RhoGDIα Acetylation at K127 and K141 Affects ... - ACS Publications

Dec 22, 2015 - Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD),...
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RhoGDIα Acetylation at K127 and K141 Affects Binding toward Nonprenylated RhoA Nora Kuhlmann, Sarah Wroblowski, Lukas Scislowski, and Michael Lammers* Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Joseph-Stelzmann-Str. 26, University of Cologne, 50931 Cologne, Germany S Supporting Information *

ABSTRACT: Rho proteins are major regulators of the cytoskeleton. As most Ras-related proteins, they switch between an active, GTP-bound and an inactive, GDP-bound conformation. Rho proteins are targeted to the plasma membrane via a polybasic region and a prenyl group attached to a C-terminal cysteine residue. To distribute Rho proteins in the cell, the molecular chaperone RhoGDIα binds to the prenylated Rho proteins forming a cytosolic pool of mainly GDP-loaded Rho. Most studies characterized the interaction of prenylated Rho proteins and RhoGDIα. However, RhoGDIα was also shown to bind to nonprenylated Rho proteins with physiologically relevant micomolar affinities. Recently, it was discovered that RhoGDIα is targeted by post-translational lysine acetylation. For one site, K141, it was hypothesized that acetylation might lead to increased levels of formation of filamentous actin and filopodia in mammalian cells. The functional consequences of lysine acetylation for the interplay with nonprenylated RhoA have not been investigated. Here, we report that lysine acetylation at lysines K127 and K141 in the RhoGDIα immunoglobulin domain interferes with the interaction toward nonprenylated RhoA using a combined biochemical and biophysical approach. We determined the first crystal structure of a doubly acetylated protein, RhoGDIα, in complex with RhoA·GDP. We discover that the C-terminus of RhoA adopts a different conformation forming an intermolecular β-sheet with the RhoGDIα immunoglobulin domain.

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electrostatic network formed in trans between the RhoGDIα Nterminus and the Rho-protein’s C-terminus has been shown to be important for the membrane delivery step of Rho proteins by RhoGDIα.10 RhoGDIα is a protein structurally and functionally charaterized in great detail up to atomic resolution.11−14 These analyses showed that RhoGDIα is able to bind to prenylated Rho proteins accommodating the Rho protein’s prenyl group in a hydrophobic cavity formed by the C-terminal immunoglobulin (IG) domain. Thereby, RhoGDIα acts as a molecular chaperone to form a cytosolic pool of mostly GDPloaded RhoA in the cytosol.14,15 The N-terminus of RhoGDIα is intrinsically unfolded and forms a helix−loop−helix conformation upon contacting the Rho proteins’ switch I and switch II regions, which are needed for nucleotide and effector binding.11,16 In contrast to PDEδ, lacking the N-terminal domain, suggested to be a universal molecular chaperone for farnesylated proteins, the RhoGDIα N-terminal region is essential for binding to prenylated Rho proteins.17,18 The formation of RhoGDIα·Rho complexes has diverse physiological functions. First, it creates a pool of Rho that can quickly be activated to produce a cellular response.14 Second, it affects

ho proteins belong to the Ras superfamily and are involved in many essential cellular processes, most of them related to the eukaryotic cytoskeleton.1,2 To this end, Rho proteins bind to effector proteins either indirectly or directly involved in intracellular transport processes, maintenance of cell architecture, cell migration, cell proliferation, cytokinesis, and diverse signal transduction processes.3 Like nearly all members of the Ras superfamily, Rho proteins act as molecular switches adopting a GTP-bound, active and a GDP-bound, inactive conformation. Effector proteins specifically bind to the active state. Rho proteins, as most other Rasrelated proteins, can fulfill this switch function in a biologically relevant form only together with proteins that accelerate the intrinsic nucleotide exchange rate, so-called GEFs (guanine nucleotide exchange factors), and the intrinsic nucleotide hydrolysis rate, so-called GAPs (GTPase-activating proteins).4 Importantly, Rho proteins are lipidated, mostly by formation of a thioether bond with a geranylgeranyl or farnesyl group, at a C-terminal cysteine residue, which is part of the so-called CaaX box (C, cysteine; a, aliphatic residue; X, any residue).5,6 This motif is essential for its biological function and is required for the binding of Rho proteins to subcellular membranes. A second motif in RhoA needed for membrane binding constists of a polybasic patch, which is located directly N-terminal of the CaaX box.7,8 It has been reported that this basic region is important for the interaction with the highly acidic, negatively charged N-terminus of a protein called RhoGDIα.9,10 This © 2015 American Chemical Society

Received: November 17, 2015 Revised: December 22, 2015 Published: December 22, 2015 304

DOI: 10.1021/acs.biochem.5b01242 Biochemistry 2016, 55, 304−312

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Biochemistry

lysine acetylation at K127 and K141 in RhoGDIα interferes with binding to nonprenylated RhoA. These data suggest that lysine acetylation of RhoGDIα is an important regulator for processes taking place before Rho proteins are irreversibly modifed by prenylation. These results suggest the potential to target steps in Rho biology preceding prenylation for the development of novel therapeutic strategies.

the Rho protein’s lifetime, because it protects the Rho protein from its proteasomal degradation.19 Third, as just three RhoGDI isoforms exist in mammals, not capable of binding all Rho proteins present in cells, RhoGDIα also determines the Rho proteins’ spectrum present in cells in a spatial and temporal manner.15,19 Most studies concentrated on the interplay of prenylated Rho and RhoGDIα, as the prenylated Rho is the dominant molecular species in cells. However, also, the interaction of RhoGDIα with nonprenylated Rho proteins is physiologically important. Moreover, the molecular events occurring before Rho prenylation by geranylgeranyltransferase I (GGTase I) and further post-translational maturation by the enzymes RceI (Rasconverting enzyme I) and Icmt (isoprenylcysteine carboxyl methyltransferase) at the ER are still unclear.20,21 It has been shown that although Rho proteins bind in the nanomolar or picomolar range to RhoGDIα if they are farnesylated or geranylgeranylated, respectively, the affinity of nonprenylated RhoA for RhoGDIα is still in the physiological range.14 Tnimov and colleagues showed recently that the presence of RhoGDIα increases the efficiency of geranylgeranylation of RhoA by GGTase I.14,22 It was reported that no ternary complex of RhoA, RhoGDIα, and GGTase I is formed in vitro.22 The exact molecular mechanism underlying this effect remains unclear. Considering that there are just three RhoGDI isoforms in mammals facing more than 30 different Rho proteins, it is likely that the cell has ways to tightly control binding to this diverse set of proteins.15 It was suggested that RhoGDIα regulates both the protein level of Rho proteins and their activation state. Depletion of RhoGDIα led to a significant reduction in inactive, cytosolic Rho proteins, whereas the amount of active, membrane-bound RhoA was nearly not affected or even increased.19 A knockdown of RhoGDIα function therefore strongly interferes with Rho protein function. Moreover, this affects the homeostasis of not only a single Rho isoform but also diverse Rho family members.19 Thereby, influencing RhoGDIα activity, its protein levels, or its binding toward Rho proteins is a powerful molecular mechanism for regulating Rho signaling.19 It turned out that RhoGDIα is targeted by post-translational phosphorylation at several sites in the N-terminal as well as in the immunoglobulin domain. Phosphorylation sites have been identified also within several Rho proteins shown to modulate its interplay with RhoGDIα, to regulate the lifetime of RhoGDI·Rho complexes, and to control RhoGDI’s specificity for binding to Rho proteins.23−27 Recent progress in quantitative proteomics shows that RhoGDIα is also extensively targeted by lysine acetylation. To date, more than eight lysine acetylation sites have been identified in RhoGDIα, which are located mostly in the immunoglobulin domain, but also in the N-terminal domain.28−30 The localization of these lysine acetylation sites in RhoGDIα suggests that it might interfere with the membrane cytosol as well as GDP/GTP cycle of Rho proteins. One study showed that mutation of lysine 141 in RhoGDIα to glutamine, which is used as a molecular mimic for lysine acetylation, leads to an increase in the level of formation of cellular filamentous actin and to more filopodia in HeLa cells, suggesting that acetylation at this site might activate RhoA and might alter the Rho protein’s specificity.31 How the interaction of nonprenylated Rho proteins and RhoGDIα is affected by lysine acetylation has not been studied so far. Using a combined synthetic biological, biochemical, and biophysical approach, we present the first analysis showing that

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EXPERIMENTAL PROCEDURES Expression and Purification of Proteins. RhoGDIα WT, RhoGDIα Δ22, and RhoA expressed in this study were all produced as GST fusion proteins using the pGEX-4T5/Tev vector derived from pGEX-4T1 (GE Healthcare) (Table S1). The acetylated RhoGDIα proteins were expressed as His6tagged fusion proteins (pRSFDuet-1; Merck Biosciences) in Escherichia coli BL21(DE3) (Table S1 and Figure S1). The E. coli cells were transformed with the vector encoding the protein of interest, and the cells were grown to an optical density of 0.6 at 600 nm (37 °C, 160 rpm). Protein expression was induced by adding 100−300 μM isopropyl β-D-thiogalactopyranoside (IPTG) and was conducted overnight (18−20 °C, 160 rpm). Afterward, the cells were harvested (4000g, 20 min) and resuspended in buffer A [50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 5 mM MgCl2, and 2 mM β-mercaptoethanol] containing 200 μM Pefabloc protease inhibitor cocktail. After cell lysis by sonication, the soluble fraction (20000g, 45 min) was applied to the equilibrated affinity chromatography column. Washing was conducted with buffer B for the GSH column [50 mM TrisHCl (pH 7.4), 300 mM NaCl, 5 mM MgCl2, and 2 mM βmercaptoethanol] and buffer C (buffer B with 10 mM imidazole) for the Ni2+-NTA column. Tev protease cleavage was performed for the GST fusion protein on the column overnight at 4 °C. The eluates after Tev cleavage or from the Ni-NTA column were concentrated by ultrafiltration and applied to a size-exclusion chromatography column (GE Healthcare). The concentrated fractions were shock-frozen in liquid nitrogen and stored at −80 °C. Protein concentrations were determined by measuring the absorption at 280 nm using the protein’s extinction coefficient. For nucleotide-bound Rho proteins, the concentration was determined by the Bradford assay (Expedeon). Incorporation of N-(ε)-Acetyllysine. We used the genetic code expansion concept to site-specifically incorporate N-(ε)acetyl-L-lysine into RhoGDIα. In brief, 10 mM N-(ε)acetyllysine (Bachem) and 20 mM nicotinamide were added to the E. coli BL21(DE3) culture at an optical density of 0.6 at 600 nm (OD600) to inhibit the only E. coli deacetylase CobB. The E. coli cells were grown for an additional 30 min at 37 °C before protein expression was induced by addition of 100−300 μM IPTG. The RhoGDIα to be lysine-acetylated was expressed using a pRSFDuet-1 vector co-expressing the synthetically evolved Methanosarcina barkeri tRNACUA (pylT gene) and the acetyl-lysyl-tRNA-synthetase as described previously (Figure S1).32−34 ITC Measurements. The interactions of nonprenylated RhoA and acetylated and nonacetylated RhoGDIα proteins and deletion mutants thereof were thermodynamically characterized by isothermal titration calorimetry on an ITC200 instrument (GE Healthcare) based on ref 35. All experiments were conducted in buffer A and performed at 20 °C; 2−4 μL of RhoA in the syringe (300 μM) was stepwise injected into the measuring cell containing acetylated/unacetylated RhoGDIα (30 μM). The heating power per injection was recorded over 305

DOI: 10.1021/acs.biochem.5b01242 Biochemistry 2016, 55, 304−312

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increased levels of filamentous actin and formation of filopodia in HeLa cells.31 K141G is located at β-strand 6 (β6) in the RhoGDIα immunoglobulin (IG) domain. K127G on the neighboring β-strand 5 was also identified as being lysineacetylated by mass spectrometry (Figure 1A). In this study, we

the time until saturation of the interaction was achieved. A onesite binding model was used to fit the data using the MicroCal software. The experiments resulted in the stoichiometry of binding (N), the change in the reaction enthalpy (ΔH), and the equilibrium association constant (KA). ΔG, ΔS, and the equilibrium dissociation constant (KD) are derived from the primary data. We used the standard EDTA-CaCl2 sample tests as described by MicroCal to assess the statistical significance of individual observations. These gave values within the manufacturer’s tolerances of ±20% in KA values and ±10% in ΔH. Crystallization. The crystals of the RhoA·GDP·RhoGDIα AcK127,141 complex belonged to orthorhombic space group P212121 with one copy of the heterodimer per asymmetric unit. The structure was refined to a final resolution of 2.75 Å; 200 nL of a protein solution at different concentrations (5 and 10 μg/ μL) was mixed with 200 nL of the reservoir solution. The crystals had a final size of approximately 50 μm × 20 μm × 50 μm and were grown in 4 M sodium formate and 0.1 M sodium acetate (pH 4.5) (buffer D) using the hanging drop vapor diffusion method. The crystals grew after 1 day at 20 °C and were shock-frozen in buffer D containing 30% (w/v) D-glucose as a cryoprotectant. This work was performed at the Diamond Light Source (Oxford, U.K.). A native data set was collected at 100 K on beamline I02 at a wavelength of 0.9791 Å using an ADSC Q315 detector. The detector distance was 342 mm; the oscillation range was 1°, and 100 frames were collected. Program suite CCP4 was used for structure determination.36 Data were indexed and integrated with mosflm version 7.0.9 and scaled with SCALA version 3.3.20.37−39 Initial phases were determined with Phaser using the Rac1·GDP·RhoGDIα [Protein Data Bank (PDB) entry 1HH4] structure as a search model.40 Coot version 0.7.1 was used to build the model into the 2Fo − Fc and Fo − Fc electron density maps in iterative rounds of refinement with REFMAC5 for RhoA·RhoGDIα Ack127,141.41,42 In the final model, 100% of all residues are in the allowed regions of the Ramachandran plot as judged by Molprobity.43,44 All structure figures presented here were made with PyMOL version 1.7.2.0.45 Data collection and refinement statistics are listed in Table 2. Examples of electron density for both structures are shown as omit maps in Figure S3. Rwork is calculated using the equation Rwork = ∑|Fo − Fc|/∑Fo. Fo and Fc are the observed and calculated structure factor amplitudes, respectively. Rfree is calculated as Rwork using the test set reflections only. Immunoblotting. Immunoblotting has been conducted using a standard protocol. The lysine-acetylated RhoGDIα proteins were stained using an anti-AcK antibody as the primary antibody (abcam ab21623, 1:1500). As a loading control, immunoblotting was performed using an anti-His6 antibody as the primary antibody (abcam ab18184, 1:2000). Bound antibodies were visualized by enhanced chemiluminescence (Roth).

Figure 1. RhoGDIα is acetylated at K127 and K141 in the immunoglobulin domain. (A) Localization of K127 and K141 in RhoGDIα as shown in a cartoon representation. Presented is RhoGDIα from the RhoGDIα·RhoA complex structure (PDB entry 4F38). K127G on β5 and K141G on β6 of the immunoglobulin domain are colored pink, and side chains are shown as sticks. (B) Site-specific incorporation of acetyl-L-lysine into RhoGDIα using the genetic code expansion concept. Shown is a sodium dodecyl sulfate−polyacrylamide gel electrophoresis gel stained with Coomassie brillant blue (CBB) to show the final purity of the proteins produced in this study. The immunoblottings using an anti-AcK and an anti-RhoGDIα antibody (loading control) reflect the successful incorporation of acetyl-L-lysine at K127 and K141 and the double incorporation at K127,141 in RhoGDIα. For wild-type RhoGDIa, we did not obtain a signal using the anti-AcK antibody. (C) Determination of the molecular weight of RhoGDIα AcK127,141 using electrospray ionization mass spectrometry (ESI-MS). The determined molecular mass of 25183.0 Da corresponds exactly to the theoretically calculated mass for doublelysine-acetylated RhoGDIα (His6-RhoGDIα, 25099.1 Da; two acetyl groups, +84 Da).

incorporated acetyl-L-lysine (AcK) site-specifically into RhoGDIα at K127 and K141 using a synthetically evolved, acetyllysyl-tRNA-synthetase/tRNACUA-pair from Methanosarcina barkeri as described earlier.32−34 Using this system, we were able to produce lysine-acetylated RhoGDIα in a high level of purity and in yields sufficient to perfom biophysical studies (Figure 1B and Table S1). All proteins behaved similar during the purification and showed the same elution profile on sizeexclusion chromatography, demonstrating that all proteins are natively folded (Figure S2). As judged by an immunoblotting using a specific antiacetyl-L-lysine antibody (anti-AcK AB), all RhoGDIα proteins were lysine-acetylated (Figure 1B). Notably, we also successfully incorporated acetyl-L-lysine (AcK) simultaneously at two positions, K127 and K141, into RhoGDIα (RhoGDIα AcK127,141) as shown by massdetermination using electrospray ionization (ESI)-mass spectrometry (Figure 1C). RhoGDIα Acetylation Impairs Binding toward Nonprenylated Rho Proteins. We analyzed the binding thermodynamics of nonprenylated RhoA toward acetylated and nonacetylated RhoGDIα by isothermal titration calorimetry (ITC). Binding of RhoA to RhoGDIα is based on electrostatic interactions as an increase in the salt concentration from 100 to 300 mM NaCl led to a 15-fold reduction in the affinity from 0.7 to 10.7 μM (Figure 2A and Table 1). Moreover, while the interaction is purely enthalpically driven in 100 mM NaCl containing buffer (ΔH = −8.7 kcal mol−1; TΔS

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RESULTS Site-Specific Incorporation of N-(ε)-Acetyllysine into RhoGDIα Using the Genetic Code Expansion Concept (GCEC). Recent quantitative proteomics screens performed with human and mouse cells and with different rat tissues showed that RhoGDIα is lysine-acetylated.28−30 For one of these sites, K141G (G, superscript RhoGDIα), Kim and colleagues showed by mutational studies that expression of RhoGDIα K141Q to mimic lysine acetylation resulted in 306

DOI: 10.1021/acs.biochem.5b01242 Biochemistry 2016, 55, 304−312

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Figure 2. Binding of RhoGDIα and nonprenylated RhoA is dependent on electrostatic interactions. (A) Isothermal titration calorimetry of the interaction of nonprenylated RhoA and RhoGDIα in buffer containing 300 mM NaCl. The reaction is exothermic, driven by an enthalpic and entropic contribution. The affinity of the interaction is nearly 15-fold reduced compared to that of the interaction in buffer containing 100 mM NaCl (300 mM NaCl, 10.7 μM; 100 mM NaCl, 0.7 μM) showing that electrostatics are important for the interaction. (B) Acetylation of RhoGDIα at K127 and K124 additively decreases the affinity for RhoA. Acetylation of RhoGDIα at K127 and K141 leads to a 2-fold reduction in affinity for nonprenylated RhoA (middle panels; KD for AcK127 = 1.4 μM, and KD for AcK141 = 1.2 μM) as compared to that for nonacetylated RhoGDIα (left panel; KD = 0.7 μM). Double acetylation of RhoGDIα at K127 and K141 additively decreases RhoA affinity to 3.3 μM (right panel), suggesting that electrostatic quenching lowers the affinity for RhoA. All reactions are driven by the reaction enthalpy (ΔH from −8.5 to −8.9 kcal mol−1). The decrease in affinity upon RhoGDIα acetylation is mainly driven by the decrease in favorable reaction entropy (TΔS).

Table 1. Thermodynamic Characterization of the Interactions of RhoGDIα with Nonprenylated RhoA As Determined by ITCa interaction with RhoGDIα RhoA

RhoA1−181

RhoA with 300 mM NaCl

WT Δ22 AcK127 AcK141 AcK127,141 WT AcK127 AcK141 AcK127,141 WT

KD (μM) 0.7 2.5 1.4 1.2 3.3 4.8 15 3.6 12.9 10.7

± ± ± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.5 1.1 2.3 0.8 2.3 0.5

ΔH (kcal mol−1) −8.7 −11.5 −8.5 −8.7 −8.9 −2.8 −2.3 −1.5 −3.1 −4.6

± ± ± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.2 0.04

TΔS (kcal mol−1)

N

−0.4 −4.0 −0.6 −0.8 −1.5 4.4 4.2 −5.8 3.5 2.0

0.9 0.8 0.9 0.9 1.1 1.0 1.0 0.9 1.1 0.9

Legend: KD, equilibrium dissociation constant; ΔH, reaction enthalpy; ΔS, reaction entropy; N, stoichiometry of the interaction. All measurements were taken at 20 °C with buffer containing 100 mM NaCl if not stated otherwise. Shown are the values measured or derived from the primary data and the standard deviation of the fits where suitable. a

= −0.4 kcal mol−1), the interaction of RhoA and RhoGDIα is driven by both favorable reaction enthalpy and reaction entropy in 300 mM NaCl containing buffer (ΔH = −4.6 kcal mol−1; TΔS = 2.0 kcal mol−1). Because lysine acetylation is known to interfere with the electrostatics, neutralizing a positive charge at the lysine’s side chain, RhoGDIα acetylation might therefore interfere with RhoA binding by electrostatic quenching. For K141G, it was shown that mutation to glutamine (K141QG) as an acetylation mimic leads to an increase in the level of filamentous actin (F-actin) and in the level of formation of filopodia in HeLa cells.31 The acetylation at K141G and K127G (AcK127G and AcK141G, respectively) both led to a nearly 2-fold reduction in the affinity for nonprenylated RhoA (KD for RhoGDIα of 0.7 μM, for RhoGDIα AcK141 of 1.2 μM, and for RhoGDIα AcK127 of 1.4 μM). Notably, the double acetylation at K127,141G (AcK127,141G), both being located at the same site of the IG domain on neighboring β-strands (AcK127, β5; AcK141, β6), additively decreases the affinity to 3.3 μM (Figure 2B and Table 1). All reactions using full-length RhoA are solely driven by a highly similar favorable enthalypic contribution [ΔH −8.5 to −8.9 kcal mol−1 (Table 1)]. The decrease in affinity is predominantly due to the increase in unfavorable reaction entropy, TΔS, from −0.4 kcal mol−1 for nonacetylated RhoGDIα to −0.6 and −0.8 kcal mol−1 for AcK127G and AcK141G, respectively, to −1.5 kcal mol−1 for

double-acetylated AcK127,141G. These data strongly suggest changes in the binding mechanism upon RhoGDIα lysine acetylation. Next, we analyzed how the acetylation of RhoGDIα at K127 and K141 alters the binding mechansim toward RhoA and performed structural analyses. Crystal Structure of Double-Acetylated RhoGDIα AcK127,141 in Complex with RhoA·GDP. To examine how acetylation of K141G leads to a reduction in the level of cellular F-actin observed by Kim et al., we determined the first crystal structure of a double-acetylated protein, RhoGDIα AcK127,141, in complex with nonprenylated full-length RhoA· GDP at a resolution of 2.75 Å (Figure 3A and Table 2).31 The complex crystallized in space group P212121 with one RhoA· GDP·RhoGDIα AcK127,141 heterodimer per asymmetric unit (Table 2). Stereofigures showing electron density for the acetylated lysines are shown in Figure S3. We used the doubleacetylated RhoGDIα for crystallization because K127G and K141G are located at the same site of the RhoGDIα IG domain on neighboring β-strands 5 and 6, and we found an additive decrease in affinity for RhoA for acetylation at these sites. K141G contributes to an electrostatic interaction network involving the highly positively charged RhoA C-terminus (polybasic region) and the RhoGDIα N-terminus. K141G forms electrostatic interactions to E19G and N18G in the RhoGDIα N-terminus as judged from the crystal structure of 307

DOI: 10.1021/acs.biochem.5b01242 Biochemistry 2016, 55, 304−312

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Figure 3. Structure of the RhoA·GDP·RhoGDIα Ack127,141 complex (PDB entry 5FR1). (A) Ribbon representation of the RhoA·GDP·RhoGDIα AcK127,141 complex. The overall conformation is altered mostly within the immunoglobulin (IG) domain of RhoGDIα. The RhoGDIα N-terminal domain contacts the RhoA nucleotide binding region. Color code: RhoA, yellow; RhoGDIα AcK127,141, gray. The AcK127 on β5 and AcK141 on β6 are shown as sticks; GDP, Mg2+, T35 from switch I (blue), switch II (green), and the P-loop (red) are highlighted. (B) Influence of RhoGDIα acetylation on the Rho−RhoGDIα electrostatic interaction network. The network is composed of the positively charged polybasic region of the Rho protein’s C-terminus (here Cdc42, K183, K184, R186, and R187), the negatively charged RhoGDIα N-terminus (E17, N18, E19, E20, D21, and E22), and the positively charged residues in the RhoGDIα IG domain (K127 and K141). K141G is within interaction distance of N18G and E19G as shown by the superposition of geranylgeranylated Cdc42·RhoGDIα (PDB entry 1DOA) and RhoA·RhoGDIα AcK127,141 (PDB entry 5FR1) presented here. (C) Interaction network of RhoGDIα AcK127 and AcK141. In the left panel, AcK127G makes a hydrogen bond with D143G on β6. I129G hydrophobically connects β5 and β6 interacting with AcK127G and AcK141G. The right panel shows the superposition of the doubleacetylated structure with the structure of geranylgeranylated Cdc42·RhoGDIα (PDB entry 1DOA). The interactions with I129G and D143G were not made in the nonacetylated structure. (D) Superposition of RhoGDIα IG domains. The IG domain of RhoGDIα AcK127,141 (PDB entry 5FR1) is rotated counterclockwise (black arrow) compared to the IG domains from RhoGDIα complexes with geranlygeranylated RhoA (PDB entry 4F38) and nonprenylated RhoA (PDB entry 1CC0) (left panel). β5 and β6 are pushed inside upon acetylation, closing the IG hydrophobic pocket (right panel, black arrow). (E) The RhoA C-terminus encompassing the CaaX box (188-SGCLVL-193) forms an additional intermolecular interaction with β7 of the RhoGDIα IG domain. D76 in RhoA makes intramolecular hydrogen bonds to the main chain carbonyl oxygen and the amide hydrogen of V192R. The question of whether this conformation has any physiological impact, for example, on the prenylation reaction catalyzed by GGTase I needs to be investigated further.22 308

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Cdc42/RhoA in complex with RhoGDIα, resulting in a closure of the IG domain (Figure 3D). These conformational differences are supported by structural alignments showing that the RhoGDIα AcK127,141·RhoA complex shows a total rmsd value of 1.5 Å from the complexes of RhoGDIα with geranylgeranylated RhoA (PDB entry 4F38) and with nonprenylated RhoA (PDB entry 1CC0), whereas the latter two are structurally much more similar, aligning with an overall rmsd value of 1.0 Å. The major differences lie within the conformation of RhoGDIα rather than RhoA (Table 3).

Table 2. Data Collection and Refinement Statistics for RhoA·RhoGDIα AcK127,141 (PDB entry 5FR1) Data Collectiona P212121

space group cell dimensions a, b, c (Å) α, β, γ (deg) resolution (Å) Rsym or Rmerge I/σI completeness (%) CC1/2 redundancy no. of reflections no. of unique reflections

53.10, 67.64, 120.23 90.0, 90.0, 90.0 60.11−2.75 (2.9−2.75) 0.097 (0.364) 46.1 (3.3) 99.8 (99.8) 0.991 (0.832) 3.5 (3.7) 41858 (6187) 11804 (1684)

Table 3. Structural Similarity of the RhoA·RhoGDIα AcK127,141 (PDB entry 5FR1) Complex Presented Here and the Previously Determined Structures of RhoA-G· RhoGDIα (PDB entry 4F38) and RhoA·RhoGDIα (PDB entry 1CC0) (G, geranylgeranylated)

Refinement resolution (Å) no. of reflections no. of “free” reflections Rwork,b Rfreec no. of atoms (non-hydogen) protein GDP/ion (Mg2+) water β-D-glucose Clashscoree Ramachandran plot (%)f favored allowed outliers B-factor (Å2) protein GDP/Mg2+ β-D-glucose water average B-factor (Å2) main chain side chain all atoms rmsd bond lengths (Å) bond angles (deg)

2.75 11763 622 19.74, 22.46

rmsda (Å) RhoA·GDP·RhoGDIα AcK127,141 (PDB entry 5FR1)

2828 28/3 15 RhoGDIα RhoA RhoA·RhoGDIα

6.33

RhoA-G· RhoGDIα (PDB entry 4F38)

RhoA-G· RhoGDIα (PDB entry 4F38)

RhoA·GDP· RhoGDIα (PDB entry 1CC0)

RhoA·GDP· RhoGDIα (PDB entry 1CC0)

1.614 0.450 1.511

1.094 0.437 1.489

1.122 0.582 0.983

a

The rmsd is the square root of the mean of the square of the distances between the matched atoms.

98.54 1.46 0

Notably, we did not obtain any electron density for the RhoA C-terminus and the RhoGDIα-N-terminus, although a structure of nonprenylated RhoA·RhoGDIα determined at a much lower resolution (5 Å, PDB entry 1CC0) shows electron density for this region. Instead, we observed additional electron density below β7 of the IG domain in which we could fit in the Cterminal RhoA CaaX box residues 188-SGCLVL-193 forming an additional intermolecular β-sheet between RhoA and RhoGDIα (Figure 3E and Figure S3). The question of whether this conformation is of any physiological importance for RhoA maturation, for example, for RhoA prenylation by geranylgeranyltransferase I (GGTase 1) needs to be investigated further. It was found recently that RhoGDIα accelerates RhoA prenylation by GGTase 1.22 Importantly, acetylation of RhoGDIα at K127G and K141G by resolving the electrostatic network and affecting the conformation of the RhoGDIα IG domain might allow the RhoA C-terminus to create these new interactions (Figure 3E). RhoGDIα Acetylation Interferes with the Formation of the Electrostatic Network between the Basic RhoA CTerminus and the Acidic RhoGDIα N-Terminus. As shown by the structural data, RhoGDIα acetylation at K127,141 interferes with the organization of the electrostatic network explaining the decrease in affinity of RhoGDIα AcK127/ AcK141 and AcK127,141 for RhoA. As a support, for the Nterminally deleted RhoGDIα Δ22, we found a 3-fold reduction in affinity for RhoA, similar to the reduction in affinity observed for AcK141,127G (Table 1). Moreover, RhoA lacking the Cterminal basic patch (RhoA1−181) binds to RhoGDIα with an affinity of 4.8 μM, a value reduced 7-fold compared to that of full-length RhoA [0.7 μM (Table 1)]. AcK141G does not lead to a further decrease in affinity for RhoA1−181 (5.3 μM), showing that it primarily acts on the electrostatic network.

28.29 39.17/(51.02) − 27.35 26.72 30.04 28.42 0.016 1.786

Values in parentheses are for the highest-resolution shell. bRwork = ∑| FO − FC|/∑FO, where FO and FC are the observed and calculated structure factor amplitudes, respectively. cRfree is claculated like Rwork using the test set reflections. dFor the definition, see REFMAC5 (www.ysbl.york.ac.uk/~garib/refmac/index.html). eFor the definition, see MolProbity version 4.1.43 The 100th percentile is the best among structures of comparable resolution; the 0th percentile is the worst. Clashscore is the number of serious steric overlaps (>0.4 Å) per 1000 atoms. For Clashscore, the comparative set of structures was selected in 2004 and for MolProbity score in 2006.43 fMolProbity version 4.1.43 a

geranylgeranylated Cdc42 in complex with RhoGDIα [PDB entry 1D0A (Figure 3B)]. Acetylation of K141G would resolve these interactions by electrostatic quenching. Moreover, acetylated K127G and K141G connect and bridge β5 and β6 of the IG domain. AcK141G on β6 forms a hydrophobic interaction with I129G on β5, which also is within interaction distance of AcK127G. Furthermore, D143G on β6 is within hydrogen bonding distance of the amide hydrogen from AcK127 (Figure 3C). The IG domain is largely rotated counterclockwise compared to nonprenylated and prenylated 309

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observed by Kim and colleagues expressing the RhoGDIα K141Q acetylation-mimetic mutant in HeLa cells, we sitespecifically lysine-acetylated RhoGDIα using the genetic code expansion concept. We analyzed the effect of RhoGDIα lysine acetylation on the thermodynamics of binding toward nonprenylated RhoA by ITC. For acetylation sites K127G and K141G, both being located at neighboring β-strands within the immunoglobulin domain, we observed an additive decrease in RhoA affintiy. The crystal structure of double-acetylated RhoGDIα in complex with RhoA showed that acetylation at K141G most likely interferes with the integrity of the electrostatic network composed of the acidic RhoGDIα Nterminus and the C-terminal basic RhoA C-terminus. Mutational studies, using C-terminally truncated RhoA1−181 lacking the C-terminal polybasic region, suggest that acetylation at K127G does affect steps in the interaction of RhoA and RhoGDIα other than acetylation at K141G. While AcK141G does not lead to a further decrease in affinity for RhoA1−181 compared to nonacetylated RhoGDIα, AcK127G further decreases RhoA1−181 binding affinity. AcK127G together with AcK141G is able to bridge β5 and β6 via interactions with I129G on β5 and D143G on β6. These interactions might interfere with the ability of the IG domain to undergo a closed-to-open transition occurring on two invariant glycine residues within the IG domain reported to be important for RhoGDIα function.46 Additionally, both acetylations increase the hydrophobicity at the surface of the IG domain, which also might contribute to the observed conformational difference compared to structures of Rho-RhoGDIα in the nonprenylated and prenylated form. This study shows that the genetic code expansion concept is powerful in analyzing the impact of lysine acetylation on protein function. We were able to site-specifically incorporate acetyl-L-lysine into RhoGDIα to obtain highly pure protein in quantities sufficient to perform biophysical studies. Notably, we were able, for the first time, to determine the first crystal structure of a natively folded protein carrying two posttranslationally relevant acetyl-L-lysines. We observed that acetylation of K127G and K141G interfered with the interaction toward nonprenylated RhoA. AcK141G leads to a decrease in RhoA affinity, mostly affecting the electrostatic network composed of the highly negatively charged RhoGDIα Nterminus and the positvely charged RhoA C-terminus. AcK127G, in turn, leads to a further decrease in affinity by mechanisms mostly affecting RhoGDIα’s conformational flexibility, in part by bridging neighboring β-strands of the RhoGDIα immunoglobulin domain. This study reveals that lysine acetylation is a post-translational modification controlling important steps during RhoA maturation preceding RhoA prenylation. Importantly, the RhoGDIα K127 and K141 acetylation sites interact with the RhoA C-terminus, on which also RhoA prenylation occurs. Therefore, RhoGDIα acetylation might also interfere with the binding toward prenylated RhoA and affect other steps in Rho signaling. However, this needs to be investigated further. The molecular events preceding RhoA prenylation are only marginally understood but might be of potential interest for the design of novel therapeutic approaches.

However, the double-acetylated RhoGDIα AcK127,141 binds to C-terminally truncated RhoA1−181 with a further reduced affinity of 12.9 μM showing that AcK127G uses distinct molecular mechanisms to interfere with RhoA binding (Table 1). This might include the observed conformational effects, i.e., the observed rotation of RhoGDIα’s IG domain, by hydrophobic shielding from the polar solvent because of the increased hydrophobicity by acetylation at K127G. Additionally, AcK127G and AcK141G both contribute to connect neighboring β-strands 5 and 6 by interaction with side chains I129G and D143G. Thereby, acetylation of RhoGDIα at K127G and K141G might interfere with the conformational flexibility of the IG domain or the relative orientation of the RhoGDIα N- and Cterminal domain known to be important for RhoGDIα function (Figure 1C,D).

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DISCUSSION Rho proteins are essential cellular proteins involved in a variety of important processes, and a dysfunction has drastic consequences on cellular function. A major player in controlling Rho function is the protein RhoGDIα. RhoGDIα has long been regarded as a housekeeping regulator binding to many different Rho proteins and targeting those to cellular membranes. Recently, it turned out that RhoGDIα has far more complex cellular roles in not only regulating the delivery and extraction of Rho proteins to and from membranes but also controlling the balance of Rho signaling by affecting Rho protein turnover.19,46 The fact that, compared to more than 80 different RhoGAPs and RhoGEFs, there are only three RhoGDI isoforms in mammals opens the question of how RhoGDIα can be regulated to achieve a high level of functional specificity. RhoGDIα was found to be tightly controlled by phosphorylation. Here we report on the regulation of RhoGDIα function by previously discovered lysine acetylation. We show that lysine acetylation affects the interplay with nonprenylated RhoA, leading to a reduction in RhoA affinity. For one site, K141 in RhoGDIα, it was reported earlier that mutation of K141 to glutamine affects the formation of F-actin and filopodia in mammalian cells.31 Most functional and structural studies report on how prenylated Rho proteins interact with RhoGDIα. However, also, nonprenylated Rho proteins were reported to bind to RhoGDIα with affinities that are physiologically relevant.14 Nonprenylated Rho proteins were shown to be active in mediating transcriptional activation of serum response factor (SRF) target genes.47 Another study showed that nonprenylated RhoA and Rac1 lead to an increase in the level of intracellular superoxide production and JNK activation upon treatment of cells with the chemical, simvastatin, impairing the interaction with RhoGDIα.48 An unresolved question is how Rho proteins were targeted to GGTase I for prenylation and which molecular events take place before prenylation. In contrast to Rab proteins, where Rab escort proteins are needed for substrate recognition by RabGGTase, for Rho proteins no escort factors have been identified.49 However, Tnimov and colleagues reported recently that the presence of RhoGDIα increases the efficiency of RhoA geranylgeranylation by GGTase I.22 We found, in agreement with previous reports, that nonprenylated RhoA binds to RhoGDIα with an affinity in the low micromolar range, suggesting that this interaction plays a physiologically important role.14 To analyze the impact of RhoGDIα lysine acetylation and to clarify the molecular mechansims underlying the phenotypes

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.5b01242. 310

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Table S1 and legends for Figures S1−S3 (PDF) Figure S1 (PDF) Figure S2 (PDF) Figure S3 (PDF) Accession Codes

The structure factors and coordinates were deposited as PDB entry 5FR1.

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AUTHOR INFORMATION

Corresponding Author

*University of Cologne, Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in AgingAssociated Diseases (CECAD), Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany. E-mail: [email protected]. Phone: +49 221-478-84308. Fax: +49 221-478-84261. Funding

This work and all collaborators were funded by the CECAD, collaborative research center 635 (SFB635), and Emmy Noether Grant LA2984/1-1 of the German Research Foundation (Deutsche Forschungsgemeinschaft). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. M. Krüger and Hendrik Nolte for conducting the proteomic experiments and Astrid Wilbrand-Hennes and René Grandjean for technical assistance (CECAD Proteomics Facility). We thank Linda Baldus for expert technical assistance. We thank the beamline groups at the at the Diamond Light Source whose outstanding efforts have made these experiments possible.

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ABBREVIATIONS Rho, Ras homologous; Ras, Rat sarcoma; RhoGDI, Rho guanine nucleotide dissociation inhibitor; IG, immunoglobulin; GEF, guanine nucleotide exchange factor; GAP, GTPaseactivating protein; AcK, acetyl-L-lysine; GTP, guanosine triphosphate; GDP, guanosine diphosphate; ER, endoplasmic reticulum; PDEδ, phosphodiesterase subunit δ; KD, equilibrium dissociation constant; AB, antibody; rmsd, root-mean-square deviation.

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