Báo cáo khoa học: Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains

Authentic Interdomain Communication in an RNA Helicase Reconstituted by Expressed Protein Ligation of Two Helicase Domains

Tác giả: Anne R. Karow, Bettina Theissen and Dagmar Klostermeier

Lĩnh vực: Biophysical Chemistry

Nội dung tài liệu: Nghiên cứu này trình bày việc tái tạo thành công một enzyme RNA helicase chức năng thông qua kỹ thuật nối protein biểu hiện (EPL). Cụ thể, enzyme RNA helicase YxiN từ Bacillus subtilis, bao gồm hai miền helicase được kết nối linh hoạt, đã được tạo ra bằng cách nối hai miền riêng biệt. Các thử nghiệm cho thấy enzyme được tái tạo này thể hiện các đặc tính tương tự như enzyme dạng hoang dã về khả năng liên kết nucleotide, hoạt động ATPase được kích thích bởi RNA và khả năng làm tách RNA. Điều này chứng minh rằng EPL có thể được sử dụng hiệu quả để tạo ra các enzyme chức năng với sự giao tiếp giữa các miền được bảo tồn, mở ra tiềm năng ứng dụng cho các protein hai miền hoặc đa miền khác.

Mục lục chi tiết:

• Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains
• Keywords
• Correspondence
• (Received 23 October 2006, accepted 13 November 2006)
• doi:10.1111/j.1742-4658.2006.05593.x
• RNA helicases mediate structural rearrangements of RNA or RNA-protein complexes at the expense of ATP hydrolysis.
• Members of the DEAD box helicase family consist of two flexibly connected helicase domains.
• They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity.
• Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding.
• The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation.
• The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein.
• Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis-Menten parameters are similar to those of the wild-type.
• Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication.
• In 1990, post-translational excision of intervening sequences from protein precursors was discovered [1,2].
• In this self-catalyzed process termed protein splicing, the intervening amino-acid sequence (intein) is excised, and the flanking sequences, the exteins, are covalently linked by a peptide bond (reviewed in [3]).
• Detailed understanding of the protein splicing mechanism has since been developed into a preparative in vitro tool for ligating proteins and peptides by peptide bonds via expressed protein ligation (EPL) [4-6].
• Here, the N-terminal ligation partner is required to be a C-terminal thioester, and the C-terminal partner usually carries an N-terminal cysteine.
• In a first, reversible step, nucleophilic substitution of the thioester by this cysteine leads to transesterification, with a high-energy thioester linking the two partners.
• In a subsequent, irreversible rearrangement, the α-amino group of the cysteine attacks the carbonyl carbon of the thioester, and, as a result, the two ligation partners are linked by a peptide bond.
• EPL has been used to link synthetic peptides to recombinantly produced proteins to introduce spectroscopic reporter groups or chemical modifications (reviewed in [4]), or for segmental isotope labeling for NMR studies [7-9], and artificial fusion proteins have been generated [10,11].
• Reconstitution of an active enzyme by ligation of large protein domains has not been reported.
• Proteins consisting of two or more flexibly linked domains are ideally suited for ligation experiments.
• One example is the family of RNA helicases, which unwind dsRNA regions in an ATP-dependent fashion, and as such are involved in many processes where structural rearrangements of RNA or RNA-protein complexes are required.
• The helicase core comprises two domains, connected by a flexible linker.
• Nine conserved sequence motifs [12,13] that mediate ATP binding and hydrolysis, RNA binding, and helicase activity line the interdomain cleft.
• From the different relative arrangements of these helicase domains in various helicase structures [14-20], it has been proposed that interdomain communication, such as closure of the cleft between the two core domains upon ATP or RNA binding, is required for efficient unwinding.
• The RNA helicase YxiN from Bacillus subtilis comprises 479 amino acids and is presumably involved in ribosome biogenesis [21].
• The N-terminal 400 amino acids constitute the catalytic core with the two helicase domains, and the C-terminal 75 amino acids confer specificity for a region in the 23S rRNA [22].
• We have used EPL to reconstitute a functional YxiN helicase from inactive precursors.
• The ligation product exhibits very similar properties to those of the wild-type enzyme with respect to nucleotide binding, RNA-stimulated ATPase activity, and RNA unwinding.
• These results for the first time demonstrate the use of EPL to generate a functional enzyme with intact interdomain communication, and predict successful application of this technique to other two-domain or multidomain proteins.
• Results and Discussion
• Choice of ligation site and purification of YxiN helicase and fragments
• For EPL, the C-terminal reaction partner needs to carry an N-terminal side chain with a strong nucleophile, typically a cysteine.
• The ligation site for EPL of the YxiN helicase domains was chosen on the basis of a homology model constructed with 3D-JIGSAW [23] using the crystal structure of the translation initiation factor eIF4A from Saccharomyces cerevisiae [16] as a template (Fig. 1).
• The linker region connecting the two helicase domains in YxiN extends from amino acids E204 to T212.
• Within the linker, the side chains of T211 and T212 could potentially act as a nucleophile in EPL.
• In addition, both positions allow the introduction of a stronger nucleophile in a conservative amino-acid exchange to a serine or cysteine.
• In vivo, C-exteins functional in protein splicing have been found with a cysteine, serine, or threonine at the N-terminus [3].
• In the homologous DEAD box helicase Vasa, the corresponding residue to T212 is a cysteine [24].
• We therefore chose to divide YxiN between T211 and T212, and constructed YxiN 1-211 as the N-terminal, and YxiN_T212-479, YxiN_S212-479, and YxiN_C212-479 as the C-terminal partners for EPL.
• Fig. 1. Homology model of YxiN.
• YxiN_1-211 and all three C-terminal constructs were produced separately as stable proteins, underlining the notion that they constitute autonomous folding units.
• YxiN_T212–479, YxiN_C212-479 and YxiN_S212-479 were produced as N-terminal intein-chitin-binding domain (CBD) fusion proteins.
• After on-column cleavage, 2-5 mg YxiN_212–479 was obtained from 1 L bacterial culture (Fig. 2) with high purities ranging from 95% to 98%.
• The N-terminal YxiN fragment, YxiN_1–211, was produced as a C-terminal intein-CBD fusion protein, and YxiN_1-211 was obtained as a C-terminal thioester by on-column cleavage with 2-mercaptoethanesulfonic acid (MESNA).
• From 1 L cell culture, 30 mg YxiN_1-211-MESNA thioester was obtained at > 95% purity (Fig. 2).
• Because of the limited stability of this high-energy compound, it was prepared directly before the ligation reactions were set up and stored at -80 °C for no more than a week.
• Wild-type YxiN helicase and the T212C mutant were recombinantly produced as an N-terminal His6-glutathione S-transferase (GST) fusion protein.
• After affinity chromatography, proteolytic cleavage and size exclusion chromatography (SEC), 4–5 mg YxiN or YxiN_T212C per liter of bacterial culture was obtained at 98% purity (Fig. 2).
• Both proteins eluted from a calibrated size exclusion column as a monomeric enzyme.
• EPL with YxiN_C212-479, YxiN_S212-479, and YxiN_T212-479
• To obtain full-length YxiN helicase, EPL was performed with YxiN_1–211 (thioester) and YxiN_C212-479.
• In the presence of MESNA, only limited amounts of ligation product were formed, with yields not exceeding 10%.
• Optimization of the reaction temperature (4 °C, 25 °C, 37 °C), pH (6.5-8.4), or MESNA concentrations (50-200 mm) did not result in further improvement (data not shown).
• In contrast, the yield of full-length ligation product was dramatically improved to 50% when thiophenol was used instead of MESNA (Fig. 3).
• As expected for a bimolecular reaction, higher concentrations of N-terminal and C-terminal fragments favored ligation.
• Whereas in vivo, C-exteins with a cysteine, serine, or threonine at the N-terminus are functional in protein splicing [3], only negligible amounts of ligation product were detected by SDS/PAGE in ligation reactions with YxiN_S212-479 and YxiN_T212–479, probably owing to the smaller nucleophilicity of serine and threonine compared to cysteine.
• Extensive variation of the reaction conditions (reaction temperatures 4, 25, and 37 °C; incubation times up to 70 h; pH between 6.5 and 10.4; addition of MESNA, thiophenol, or glycerol) did not lead to increased ligation efficiencies.
• This shows that, in vitro, a cysteine is absolutely required as the nucleophile for linking large protein domains with high efficiency.
• Consequently, the YxiN_C212–479 fragment was used as the C-terminal ligation partner in all subsequent ligation reactions.
• In the following, the terms ‘ligation product’ and ‘ligated YxiN’ refer to YxiN_T212C prepared by EPL.
• To distinguish effects caused by the ligation reaction from effects due to the T212C mutation, the YxiN_T212C mutant was prepared as a control, and the biochemical properties of wild-type YxiN, YxiN_T212C, and ligated YxiN were compared.
• Purification and characterization of the ligation product
• The ligation product was purified from the YxiN fragments by SEC (Fig. 3).
• Ligated YxiN is eluted at the same volume as wild-type YxiN and YxiN_T212C, indicating that aggregation as a possible side reaction of EPL does not occur and that the ligation product has the same globular shape as the wild-type.
• Far-UV CD spectra of ligated and wild-type YxiN are nearly identical (Fig. 4), showing that the secondary structure content is the same.
• The minima at 208 nm and 222 nm indicate a high α-helical content, which is similar to the CD spectrum of RecA [25] and thus consistent with the RecA folds present in the YxiN helicase.
• YxiN_T212C exhibits a similar spectrum to the wild-type, confirming that the T212C substitution does not interfere with folding.
• Interestingly, a mixture of the two YxiN helicase domains also shows similar CD properties.
• This confirms that the two helicase domains are autonomous folding units and suggests that the CD signal of YxiN is mainly caused by the secondary structure of the two RecA-like domains, with little contribution from interactions between them.
• Nucleotide binding: 2′(3′)-O-(N-methylanthraniloyl) (mant)-ADP, ADP, and ATP
• As a critical test whether the ligation product is functional with respect to nucleotide recognition, we compared the nucleotide-binding properties of ligated and wild-type YxiN and YxiN_T212C in fluorescence equilibrium titrations using mant-ADP (Fig. 5).
• Upon addition of YxiN to mant-ADP, a twofold increase in mant fluorescence was observed.
• This increase was reversed by adding an excess of ADP as a competitor, proving specific binding of (mant-)ADP to the nucleotide binding site.
• All titration curves were well described by a simple 1 : 1 binding model, consistent with a single nucleotide-binding site.
• The Ka values for the mant-ADP complexes of YxiN, ligated YxiN, and YxiN_T212C were similar: 15 ± 0.3 μμ (wild-type), 30 ± 1.7 μμ (ligation product), and 22 ± 1.9 μμ (YxiN_T212C), respectively.
• For the ADP complexes, virtually identical Ka values of 55 ± 4 μμ (wild-type), 58 ± 4 μμ (ligation product), and 61 ± 13 μμ (YxiN_T212C) were obtained in competitive titrations of the respective mant-ADP-YxiN complexes with ADP (Table 1).
• Table 1. Dissociation constants of adenine nucleotide complexes of wild-type YxiN, YxiN_T212, and ligated YxiN.
• Encouraged by the extremely slow ATP hydrolysis by YxiN in the absence of RNA, we determined the Ka values for the ATP complexes in competitive fluorescence titrations.
• From the ATP hydrolysis rates of YxiN in the absence of RNA substrate (see below), it can be estimated that less than 15% of the ATP in the solution would be hydrolyzed in the course of the experiments.
• The Ka values obtained were 346 ± 29 μμ (wild-type), 364 ± 27 μμ (ligation product), and 350 ± 43 μμ (YxiN_T212).
• Thus, both YxiN_T212C and ligated YxiN are wild-type-like with respect to ADP and ATP binding.
• Interestingly, no nucleotide binding was observed with a mixture of the two helicase domains under identical conditions (data not shown), which puts a lower limit for the Ka value of ≈ 0.7 mm.
• This demonstrates that, even though most of the nucleotide-binding motifs are located in the N-terminal helicase domain, the C-terminal domain contributes significantly to tight nucleotide binding, consistent with the observation of a network of interactions between residues from both domains in the structures of the homologous DEAD box helicases Vasa [24] and eIF4A-III [26] in complex with ADPNP and RNA.
• For the homologous RNA helicase DbpA from Escherichia coli, virtually identical dissociation constants have been reported (Ka = 50 µm for the DbpA-ADP complex and Ka = 400 µm for the DbpA-ATP complex [27]), suggesting that the nucleotide cycle of these homologous helicases is similar.
• Interaction with RNA and interdomain communication: steady-state ATPase activity
• We next tested whether the low intrinsic ATPase activity of the ligation product is stimulated by RNA, which requires authentic interdomain communication.
• As a model RNA substrate, a 154-mer comprising nucleotides 2481-2634 of the B. subtilis 23S rRNA was used [21].
• At saturating ATP concentrations, this RNA substrate stimulates the ATPase activity of wild-type and ligated YxiN and YxiN_T212C in a concentration-dependent manner (Fig. 6).
• All enzymes exhibit Michaelis-Menten behavior with a kcat of 4.1 ± 0.1 s⁻¹ (wild-type), 3.9 ± 0.1 s⁻¹ (ligation product), and 2.6 ± 0.1 s⁻¹ (YxiN_T212C) at 37 °C.
• The apparent Km values for RNA are 33 ± 3.3 nm (wild-type), 27 ± 3.7 nm (