly 60. This domain contains a short \(\alpha\)-helical segment containing residues 67-73, called the \(\alpha\)2 helix, which can interact with another \(\alpha\)-helical domain, the \(\alpha\)3 helix, involving residues 89-103. The disposition of the two switch domains and the \(\alpha\)3 helix can give rise to drug-accessible pockets, enabling specific agents, including peptides, to lock oncogenic RAS-p21 in inactive states, mainly affecting the conformation of the switch 1 domain, resulting in the blockade of uncontrolled cell division, as discussed in ensuing sections. In the structure of the activated wild-type RAS-p21 bound to GppNHp, guanine ring and pendant atoms form hydrogen bonds with Asp 119, Ser 145 and Ala 146; the ribofuranose ring forms hydrogen bonds with Val 29 and Asp 30 and Lys 117. Importantly, all three phosphate residues form a network of hydrogen bonds with P-loop residues, i.e., in the domain involving residues 4-20: the \(\alpha\)-phosphate oxygens form multiple hydrogen bonds with the main chain and -OH side chain of Ser 17 and main chain of Ala 18, while the connecting \(\alpha\)-\(\beta\) oxygen in this phosphate forms a hydrogen bond with the backbone of Gly15. Val 14, Gly 15, Ser 17 and Lys 16 all form hydrogen bonds to the \(\beta\)-phosphate atoms. Gly 13 forms a hydrogen bond to the \(\beta\)-\(\Upsilon\) nitrogen of the \(\Upsilon\)-phosphoroamidate nitrogen. Interestingly, residues from the P-loop, switch 1 and switch 2 domains (the latter containing residues 59-64 called the L4 loop) form extensive hydrogen bonding interactions with the \(\Upsilon\)-phosphate including Lys 16, Tyr 32, Thr 35 and Gly 60. All three of these domains are involved in the critical conformational changes that occur in the activation of RAS-p21. Effects of GTP Binding and Oncogenic Amino Acid Substitutions on the Structure and Function of RAS-p21  
As noted in Section 1.1 above, arbitrary non-cyclic-L-amino acid substitutions for Gly 12 cause the RAS-p21 protein to become oncogenic. A comparison of the X-ray crystal structures of wild-type RAS-p21 with oncogenic forms of RAS-p21 including G12R, G12V, Q61 His, Q61L between 2 and 2.6 A resolution indicated no major structural changes between the wild-type and mutant forms [13]. Rather, each amino acid substitution at both the 12 and 61 positions could likely cause the loss of GTPase activity due to a disruption of the positioning of functional groups (like the carboxamido group of Gln 61) that would be involved in GTP hydrolysis, resulting in the prolonged activation of the mutant protein. For example, hydrogen bonding of the backbone NH of Gly 12 to a \(\Upsilon\)-phosphate oxygen might be disrupted by a side chain of a substituted amino acid while the loss of the carboxamidogroup of Gln 61 would prevent a similar type of interaction with this phosphate moiety in the RAS-p21 active site. In addition, these substitutions were considered to lower GAP activity in promoting \(\beta\)-\(\Upsilon\)-phosphate hydrolysis [13]. On the other hand, Pro 12, which has no backbone NH, is non-oncogenic.