Real-time NMR study of guanine nucleotide exchange and activation of RhoA by PDZ-RhoGEF

Abstract

Small guanosine triphosphatases (GTPases) become activated when GDP is replaced by GTP at the highly conserved nucleotide binding site. This process is intrinsically very slow in most GTPases but is significantly accelerated by guanine nucleotide exchange factors (GEFs). Nucleotide exchange in small GTPases has been widely studied using spectroscopy with fluorescently tagged nucleotides. However, this method suffers from effects of the bulky fluorescent moiety covalently attached to the nucleotide. Here, we have used a newly developed real-time NMR-based assay to monitor small GTPase RhoA nucleotide exchange by probing the RhoA conformation. We compared RhoA nucleotide exchange from GDP to GTP and GTP analogues in the absence and presence of the catalytic DH-PH domain of PDZ-RhoGEF (DH-PH(PRG)). Using the non-hydrolyzable analogue guanosine-5'-O-(3-thiotriphosphate), which we found to be a reliable mimic of GTP, we obtained an intrinsic nucleotide exchange rate of 5.5 x 10(-4) min(-1). This reaction is markedly accelerated to 1179 x 10(-4) min(-1) in the presence of DH-PH(PRG) at a ratio of 1:8,000 relative to RhoA. Mutagenesis studies confirmed the importance of Arg-868 near a conserved region (CR3) of the Dbl homology (DH) domain and revealed that Glu-741 in CR1 is critical for full activity of DH-PH(PRG), together suggesting that the catalytic mechanism of PDZ-RhoGEF is similar to Tiam1. Mutation of the single RhoA (E97A) residue that contacts the pleckstrin homology (PH) domain rendered the mutant 10-fold less sensitive to the activity of DH-PH(PRG). Interestingly, this mutation does not affect RhoA activation by leukemia-associated RhoGEF (LARG), indicating that the PH domains of these two homologous GEFs may play different roles.

FIGURE 1.

RhoA nucleotide exchange in the presence of DH-PH^PRG monitored by NMR. A, schematic of RhoA nucleotide exchange mediated by DH-PH^PRG. ¹⁵N-labeled RhoA (blue, 20.4 kDa) with GDP and Mg²⁺ (purple) bound is in an inactive state. DH-PH^PRG (green, 43 kDa) interacts with residues in the RhoA switch regions, promoting the release of GDP. Subsequently, GTP binds RhoA, and the GEF is released to produce the activated form of RhoA. B, kinetics of RhoA-GDP nucleotide exchange to GTP (green dashed line) and GTPγS (blue) measured by the real-time NMR assay. A theoretical GTP exchange curve corrected for hydrolysis (see supplemental material) is also presented (green solid line) Error bars indicate S.D. for values reported by multiple peaks. C, snapshots of ¹H-¹⁵N HSQC spectra during the 75-min time course of RhoA-GDP to GTPγS nucleotide exchange, in the presence of DH-PH^PRG (6 nm). Black and blue boxes indicate the positions of the RhoA-GDP and RhoA-GTPγS cross peaks, respectively, for Gly-14 and Ser-73. D, RhoA-GDP to GTPγS nucleotide exchange, with increasing concentration of DH-PH^PRG. The intrinsic nucleotide exchange rate is shown in black. Nucleotide exchange rates k (10⁻⁴ min⁻¹) are 5.5, 19, 24, 39, 130, 267, 371, and 1,179 for GEF concentrations of 0, 0.36, 0.51, 0.9, 1.8, 4, 6, 25 nm, respectively. Lower panel, the hyperbolic (green curve) dependence, on GEF concentration, of the nucleotide exchange rate is indicative of a two-step binding model.

FIGURE 2.

Sequence alignment of DH-PH domains of human RhoGEFs. PDZ-RhoGEF (O15085, residues 734–923), LARG (Q9NZN5, residues 787–977), p115-RhoGEF (Q92888, residues 416–605), Dbl-GEF (Q92974,, residues 235–432), p63-RhoGEF (Q86VW2, residues 160–336), Dbs-GEF (O15068, residues 631–811), and Tiam1 (Q13009, residues 1,040–1,234) were aligned using Clustal_W2. Hydrophobic, acidic, basic, and polar residues are indicated in red, blue, magenta, and green, respectively. The stars mark residues conserved throughout Rho guanine nucleotide exchange factors. Colons correspond to conserved substitutions and, periods correspond to semiconserved substitutions. Conserved residues examined in this study (Glu-741, Arg-868, and Ser-1065) are boxed. CR1, CR2, and CR3 are three highly conserved regions of the DH domain.

FIGURE 3.

PDZ-RhoGEF preferentially catalyzes nucleotide exchange in the direction of activation (GDP to GTPγS). Plots of the nucleotide exchange for RhoA-GDP to GTPγS (circles) and RhoA-GTPγS to GDP (triangles) (intrinsic, open; GEF-mediated, filled) are shown. A 5-fold molar excess of GTPγS was added to ¹⁵N-labeled RhoA-GDP, and a 50-fold molar excess of GDP was added to ¹⁵N-labeled RhoA-GTPγS. Error bars indicate S.D. for values reported by multiple peaks.

FIGURE 4.

Mutations in conserved regions of DH-PH^PRG severely decrease GEF activity, and E97A mutation decreases the activation of RhoA. A, RhoA nucleotide exchange (GDP to GTPγS) stimulated by wild-type (wt) DH-PH^PRG (1.8 nm) versus mutants E741D, E741A, and R868G. Rates derived from these curves are displayed in a histogram in the right panel. The experiments were also performed with 4 nm DH-PH^PRG, in which wild-type, E741D, E741A, R868G, and intrinsic nucleotide exchange rates are 207 × 10⁻⁴ ± 3.3 × 10⁻⁵ min⁻¹, 6.4 × 10⁻⁴ ± 1 × 10⁻⁴ min⁻¹, 14.4 × 10⁻⁴ ± 1 × 10⁻⁴ min⁻¹, 11.1 × 10⁻⁴ ± 1 × 10⁻⁴ min⁻¹ and 4.4 × 10⁻⁴ ± 2 × 10⁻⁴ min⁻¹, respectively. B, interface of PDZ-RhoGEF DH (red) and PH domains (cyan) with RhoA (green). DH-PH^PRG Glu-741 (in CR1), Arg-868 (near CR3), and RhoA switch I residues Tyr-34, Thr-37, and Val-38 are highlighted (PDB code: 1XCG). PyMOL software was used to create the schematic representations. C, mutation of Glu-97 reduces the ability of DH-PH^PRG to activate RhoA. RhoA nucleotide exchange to GTPγS in the absence (circles) or presence (triangles) of DH-PH^PRG (4 nm). Black and blue curves correspond to wild-type RhoA and RhoA-E97A, respectively. Right panel, RhoA Glu-97 forms hydrogen bonds with Ser-1065 and Asn-1068 of the PH domain. In A and C, error bars indicate S.D. for values reported by multiple peaks.

FIGURE 5.

Kinetics of RhoA nucleotide exchange with GTP analogues and competition assay. A, kinetics of RhoA-GDP nucleotide exchange to GTPγS (blue), GMPPNP (red), and mant-GTP (magenta) measured individually using our NMR-based assay. The rates (10⁻⁴ min⁻¹) are displayed with a histogram (inset) for each curve. B, overlay of five ¹H-¹⁵N HSQC spectra illustrating the distinct chemical shifts of Gln-29 exhibited by RhoA when bound to GDP (black), GMPPNP (red), GTP (green), GTPγS (blue), and mant-GTP (magenta). C, Gln-29 amide proton chemical shift changes (Δδ¹H, relative to RhoA-GDP) versus exchange rates for each nucleotide (colored as above).In A and C, error bars indicate S.D. D, competition assay for binding of nucleotide analogues to RhoA. Snapshots of ¹H-¹⁵N HSQC spectra collected before and after the addition of premixed GTP, GTPγS, GMPPNP, and mant-GTP, each at a 5-fold molar excess over RhoA.