Biochimica et Biophysica Acta (BBA) - General Subjects
ReviewLessons from computer simulations of Ras proteins in solution and in membrane
Introduction
Ras (Rat Sarcoma) protein was discovered more than four decades ago as the first oncogene product [1], [2]. Subsequent discoveries of many other related genes gave rise to the Ras family of proteins, a group of lipid-modified and membrane-associated intracellular switches that regulate cell growth, proliferation and differentiation [3], [4]. The switching function of Ras involves cycling between a GDP-bound ‘off’ and GTP-bound ‘on’ conformational states [5], [6], [7], [8]. However, this binary on/off picture is being challenged by the discovery of other (intermediate) conformational states in recent years [9], [10], [11], [12], [13], [14]. Efficient cycling between the on/off states of Ras requires GDP release and GTP hydrolysis facilitated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) [7], [15], respectively. For instance, GAP increases the very slow intrinsic ability of Ras to hydrolyze GTP (kcat = 0.028 min− 1 [16]) by about 105-fold [17]. Therefore, interaction with GAPs is crucial for turning off signal transmission. Somatic or germline Ras mutations that interfere with its intrinsic and/or GAP-assisted ability to hydrolyze GTP can result in uncontrolled cell growth or cancer [18]. In fact, Ras is found mutated in about 15% of all human tumors and in up to 90% of cases in specific tumor types [19], as well as in a number of developmental disorders [20], [21]. Therefore, a staggering number of biochemical (e.g., [22], [23], [24]), structural [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], spectroscopic [14], [35], [36], [37], [38] and theoretical [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55], [56] studies have been devoted to investigating the mechanistic aspects of the Ras GTPase function.
Our goal here is to summarize the contribution of molecular simulations to our current understanding of normal and aberrant Ras function. We focus on lessons from molecular mechanical simulations in aqueous and membrane environments. Though ab initio simulations with various flavors of quantum mechanics continue to play a central role in studying the catalytic process within the active site of Ras [57], [58], [59], [60], [61], [62], [63], [64], [65], [66], [67], [68], [69], [70], they are beyond the scope of the current review. The review is organized as follows. As a background, we first provide a general overview of the accumulated knowledge on Ras. We then turn to simulations of the soluble catalytic domain in aqueous media, followed by the isolated lipid anchor and the full-length protein in lipid bilayers. Given the large number of reports in the field, we could not cite them all and apologize to those authors whose work was left out due to space limitation.
Section snippets
Overview of Ras biology, biochemistry and structure
There are three major Ras isoforms in humans: N-, H- and K-Ras. These isoforms share a nearly identical water-soluble catalytic domain [7], [71] comprising the N-terminal residues 1–166. The catalytic domain can be subdivided into two lobes [42]. Lobe1 (residues 1–86) is strictly conserved across the Ras family and contains the functionally critical switch regions (switch1: residues 25–40 and switch2: residues 57–75), as well as the phosphate binding P-loop (residues 10–17) (Fig. 1, also see
Classical MD simulations of Ras in solution
The earliest unbiased MD simulations of Ras [98], [99], [100], [101], [102] were very short (typically 100–500 ps) by current standards but they were able to elucidate the flexible nature of the nucleotide-binding loops (for illustration, see Fig. 2 from more recent simulations). These initial studies thus helped explain why the conformation of these loops, if observed at all, differ between X-ray structures of Ras solved with GDP and GTP analogues [25], [27], [82], [83]. Another slightly longer
Enhanced and biased simulations of Ras in solution
The atomically detailed classical MD (cMD) simulations described in the previous section provided invaluable insights into the dynamics of wild type and mutant Ras. However, computational cost rarely allowed for running cMD simulations long enough to sample large timescale global motions. Such motions have been probed by enhanced or biased simulation approaches such as accelerated MD (aMD) [117] and targeted MD (tMD) [118], [119]. For instance, nucleotide-dependent spontaneous transition
Atomistic simulations of Ras in membranes
In addition to characterizing the dynamics of the Ras catalytic domain in solution, cMD has played a central role in providing structural insights into bilayer-bound Ras [40], [41], [43], [44], [45], [47], [129], [130]. The simulation results were generally consistent with the available, albeit limited, experimental data from solid state NMR and other spectroscopic techniques [130], [131], [132], [133]. One of the important observations from simulations of N- [45], [129], [130], H- [40], [41]
Coarse-grained simulations of Ras in membrane
Experiments in intact plasma membrane sheets [75] and synthetic bilayers [142], [143] have shown that Ras proteins assemble into dynamic clusters on membrane surfaces [72], [74], [75]. These nano-sized subdomains, or nanoclusters, are small (6–20 nm radius) and contain about 7 proteins per cluster [72]. Different Ras isoforms form distinct and non-overlapping nanoclusters [94], with clusters of active H-Ras and K-Ras, as well as the isolated lipid anchor of K-Ras (tK), being localized at
Perspective: simulations can aid in anti-Ras inhibitor design
Decades of efforts by academia and industry have failed to yield selective Ras inhibitors. Complicating factors to directly targeting Ras include the conservation of the active site in a large number of small G-proteins and the high concentration and affinity of cellular GTP for Ras. Another reason could be lack of molecular-level insight into the protein–lipid interactions underlying the distinct spatiotemporal membrane-organization of different Ras proteins. The ultimate goal of the
Acknowledgement
P.P. is supported by a postdoctoral training fellowship funded by the CPRIT Computational Cancer Biology Training Program (CCBTP) from the Cancer Prevention and Research Institute of Texas (CPRIT) (Grant No: RP101489). We thank the Texas Advanced Computing Center (TACC) for computational resources. This work is supported in part by grant from the National Institutes of Health General Medical Sciences (grant number R01GM100078).
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