A GAP-GTPase-GDP-Pi Intermediate Crystal Structure Analyzed by DFT Shows GTP Hydrolysis Involves Serial Proton Transfers

Cell signaling by small G proteins uses an ON to OFF signal based on conformational changes following the hydrolysis of GTP to GDP and release of dihydrogen phosphate (P_i). The catalytic mechanism of GTP hydrolysis by RhoA is strongly accelerated by a GAP protein and is now well defined, but timing of inorganic phosphate release and signal change remains unresolved. We have generated a quaternary complex for RhoA-GAP-GDP-P_i. Its 1.75 Å crystal structure shows geometry for ionic and hydrogen bond coordination of GDP and P_i in an intermediate state. It enables the selection of a QM core for DFT exploration of a 20 H-bonded network. This identifies serial locations of the two mobile protons from the original nucleophilic water molecule, showing how they move in three rational steps to form a stable quaternary complex. It also suggests how two additional proton transfer steps can facilitate P_i release.     

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

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 O^3G and thence to P^G. 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 RhoGAP_Arg85′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.     

Dietary genistein enhances phosphorylation of regulatory myosin light chain in the myocardium of ovariectomized mice

There is evidence that isoflavones, such as genistein, can directly or indirectly improve lipid profile and lower blood pressure and hence exert cardiovascular protection. It is further believed, that genistein attenuates vascular contraction and thus vascular tone and blood pressure through altering the phosphorylation of the regulatory myosin light chain (MLC) probably via the myosin light chain kinase (MLCK) or the RhoA pathway. However, the direct role of genistein in the myocardium is poorly reviewed. In this study, we investigated the impact of genistein on the cardiac proteome in ovariectomized female mice using a 2DE-MS approach. Dietary genistein intake considerably changed the abundance of several cytoskeletal and contractile proteins and enhanced the phosphorylation of MLC. The MLC phosphorylation was mediated through increased abundance of MLCK and inhibition of myosin light chain phosphatase latest known to be inversely regulated by RhoA. Contrary to others, in our model genistein did neither inhibit the cardiac MLCK, nor the cardiac RhoA pathway in vivo.     

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

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 O^3G and thence to P^G. 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 RhoGAP_Arg85′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.     

p190RhoGAP and Rap-dependent RhoGAP (ARAP3) inactivate RhoA in response to nerve growth factor leading to neurite outgrowth from PC12 cells

Rat pheochromocytoma (PC12) cells have been used to investigate neurite outgrowth. Nerve growth factor (NGF) has been well known to induce neurite outgrowth from PC12 cells. RhoA belongs to Ras-related small GTP-binding proteins, which regulate a variety of cellular processes, including cell morphology alteration, actin dynamics, and cell migration. NGF suppressed GTP-RhoA levels after 12 h in PC12 cells and was consistently required for a long time to induce neurite outgrowth. Constitutively active (CA)-RhoA suppressed neurite outgrowth from PC12 cells in response to NGF, whereas dominant-negative (DN)-RhoA stimulated it, suggesting that RhoA inactivation is essential for neurite outgrowth. Here, we investigated the mechanism of RhoA inactivation. DN-p190RhoGAP abrogated neurite outgrowth, whereas wild-type (WT)-p190RhoGAP and WT-Src synergistically stimulated it along with accelerating RhoA inactivation, suggesting that p190RhoGAP, which can be activated by Src, is a major component in inhibiting RhoA in response to NGF in PC12 cells. Contrary to RhoA, Rap1 was activated by NGF, and DN-Rap1 suppressed neurite outgrowth, suggesting that Rap1 is also essential for neurite outgrowth. RhoA was co-immunoprecipitated with Rap1, suggesting that Rap1 interacts with RhoA. Furthermore, a DN-Rap-dependent RhoGAP (ARAP3) prevented RhoA inactivation, abolishing neurite formation from PC12 cells in response to NGF. These results suggest that NGF activates Rap1, which, in turn, up-regulates ARAP3 leading to RhoA inactivation and neurite outgrowth from PC12 cells. Taken together, p190RhoGAP and ARAP3 seem to be two main factors inhibiting RhoA activity during neurite outgrowth in PC12 cells in response to NGF.