Structure, Catalytic Mechanism, and Membrane Interaction of the mTOR Activator Rheb

The activator of mammalian target of rapamycin complex 1 (mTORC1), Ras homolog enriched in brain (Rheb), is a membrane-associated protein belonging to the Ras subfamily of small GTPases. Rheb's slow GTPase activity is stimulated by the GTPase activating protein (GAP) domain of tuberous sclerosis complex 1 and 2 (TSC1/2). Rheb hyperactivation, through its overexpression or loss of TSC1/2 GAP function, results in hyperactivated mTORC1 signaling culminating in tumourigenesis. The molecular details of Rheb GTP hydrolysis and the effect of membrane association on Rheb structure, dynamics and its GTPase function are currently not fully understood. The studies presented in this thesis focus on two key determinants of Rheb function i) the mechanism of Rheb GTP hydrolysis and ii) the structural and functional consequence of Rheb-bilayer membrane interaction. Through studies of fluorescent nucleotides, we revealed that the conserved G2-box residue Tyr35 auto-inhibits GTP hydrolysis in Rheb. We demonstrated that a non-canonical catalytic residue, Asp65, in the switch II region of Rheb, contributed more to the GTP hydrolysis rate than Gln64, which corresponds to the canonical Ras Gln61. This non-canonical auto-inhibited mechanism of GTP hydrolysis was required for optimal mTORC1 regulation. These structural insights were then used to guide the design of novel gain- and loss-of function mutants by substitutions of the ultra-conserved G3-box Gly63 of Rheb. Finally, using solution NMR spectroscopy, we monitored the Rheb GTPase cycle and characterized its nucleotide-dependent membrane orientations on nanodisc-based phospholipid bilayers. Rheb was shown to sample two orientations in which its C-terminal helix was semi-perpendicular or semi-parallel with respect to the bilayer plane. The semi-parallel orientation, where switch II residues critical for mTORC1 communication are accessible, was favored in the GTP bound conformation, suggesting that membrane-tethering modulates Rheb function. These structural insights into the catalytic machinery of Rheb and its membrane interface suggest new approaches to modulate these key determinants of Rheb function through small molecules towards development of therapeutic avenues for Rheb-mediated pathogenesis such as tuberous sclerosis or cancer.Ph.D.2016-06-16 00:00:0

The Auto-Inhibitory Role of the EPAC Hinge Helix as Mapped by NMR

<div><p>The cyclic-AMP binding domain (CBD) is the central regulatory unit of exchange proteins activated by cAMP (EPAC). The CBD maintains EPAC in a state of auto-inhibition in the absence of the allosteric effector, cAMP. When cAMP binds to the CBD such auto-inhibition is released, leading to EPAC activation. It has been shown that a key feature of such cAMP-dependent activation process is the partial destabilization of a structurally conserved hinge helix at the C-terminus of the CBD. However, the role of this helix in auto-inhibition is currently not fully understood. Here we utilize a series of progressive deletion mutants that mimic the hinge helix destabilization caused by cAMP to show that such helix is also a pivotal auto-inhibitory element of apo-EPAC. The effect of the deletion mutations on the auto-inhibitory apo/inactive <em>vs.</em> apo/active equilibrium was evaluated using recently developed NMR chemical shift projection and covariance analysis methods. Our results show that, even in the absence of cAMP, the C-terminal region of the hinge helix is tightly coupled to other conserved allosteric structural elements of the CBD and perturbations that destabilize the hinge helix shift the auto-inhibitory equilibrium toward the apo/active conformations. These findings explain the apparently counterintuitive observation that cAMP binds more tightly to shorter than longer EPAC constructs. These results are relevant for CBDs in general and rationalize why substrates sensitize CBD-containing systems to cAMP. Furthermore, the NMR analyses presented here are expected to be generally useful to quantitatively evaluate how mutations affect conformational equilibria.</p> </div

SVD analysis of the chemical shifts measured for the C-terminal truncation mutants de305, de310 and de312.

<p><b>a</b>) This panel shows the PC1 vs. PC2 plot with three sets of loadings (diamonds) for each of the C-terminal hinge helix deletion mutants: de312 (red), de310 (blue) and de305 (green). There are four loadings per mutant with each loading corresponding to a state referenced to Rp-cAMPS, as labelled in the figure. The smaller arrows correspond to the separation along PC1 between the Wt(apo) and the mutant(apo) state. The large arrows correspond to the separation along PC1 between the Wt(apo) and the cAMP-bound Wt(holo). <b>b</b>) The percentage ratio of the two separations measured in panel (a) (<i>i.e.</i> relative magnitude of the two arrows), provides a quantitative measure of the overall fractional shift toward activation caused by the mutation.</p

Chemical shift projection analysis to map the effects of the apo truncation mutants de312 (red), de310 (blue) and de305 (green) relative to Wt(apo).

<p>The dashed lines represent the secondary structure of the apo-EPAC (PDB ID: 2BYV). The grey highlights are regions subject to some of the most significant cAMP-dependent changes on the Wt(apo). (<b>a</b>) The compounded chemical shift profile of the apo-mutants relative to apo-Wt, that is the magnitude of vector A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048707#pone-0048707-g002" target="_blank">Figure 2A</a>. (<b>b</b>) Fractional shift toward activation achieved by the mutations in the absence of cAMP and with compounded chemical shifts greater than 0.05 ppm between the Wt(apo) and Wt(holo) state. (<b>c</b>) Cosine values for the projection angle, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048707#pone-0048707-g002" target="_blank">Figure 2A</a>, which is also an indicator of the direction of chemical shift movement along the activation path (vector B in Fig. 2A).</p

The CBD of EPAC and the domain organization.

<p><b>a</b>) The regulatory region consists of the DEP (disheveled Egl-10 pleckstrin) domain and the cAMP binding domain (CBD), colored grey. The catalytic region includes the CDC25 homology domain (CDC25HD), the Ras exchange motif (REM), and Ras association (RA) domain. The dashed red lines illustrate an expanded view of the sequence alignment of CBDs for the regions spanning the PBC α5 helix to the Hinge Helix. The corresponding secondary structure is shown above the sequence. The asterisks mark the site of termination in the deletion mutants. <b>b</b>) The structure of the CBD of apo-EPAC is shown in grey, whereas the major changes caused by cAMP (black spheres) binding are shown in orange. The curved black arrow illustrates the transition of the hinge helix from the apo form (grey; PDB ID: 2BYV) to the holo form (orange; PDB ID:3CF6). <b>c</b>)The thermodynamic cycle of cAMP dependent EPAC activation. Dashed lines encircle the equlibrium between the apo/inactive and apo/active states, <i>i.e.</i> the auto-inhibitory equilibrium. <b>d</b>) The hydrophobic “spine”, a network of residues involving the hydrophobic contacts between the hinge helix and adjacent helices (α4 and α5).</p

Total Variance Breakdown in the SVD Analysis of Deletion Mutants and L273W.

*<p>The percentages reported in parentheses are the cumulative contribution of PC1 and PC2 for each SVD analysis involving a mutant.</p

Chemical shift projection analysis (CHESPA) using mutations as perturbations.

<p><b>a</b>) Schematic of CHESPA. Open circles indicate HSQC peaks of the apo forms, whereas the filled circle represents the holo form (cAMP bound) HSQC peak. The green open circle represents the apo-mutant. The compounded chemical shift between the Wt(apo) and Wt(holo) was computed as the magnitude of the vector B, |B|. Similarly the compounded chemical shift between the Wt(apo) and Mutant(apo) was calculated as |A|. The magnitude of vectors A and B define the radii of the dashed circles centered on the Wt(apo) peak (<b>b</b>) Representative regions of the [¹⁵N-¹H] HSQC spectra of Wt(apo) (grey) and cAMP-bound, Wt(holo) (black) overlaid with the [¹⁵N-¹H] HSQC spectra of apo-Mutants: de312 (red), de310 (blue), de305 (green). Arrows indicate the direction of shift toward activation and dashed contour lines enclose peaks of the same residues.</p

Binding isotherms for the titration of cAMP into an NMR sample with 15

<p> <b>µM de305 (green) and 25 </b><b>µM Wt (grey) in 20 </b><b>mM phosphate buffer, pH </b><b>7.6, 50 </b><b>mM NaCl, 99.9% D₂O, and at 25°C.</b> The binding of cAMP to de305 and Wt was monitored through the STD amplification factor (STDaf) normalized to the plateau value and plotted versus the total cAMP concentration. The binding of cAMP to the Wt construct, Epac1_149–318 was measured here to ensure an unbiased comparison to de305 since previous measurements <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048707#pone.0048707-Rehmann2" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048707#pone.0048707-Kraemer1" target="_blank">[34]</a> were on Epac1_149–317 and used different experimental conditions and methods.</p