Analysis of Binding Site Hot Spots on the Surface of Ras GTPase

Abstract

We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the “off” and “on” allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond the active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to well-ordered locations that should be easier to target.

The Ras–Membrane Interface: Isoform-Specific Differences in the Catalytic Domain

“…The interaction between the G domain and the plasma membrane in this active orientation places positively charged helix 4 residues R128 and R135 close to the membrane, promoting specific electrostatic attraction to abundant negatively charged head groups or nonspecific interactions with phosphate groups in the plasma membrane (3,54). Computational solvent mapping, known as FTMap, indicates that residues surrounding R128 and R135 form "hot spots" of protein-protein or protein-ligand interactions on the solvent-accessible surface (10). It is possible that specific membrane lipid head groups interact with H-Ras residues at these hot spots.…”

“…An H-Ras surface pocket identified by the multiple solvent crystal structures (MSCS) technique consisting of residues H94, L133, S136, and Y137 (referred to as cluster 2, see Fig. 5), which is situated between helices 3 and 4 and also lies near the membrane when H-Ras is signaling active, indicates another venue of protein-membrane communication (10). Interestingly, crystal structures obtained with organic molecules bound to Ras show that polar and hydrophobic interactions mediate protein-ligand binding (10).…”

“…5), which is situated between helices 3 and 4 and also lies near the membrane when H-Ras is signaling active, indicates another venue of protein-membrane communication (10). Interestingly, crystal structures obtained with organic molecules bound to Ras show that polar and hydrophobic interactions mediate protein-ligand binding (10). One can imagine that phospholipid head groups interact with the Ras isoforms using comparable mechanisms.…”

“…Despite only having a crystal structure of H-Ras indicating the importance of allosteric modulation, this modulation could be particularly important in regulating K-Ras, which is natively surrounded by a higher amount of negatively charged lipid head groups and also happens to be the most potent Raf activator (65). Regardless of whether switch II is ordered in the R (reactive) state or disordered in the T (tardy) state, FTMap results show that the R128, R135, allosteric, and cluster 2 sites are hot spots for small-molecule interactions (10). This supports the idea that the membrane plays a structural role in mediating Ras function.…”

“…Despite this fact, each isoform promotes distinct, yet overlapping, signaling outputs to reduce functional redundancy (7,8). These differences have been attributed primarily to the C-terminal HVR, but significant differences among the isoforms also occur in the allosteric lobe (residues 87-166) where structural biology studies have begun to focus (9,10). This shift has paralleled the recent rise of powerful cell biology techniques capable of obtaining real-time protein-membrane interaction information (11).…”