Yao-Wen Wu

Analysis of the eukaryotic prenylome by isoprenoid affinity tagging

Protein prenylation is a widespread phenomenon in eukaryotic cells that affects many important signaling molecules. We describe the structure-guided design of engineered protein prenyltransferases and their universal synthetic substrate, biotin-geranylpyrophosphate. These new tools allowed us to detect femtomolar amounts of prenylatable proteins in cells and organs and to identify their cognate protein prenyltransferases. Using this approach, we analyzed the in vivo effects of protein prenyltransferase inhibitors. Whereas some of the inhibitors displayed the expected activities, others lacked in vivo activity or targeted a broader spectrum of prenyltransferases than previously believed. To quantitate the in vivo effect of the prenylation inhibitors, we profiled biotin-geranyl-tagged RabGTPases across the proteome by mass spectrometry. We also demonstrate that sites of active vesicular transport carry most of the RabGTPases. This approach enables a quantitative proteome-wide analysis of the regulation of protein prenylation and its modulation by therapeutic agents.

RabGEFs are a major determinant for specific Rab membrane targeting

Analysis of three different Rab-RabGEF pairs reveals that RabGEFs contain the minimal targeting machinery for recruiting Rabs to specific membranes.

Membrane targeting mechanism of Rab GTPases elucidated by semisynthetic protein probes

Post-translationally isoprenylated proteins represent major hubs in most membrane-connected signaling networks. GDP dissociation inhibitors (GDIs) are molecular chaperones that shuttle geranylgeranylated GTPases between membranes and the cytosol. Despite numerous studies, the mechanism of targeted membrane delivery of GTPases remains unknown. Here we have combined chemical synthesis and expressed protein ligation to generate fluorescent lipidated RabGTPase-based sensor molecules. Using these protein probes, we have demonstrated that RabGDI and the related Rab escort protein REP show a three-order-of-magnitude greater affinity for GDP-bound Rab GTPase than for the GTP-bound state. Combined with a relatively high dissociation rate of the Rab-GDI complex, this would enable guanine nucleotide exchange factors (GEFs) to efficiently dissociate the complex and promote membrane attachment of the GTPase. The findings suggest strongly that GEFs are necessary and sufficient for membrane targeting of GTPases and that the previously proposed GDI displacement factors (GDFs) are not thermodynamically required for this process.

Interaction analysis of prenylated Rab GTPase with Rab escort protein and GDP dissociation inhibitor explains the need for both regulators

Prenylated Rab GTPases regulate intracellular vesicle trafficking in eukaryotic cells by associating with specific membranes and recruiting a multitude of Rab-specific effector proteins. Prenylation, membrane delivery, and recycling of all 60 members of the Rab GTPase family are regulated by two related molecules, Rab escort protein (REP) and GDP dissociation inhibitor (GDI). Biophysical analysis of the interaction of prenylated proteins is complicated by their low solubility in aqueous solutions. Here, we used expressed protein ligation to construct a semisynthetic fluorescent analogue of prenylated Rab7, Rab7-NBD-farnesyl. This molecule is soluble in the absence of detergent but is otherwise similar in its behavior to naturally prenylated Rab7 GTPase. To obtain information on the interaction of natively mono- and diprenylated Rab7 GTPases with REP and GDI molecules, we stabilized the former molecules in solution by using the β-subunit of Rab geranylgeranyl transferase, which we demonstrate to function as an unspecific chaperone of prenylated proteins. Using competitive titrations of mixtures of natively prenylated and fluorescent Rab, we demonstrate that monogeranylgeranylated Rab7 binds to the REP protein with a Kd value of ≈70 pM. The affinity of doubly prenylated Rab7 is ≈20-fold weaker. In contrast, GDI binds both prenylated forms of Rab7 with comparable affinities (Kd = 1–5 nM) but has extremely low affinity to unprenylated Rab molecules. The obtained data allow us to formulate a thermodynamic model for the interaction of RabGTPases with their regulators and membranes and to explain the need for both REP and GDI in Rab function.

A Highly Efficient Strategy for Modification of Proteins at the C Terminus

Site-specific protein modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins both in biochemical and in cellular investigations. Although many chemical reactions are applicable in principle, methods for the site-specific modification of proteins remain in high demand, and there is a requirement for readily available ligation reagents and mild reaction conditions. Oxime-based reactions have found wide application in the conjugation of biomolecules on account of the absence of oxyamino groups in proteins and their orthogonal reactivity with ketones to give stable oximes. The oxyamine–ketone bioorthogonal reaction has been exploited in protein modification mainly by means of incorporating ketone groups into proteins by various chemical, enzymatic, and molecular biological methods. To expand the application of this efficient methodology to protein ligation, we planned the development of simple and general methods to incorporate oxyamino groups into proteins. In an earlier approach to the use of auxiliary groups in native chemical ligation, it was shown that the nitrogen atom of oxyamines can react with a thioester group intramolecularly. On the other hand, aminolysis of peptide and protein thioesters has been successfully reported in some studies. We reasoned that in theory, highly nucleophilic oxyamines could also react with a thioester moiety in a protein intermolecularly. If this reaction would be performed with a linker carrying two oxyamino groups, then one oxyamine could form a hydroxamic acid bond to the protein and the second would still be available for a subsequent ligation reaction with the selectively functionalized protein. Herein, we describe how a bis(oxyamine) molecule was first incorporated at protein C termini to produce oxyamino-modified proteins, and how the subsequent efficient and specific reaction of the second oxyamino group with ketones was achieved to modify proteins site-specifically under mild conditions. The unique position and chemistry of protein C termini has stimulated efforts to target this location for site-selective protein modification. In one approach, a protein tag is appended to a target protein, and an enzymatic reaction is used to covalently introduce a C-terminal modification onto a protein. However, the protein tag is still retained in the labeled protein, which may interfere with protein function. An intein-based protein-cleavage reaction has generated very useful approaches to the C-terminal modification of proteins, mainly based on thioester-mediated ligation chemistry. A limitation of this method is that it generally leads to introduction of a cysteine residue into the target protein, regardless of whether this corresponds to the native structure or not. In addition, the thioester reaction frequently proceeds at relatively slow ligation rates and requires a relatively high protein concentration. Recently, it was reported that the Cterminal carboxylate can also be transformed into a thioacid, followed by C-terminal modification of the protein by means of thioacid/azide amidation in the presence of 6m guanidine hydrochloride (Gdn-HCl) containing 3 mm 2,6-lutidine. The modified conditions are too harsh to maintain the proper folding of a protein and the thioacid group is prone to hydrolysis. In contrast to these reports, we could introduce an oxyamino group into the C terminus of protein in phosphate buffer (pH 7.5), and the modified protein can subsequently undergo fast and chemoselective oxime ligation on ice. The synthesis of 1,2-bis(oxyamino)ethane (1) started from the commercially available and inexpensive reagents Nhydroxyphthalimide and 1,2-dibromoethane (Figure 1). Bis(oxyamine) 1 was obtained in a convenient manner without chromatography (> 20% yield over two steps; for details see the Supporting Information). The facile and economic synthesis of 1 is important for the wide use of the method reported herein. Rab1bD3-thioester (Ras-related GTPase) generated through intein-mediated partial protein splicing was chosen as the model protein. In order to introduce an oxyamino group into the protein, the reaction of Rab1bD3-thioester and 1 was performed on ice for 4 h (quantitative conversion). As shown in Figure 1, the bis[*] L. Yi, Dr. Y.-W. Wu, Prof. R. S. Goody Department of Physical Biochemistry Max-Planck-Institut f r molekulare Physiologie Otto-Hahn-Strasse 11, 44227 Dortmund (Germany) Fax: (+ 49)231-133-2399 E-mail: roger.goody@mpi-dortmund.mpg.de L. Yi, Dr. H. Sun, Dr. G. Triola, Prof. H. Waldmann Department of Chemical Biology Max-Planck-Institut f r molekulare Physiologie Otto-Hahn-Strasse 11, 44227 Dortmund (Germany) and Chemische Biologie, Fachbereich Chemie Universit t Dortmund, 44227 Dortmund (Germany) Fax: (+ 49)231-133-2499 E-mail: herbert.waldmann@mpi-dortmund.mpg.de [] These authors contributed equally to this work.

One-Pot Dual-Labeling of a Protein by Two Chemoselective Reactions†

Multicolor labeling is a valuable technique for the characterization of proteins with respect to their structure, folding, and interactions both as single molecules and in cellular investigations. The key technique for such studies is based on fluorescence resonance energy transfer (FRET). FRET applications require the attachment of donor (D) and acceptor (A) molecules to specific sites of a given protein or proteins. Such labeling is typically achieved through conjugation at cysteine residues or amino groups or by genetic fusion to different fluorescent proteins. Recent advancements in chemical methods have substantially expanded the tools that are available for site-specific modification of proteins. However, site-specific incorporation of multiple fluorophores into a single protein remains a considerable challenge. Dual labeling of a single protein has been achieved using multistep reactions. For example, sortases with different substrate specificity were used for site-specific Cand Nterminal labeling of a single protein. Muir and Cotton reported a method for producing a dual-labeled protein through a multistep expressed protein ligation approach. Recently, Yang and Yang used a three-step strategy based on split inteins for site-specific two-color protein labeling. Herein, we report a facile and efficient method for duallabeling of proteins based on chemoselective reactions. Frequently used chemoselective reactions include native chemical ligation (NCL), Staudinger ligation, Huisgen 1,3dipolar cycloaddition (click chemistry), oxime ligation, strainpromoted cycloaddition, and Diels–Alder ligation. We reasoned that by employing two chemoselective reactions for protein labeling, it should be possible to obtain two-color labeled proteins in a one-pot reaction in a straightforward fashion. Recently, we reported a method for intein-mediated incorporation of a (bis)oxyamine moiety into the C terminus of proteins, making them amenable to efficient conjugation with a keto fluorophore under mild conditions. For Nterminal labeling, a protein containing an N-terminal cysteine can undergo NCL with thioester probes. The exposure of an N-terminal cysteine can be achieved by TEV (tobacco etch virus) protease cleavage. Hence, we speculated that both NCL and oxime ligation could be employed for one-pot twocolor labeling of a given protein. Herein we present a strategy for constructing a dual-labeled Rab7 GTPase in a one-pot reaction and illustrate the use of the method for studying protein refolding and protein–protein interactions. To generate Rab7 with an N-terminal cysteine, we fused a peptide sequence that provides a TEV cleavage site (ENLYFQ:C; the dotted line indicates the cleavage site) to the N terminus of the Rab7D3 protein. Rab7D3 fused Nterminally to an engineered Mxe GyrA intein domain can undergo initial N!S acyl transfer and be subsequently cleaved by thiol reagents (such as 2-mercapoethanesulfonic acid) by an intermolecular transthioesterification reaction, releasing a a-thioester-tagged protein. Subsequently, the Rab7D3-thioester (10 mgmL , 400 mm) was treated with 500 mm (bis)oxyamine at pH 7.5 to produce the oxyamine protein derivative. TEV protease was then added to cleave the N-terminal protection sequence (Scheme 1). This led to a doubly functionalized Rab7 protein with an N-terminal cysteine and a C-terminal oxyamine, N-Cys-Rab7D3-ONH2, for chemoselective reactions. Herein, we chose coumarin thioester (quantum yield of 0.27) as FRET donor and keto fluorescein (quantum yield of 0.97) as FRET acceptor, both of which can be easily prepared from commercially available reagents (for details, see the Supporting Information). R0 for the coumarin and fluorescein pair is 47 . Our first goal was to achieve quantitative conversion for both reactions. The NCL reaction for Nterminal modification was not complete after incubation for three days with 2-mercaptoethanesulfonate (MESNA) as a thiol cofactor (data not shown). The addition of 100–200 mm (4-carboxymethyl)thiophenol (MPAA) significantly accelerated the reaction, and the reaction of N-Cys-Rab7D3ONH2 (1 mgmL , 43 mm) with 0.7 mm coumarin thioester was complete in 2–3 h at pH 7.0 at room temperature or 12 h on ice with quantitative conversion (see the Supporting Information). The C-terminal oxime ligation of N-Cys[*] L. Yi, Dr. A. Itzen, Prof. R. S. Goody, Dr. Y. W. Wu Department of Physical Biochemistry Max-Planck-Institut f r molekulare Physiologie Otto-Hahn-Strasse 11, 44227 Dortmund (Germany) E-mail: yaowen.wu@mpi-dortmund.mpg.de roger.goody@mpi-dortmund.mpg.de L. Yi, Dr. H. Sun, Dr. G. Triola, Prof. H. Waldmann Department of Chemical Biology Max-Planck-Institut f r molekulare Physiologie Otto-Hahn-Strasse 11, 44227 Dortmund (Germany) and Chemische Biologie, Fachbereich Chemie Technische Universit t Dortmund 44227 Dortmund (Germany) [] These authors contributed equally to this work.

Structures of RabGGTase-substrate/product complexes provide insights into the evolution of protein prenylation.

Post‐translational isoprenylation of proteins is carried out by three related enzymes: farnesyltransferase, geranylgeranyl transferase‐I, and Rab geranylgeranyl transferase (RabGGTase). Despite the fact that the last one is responsible for the largest number of individual protein prenylation events in the cell, no structural information is available on its interaction with substrates and products. Here, we present structural and biophysical analyses of RabGGTase in complex with phosphoisoprenoids as well as with the prenylated peptides that mimic the C terminus of Rab7 GTPase. The data demonstrate that, unlike other protein prenyl transferases, both RabGGTase and its substrate RabGTPases completely ‘outsource’ their specificity for each other to an accessory subunit, the Rab escort protein (REP). REP mediates the placement of the C terminus of RabGTPase into the active site of RabGGTase through a series protein–protein interactions of decreasing strength and selectivity. This arrangement enables RabGGTase to prenylate any cysteine‐containing sequence. On the basis of our structural and thermodynamic data, we propose that RabGGTase has evolved from a GGTase‐I‐like molecule that ‘learned’ to interact with a recycling factor (GDI) that, in turn, eventually gave rise to REP.

The role of the hypervariable C-terminal domain in Rab GTPases membrane targeting

Significance Rab GTPases are key regulators of intracellular vesicular transport in eukaryotic cells, for example in neurotransmission and endocytosis. Each of more than 60 Rabs in human cells regulates these processes at a specific subcellular membrane. The hypervariable C-terminal domain (HVD) of Rab has been proposed to contain a targeting signal, but recent studies have argued this model. However, the role of the Rab HVD and the mechanism of Rab membrane targeting remain elusive. In this study, using a combination of unique synthetic protein probes and cell biological tools, we dissect the role of the HVD for subcellular Rab targeting. We find that the HVD can act as a structurally flexible spacer, a subcellular localization determinant, and/or an enhancer of membrane affinity. Intracellular membrane trafficking requires correct and specific localization of Rab GTPases. The hypervariable C-terminal domain (HVD) of Rabs is posttranslationally modified by isoprenyl moieties that enable membrane association. A model asserting HVD-directed targeting has been contested in previous studies, but the role of the Rab HVD and the mechanism of Rab membrane targeting remain elusive. To elucidate the function of the HVD, we have substituted this region with an unnatural polyethylenglycol (PEG) linker by using oxime ligation. The PEGylated Rab proteins undergo normal prenylation, underlining the unique ability of the Rab prenylation machinery to process the Rab family with diverse C-terminal sequences. Through localization studies and functional analyses of semisynthetic PEGylated Rab1, Rab5, Rab7, and Rab35 proteins, we demonstrate that the role of the HVD of Rabs in membrane targeting is more complex than previously understood. The HVD of Rab1 and Rab5 is dispensable for membrane targeting and appears to function simply as a linker between the GTPase domain and the membrane. The N-terminal residues of the Rab7 HVD are important for late endosomal/lysosomal localization, apparently due to their involvement in interaction with the Rab7 effector Rab-interacting lysosomal protein. The C-terminal polybasic cluster of the Rab35 HVD is essential for plasma membrane (PM) targeting, presumably because of the electrostatic interaction with negatively charged lipids on the PM. Our findings suggest that Rab membrane targeting is dictated by a complex mechanism involving GEFs, GAPs, effectors, and C-terminal interaction with membranes to varying extents, and possibly other binding partners.

Quantitative Analysis of Prenylated RhoA Interaction with Its Chaperone, RhoGDI.

Background: RhoGDI is a key regulator and a chaperon of Rho GTPases. Results: RhoGDI strongly discriminates between GDP- and GTP-bound forms of prenylated RhoA, although both complexes are of high affinity. Conclusion: We provide direct evidence for the existence of two populations of the RhoGDI·RhoA complexes in the cell, characterized by different lifetimes. Significance: The obtained data allows us to formulate the model for membrane delivery and extraction of Rho GTPases. Small GTPases of the Rho family regulate cytoskeleton remodeling, cell polarity, and transcription, as well as the cell cycle, in eukaryotic cells. Membrane delivery and recycling of the Rho GTPases is mediated by Rho GDP dissociation inhibitor (RhoGDI), which forms a stable complex with prenylated Rho GTPases. We analyzed the interaction of RhoGDI with the active and inactive forms of prenylated and unprenylated RhoA. We demonstrate that RhoGDI binds the prenylated form of RhoA·GDP with unexpectedly high affinity (Kd = 5 pm). The very long half-life of the complex is reduced 25-fold on RhoA activation, with a concomitant reduction in affinity (Kd = 3 nm). The 2.8-Å structure of the RhoA·guanosine 5′-[β,γ-imido] triphosphate (GMPPNP)·RhoGDI complex demonstrated that complex formation forces the activated RhoA into a GDP-bound conformation in the absence of nucleotide hydrolysis. We demonstrate that membrane extraction of Rho GTPase by RhoGDI is a thermodynamically favored passive process that operates through a series of progressively tighter intermediates, much like the one that is mediated by RabGDI.

Psoromic Acid is a Selective and Covalent Rab-Prenylation Inhibitor Targeting Autoinhibited RabGGTase

Post-translational attachment of geranylgeranyl isoprenoids to Rab GTPases, the key organizers of intracellular vesicular transport, is essential for their function. Rab geranylgeranyl transferase (RabGGTase) is responsible for prenylation of Rab proteins. Recently, RabGGTase inhibitors have been proposed to be potential therapeutics for treatment of cancer and osteoporosis. However, the development of RabGGTase selective inhibitors is complicated by its structural and functional similarity to other protein prenyltransferases. Herein we report identification of the natural product psoromic acid (PA) that potently and selectively inhibits RabGGTase with an IC(50) of 1.3 μM. Structure-activity relationship analysis suggested a minimal structure involving the depsidone core with a 3-hydroxyl and 4-aldehyde motif for binding to RabGGTase. Analysis of the crystal structure of the RabGGTase:PA complex revealed that PA forms largely hydrophobic interactions with the isoprenoid binding site of RabGGTase and that it attaches covalently to the N-terminus of the α subunit. We found that in contrast to other protein prenyltransferases, RabGGTase is autoinhibited through N-terminal (α)His2 coordination with the catalytic zinc ion. Mutation of (α)His dramatically enhances the reaction rate, indicating that the activity of RabGGTase is likely regulated in vivo. The covalent binding of PA to the N-terminus of the RabGGTase α subunit seems to potentiate its interaction with the active site and explains the selectivity of PA for RabGGTase. Therefore, psoromic acid provides a new starting point for the development of selective RabGGTase inhibitors.