Conformational control of small GTPases by AMPylation

Abstract

Posttranslational modifications (PTMs) are important physiological means to regulate the activities and structures of central regulatory proteins in health and disease. Small GTPases have been recognized as important molecules that are targeted by PTMs during infections of mammalian cells by bacterial pathogens. The enzymes DrrA/SidM and AnkX from Legionella pneumophila AMPylate and phosphocholinate Rab1b during infection, respectively. Cdc42 is AMPylated by IbpA from Histophilus somni at tyrosine 32 or by VopS from Vibrio parahaemolyticus at threonine 35. These modifications take place in the important regulatory switch I or switch II regions of the GTPases. Since Rab1b and Cdc42 are central regulators of intracellular vesicular trafficking and of the actin cytoskeleton, their modifications by bacterial pathogens have a profound impact on the course of infection. Here, we addressed the biochemical and structural consequences of GTPase AMPylation and phosphocholination. By combining biochemical experiments and NMR analysis, we demonstrate that AMPylation can overrule the activity state of Rab1b that is commonly dictated by binding to guanosine diphosphate or guanosine triphosphate. Thus, PTMs may exert conformational control over small GTPases and may add another previously unrecognized layer of activity control to this important regulatory protein family.

Keywords: NMR; posttranslational modifications; protein dynamics; small GTPases.

Fig. 1.

PTMs of Rab1b and Cdc42 by bacterial pathogens. (A) AMP transferases (ATase) DrrA, VopS, and IbpA utilize ATP to transfer AMP covalently to Tyr77 of Rab1b, Thr35 of Cdc42, and Tyr32 of Cdc42, respectively. The deAMPylase SidD can hydrolytically cleave the AMP group from AMP_Y77–Rab1b. The phosphocholinase AnkX utilizes CDP–choline to transfer a phosphocholine group (PC) to Ser76 of Rab1b. The dephosphocholinase Lem3 hydrolytically cleaves the PC group. PP_i: pyrophosphate, CMP: cytidine monophosphate. (B) AMPylation (Rab1b or Cdc42) and phosphocholination (Rab1b) occur in the switch I and switch II regions of GTPases. In the inactive GDP state (Left), the switch regions are conformationally flexible, but they become highly structurally ordered in the active GTP state (Right). Green spheres: positions of amino acids with indicated PTMs, sticks: GDP (Left) and nonhydrolyzable GTP-analog GpNHp (Right). The position of posttranslationally modified amino acids in Rab1b and Cdc42 are indicated based on the Rab1-structures only, since these positions are homologous among the proteins (PDB code 3NKV, Right; 4LHV, Left) (11).

Fig. 2.

AMPylation or phosphocholination stabilize small GTPases. (A) The rate of intrinsic nucleotide release of Cdc42 is affected moderately by AMPylation. The rates of intrinsic GDP and GTP release have been determined by monitoring the decrease in fluorescence of nucleotide mant-derivatives (progress curves are shown in SI Appendix, Fig. S1). (B) The rate of intrinsic GTP hydrolysis of Cdc42 is affected significantly by AMPylation. GTP-hydrolysis rates of indicated Cdc42 variants loaded preparatively with GTP have been determined from quantification of GTP contents using reversed-phase HPLC (progress curves are shown in SI Appendix, Fig. S2). (C) AMPylation or phosphocholination of Rab1b or AMPylation of Cdc42 stabilize the GTPases. The melting temperatures of indicated GTPase (GDP and GTP states, modified and nonmodified) have been determined from thermal unfolding by using circular dichroism signal changes as an indicator for thermal denaturation. (D) Circular dichroism signal changes in dependence of temperature (with respect to Fig. 2C) indicate thermal unfolding of modified and nonmodified Rab1b (Left) and Cdc42 (Right). The melting temperature is determined from the point of inflection of the data traces (the original spectra are shown in SI Appendix, Fig. S3).

Fig. 3.

AMPylation-induced conformational activation of Rab1b:GTP and Rab1b:GDP characterized by NMR spectroscopy. (A) Overlay of the ¹H,¹⁵N-HSQC NMR spectra of the active GTP-bound (black) and the inactive GDP-bound state of Rab1b (blue). Changes in the structural environment are characterized by CSP and plotted onto the crystal structure of AMP_Y77–Rab1b:GppNHp (PDB code: 3NKV). (B and C) Effect of AMPylation on the activity state of Rab1b:GTP and Rab1b:GDP. Overlay of the ¹H,¹⁵N-HSQC NMR spectra of AMP_Y77–Rab1b:GTP (black) and AMP_Y77–Rab1b:GDP (red). (C) CSP calculated for Rab1b:GTP vs. Rab1b:GDP (blue) and AMPylated AMP_Y77–Rab1b:GTP vs. AMP_Y77–Rab1b:GDP (red). (D) Overlay of ¹H,¹⁵N-HSQC NMR spectra of Rab1b:GTP (red) and AMP_Y77–Rab1b:GTP (black). Changes in the structural environment are characterized by CSP and plotted onto the crystal structure of AMP_Y77–Rab1b:GppNHp (PDB code: 3NKV; ref. 11). Spectra were recorded at 25 °C in 20 mM Hepes, pH 7.5 in 50 mM NaCl, 1 mM MgCl₂, 2 mM DTE, 10 µM GDP, 100 µM DSS. The total protein concentration was 300 µM.

Fig. 4.

Phosphocholination-induced effects on Rab1b:GTP and Rab1b:GDP characterized by NMR spectroscopy. (A) Overlay of the ¹H,¹⁵N-HSQC NMR spectra of Rab1b: GTP (red) and PC_S76–Rab1b:GTP (black). Changes in the structural environment are characterized by CSP and plotted onto the crystal structure of AMP_Y77–Rab1b:GppNHp (PDB code: 3NKV; ref. 11). Residue S76 bearing the PC moiety is indicated as a green sphere. (B) Overlay of the ¹H,¹⁵N-HSQC NMR spectra of the Rab1b:GDP (red) and PC_S76–Rab1b:GDP (black). Spectra were recorded at 25 °C in 20 mM Hepes, pH 7.5 in 50 mM NaCl, 1 mM MgCl₂, 2 mM DTE, 10 µM GDP, 100 µM DSS. The total protein concentration was 300 µM.

Fig. 5.

AMPylation-induced effects on Cdc42:GTP characterized by NMR spectroscopy. (A) Conformational effects of AMPylation on AMP_T35–Cdc42 in different activity states. Overlay of the ¹H,¹⁵N-HSQC NMR spectra of AMP_T35–Cdc42 in the GTP (black) and GDP (blue) states. Changes in the vicinity of the covalent modification are characterized by CSP for residues Thr35 and plotted onto the crystal structure of Cdc42:GppNHp (PDB code: 1NF3). (B) Cdc42–AMPylation at Tyr32 or Thr35 affects the active state. Overlay of the ¹H,¹⁵N-HSQC NMR spectra Cdc42:GTP (black), AMP_T35–Cdc42:GTP (blue), and AMP_Y32–Cdc42:GTP (red). Changes in the structural environment are characterized by CSP for the modification at Thr35 and plotted onto the crystal structure of Cdc42:GppNHp (PDB code: 1NF3). (C) Cdc42–AMPylation at Tyr32 or Thr35 affects the inactive state. Overlay of the ¹H,¹⁵N-HSQC NMR spectra of Cdc42:GDP (black), AMP_T35–Cdc42:GDP (blue), and AMP_Y32–Cdc42:GDP (red). (D) CSPs calculated for Cdc42:GTP vs. AMP_T35–Cdc42:GTP (gray), Cdc42:GDP vs. AMP_Y32–Cdc42:GDP (red), and Cdc42:GDP vs. AMP_T35–Cdc42:GDP (blue).

Fig. 6.

PTMs affect GTPase conformations differently (conformationally identical or nonidentical states are indicated by an equal sign or unequal sign, respectively). Classically, GTPases such as Rab1b differ strongly in their active, GTP-bound and inactive, GDP-bound conformations. Tyr77–AMPylation of Rab1b results in a redistribution of conformational states in favor of the active form. That is, AMP_Y77–Rab1b:GDP is conformationally identical to active Rab1b:GTP but not inactive Rab1b:GDP. Ser76 phosphocholination of Rab1b, instead, retains the normal distribution of conformational states (i.e., GDP bound is inactive, GTP state is active). In contrast, Tyr32 and Thr35 AMPylation of Cdc42:GDP and Cdc42:GTP results in the formation of two additional conformational states. Here, the AMP-modified states do not resemble either the active or the inactive states (green, yellow, gray: active, inactive, and intermediate states, respectively; blue and red: equal and nonidentical conformations, respectively).