Assessing the Influence of Mutation on GTPase Transition States by Using X‐ray Crystallography, 19F NMR, and DFT Approaches

Abstract We report X‐ray crystallographic and 19F NMR studies of the G‐protein RhoA complexed with MgF3 −, GDP, and RhoGAP, which has the mutation Arg85′Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X‐ray data show how Tyr34 maintains solvent exclusion and the core H‐bond network in the active site by relocating to replace the missing Arg85′ sidechain. The 19F NMR data show deshielding effects that indicate the main function of Arg85′ is electronic polarization of the transferring phosphoryl group, primarily mediated by H‐bonding to O3G and thence to PG. DFT calculations identify electron‐density redistribution and pinpoint why the TS for guanosine 5′‐triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAPArg85′Ala relative to wild‐type (WT) RhoGAP. This study demonstrates that 19F NMR measurements, in combination with X‐ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site‐specific modifications.


Data Collection and Structure Solution
Diffraction data from RhoA/GAP Arg85'Ala -GDP-MgF 3 -TSA complexes crystals were collected at 100K to 2.2 Å resolution at beamline ID29 at the ESRF, Grenoble. [1] The structure of the complex was solved by molecular replacement using the previous structure RhoA/GAP wt -GDP-AlF 4 -TSA complexes (PDB:1tx4) as a search model with the bound ligands and water molecules removed. [2] Data were processed with XDS, [3] and further scaled using AIMLESS. [4] Ligands were included after a few refinement cycles. Refinement was carried out alternately using REFMAC5 [5] and manual rebuilding with COOT. [6] Diffraction data from RhoA/GAP Arg85'Ala -GDP-MgF 3 -TSA complex crystals at 2.4 Å were collected at 100 K at beam line 14.2 at the BESSY Synchrotron, Berlin. The structure was solved in a similar manner to RhoA/GAP Arg85'Ala -GDP-MgF 3 -, using PDB: 1ow3 as a search model without ligands. [7] Models were validated using Molprobity [8] and structure figures were produced with PyMol. [9][3] and further scaled using AIMLESS. Ligands were included after a few refinement cycles. Refinement was carried out alternately using REFMAC5 and manual rebuilding with COOT. Diffraction data from RhoA/GAP Arg85'Ala -GDP-MgF 3 -TSA complex crystals at 2.4 Å were collected at 100 K at beam line 14.2 at the BESSY Synchrotron, Berlin. The structure was solved in a similar manner to RhoA/GAP Arg85'Ala -GDP-MgF 3 -, using PDB: 1ow3 as a search model without ligands. Models were validated using Molprobity and structure figures were produced with PyMol.

NMR Measurements
The 1D 19 F NMR spectra were recorded on a Bruker Avance 500 MHz spectrometer equipped with a 5 mm cryo 1 H/ 19 F probe. Typically, 1024 scans were acquired over a spectral width of 200 ppm with the carrier frequency set to -140 ppm. Selective 19 F irradiation was achieved with a continuous wave at a power level of 42 dB applied over the 1 s recycle delay at the frequency of free Fpeak. All spectra were recorded at 25 °C. Since F 3 has two exchangeable H-bond partners, the peaks that resolve into a pseudotriplet at 50% D 2 O are assigned to this atom. Hence, F 3 is the middle resonance in the Arg85´Ala complex spectrum (Fig 2), and so removal of the arginine finger causes the resonance of F 3 to move markedly upfield (by 11.7 ppm), while the resonance of F 2 moves downfield by 3.5 ppm. The SIIS values for both F 2 and F 3 marginally decrease (by 0.2 ppm) compared to WT, implying that MgF 3 is coordinated less tightly in the mutant than in the WT TSA complex. shows this behavior, confirming its assignment as F 2 . [1h] The same behavior is observed for the most downfield peak of the Arg85´Ala complex, endorsing its assignment as F 2 . Since F 3 has two H-bond partners, the peaks that form a pseudotriplet at 50% D 2 O are assigned to this atom. Hence for Arg85´Ala, F 3 is the middle peak (Fig. 2). Removal of the arginine finger causes the resonance of F 3 to move markedly upfield (11.7 ppm), while that of F 2 moves downfield (3.5 ppm). The SIIS values for both F 2 and F 3 marginally decrease (by 0.2 ppm) compared to WT, implying that MgF 3 is coordinated less tightly in the mutant than in the WT TSA complex.

Computational Data for Mutant Transition State
Our model for the transition state (TS) of the γ-phosphate hydrolysis reaction was obtained using Kohn-Sham Density Functional Theory (KS-DFT). We used the M06-2X functional formulation of KS-DFT. [10] A cc-pVDZ basis set was used to represent single-particle wave-functions [11] for carbon, hydrogen, oxygen, and nitrogen atoms, while the cc-pVTZ basis was used for magnesium and phosphorus atoms. [11] The active site (cluster) model ( Figure 3, main text) was constructed so as to maintain all key hydrogen bonding capable of stabilizing the transition state. The initial geometry about the γ-phosphorus atom was obtained by replacing the tbp magnesium by phosphorus and the three fluorines by oxygens in the high-resolution X-ray structure (PDB: 2ngr). More specifically, we included atoms in residues 12-20B, 34B, 36-38B, and 59-63B (RhoA) and 85-86A from the crystal structure designation. All amino acid hydrogen bonds stabilizing the attacking water, the γ-phosphoryl group, or leaving group were included. Waters bonded to the catalytic magnesium were also retained. Where opportune, we truncated amino acid residues with a methyl group in which the carbon was fixed at the crystallographic coordinates of the cognate atom in the X-ray crystal structure. Initially, the TS search utilized cc-pVDZ for all atoms and an integration grid consisting of 99 radial points and 590 solid-angle points in the Lebedev grid. Upon calculating an initial TS structure in this manner, we increased the quality of the calculation for greater accuracy and to eliminate spurious small imaginary frequencies. We added basis functions in the manner most conducive of better representing the active site. We improved the phosphorus basis set to cc-pVTZ so as to represent its polarizable electron density with more accuracy. We also increased the magnesium basis set given its strong electron polarization. All oxygen atoms in the phosphate chain used aug-cc-pVDZ, aside the nucleophilic water oxygen, gamma oxygens, and O 3B which had aug-cc-pVTZ given the increased importance. [12] The final refined integration grid had 130 radial points, 700 solid angle points. The structure was considered optimized when the force on all nuclei fell below 1 μHartree/Bohr. The SCF was considered converged when the density matrix residual was less than 10 -6 . After decreasing the initial 1.91 Å Mg-F distance to a value of 1.71 Å for the three new P-O bonds, we optimized the geometry of the resulting active site model (175 atoms) to obtain the TS using standard algorithms, [13] as implemented in the Gaussian09 software package. [14] All methyl carbons were fixed at their initial locations, which did not introduce any significant error into the calculation. This procedure gave a converged TS model with a harmonic vibrational value of 198i cm -1 corresponding to motion along the reaction coordinate (see movie S1).
However, in freezing the Cartesian coordinates associated with the terminating methyl groups, there were a small number of non-relevant imaginary frequencies associated methyl group librations (37i cm -1 , 27i cm -1 , 17i cm -1 , and 11i cm -1 ).

Obtaining the Calculated Active Site Model for RhoA/GAP Arg85'Ala -GDP-MgF 3 complex
An active site model for the RhoA/GAP Arg85'Ala -GDP-MgF 3 complex was obtained from the atomic coordinates of the TS model except that the P and O atoms in the γ-phosphoryl group were replaced by Mg and F, respectively. The optimized structure was obtained using a similar computational protocol to that used for the TS model except that standard optimization algorithms were used to find the ground state structure. Given the electronegativity of fluorine, it was necessary to add diffuse functions in the form of an augcc-pVTZ basis on the fluorine atoms.  (Table 3). [15] References:

NMR Chemical Shift Calculations
[  where S is an integer value that ranges from 2 to 100. Hence, A ranges from 0.2-1.5 Å.      , and respectively). . (A) At the highest electron surface density, pairs of covalently bonded atoms are linked by intact electron surface density but there is no bonding from P G to O 3w nor to O 3B ; while there is clear intact surface density from O w3 to its two hydrogens. (B) At intermediate surface density, there is bonding from P G to O 3w but not to O 3B . (C & D) Progressive lowering of the electron surface density shows partial bonding to O3B as well as good H-bonding from the two hydrogens of Wat3 to the carbonyl oxygens of Thr37 (top left) and Gln63 (top right). (Atom coloring: hydrogen, white; carbon, silver; nitrogen, blue; oxygen, red; phosphorus, orange; magnesium, lime green).