Báo cáo khoa học: Identification of a domain in the a-subunit of the oxaloacetate decarboxylase Na+ pump that accomplishes complex formation with the c-subunit pot

Identification of a domain in the a-subunit of the oxaloacetate decarboxylase Na + pump that accomplishes complex formation with the c-subunit Pius Dahinden, Klaas M. Pos* and Peter Dimroth Institute of Microbiology ETH Zu ¨ rich, ETH Ho ¨ nggerberg, Zu ¨ rich, Switzerland Oxaloacetate decarboxylase is a member of the sodium ion transport decarboxylase (NaT-DC) enzyme family which also includes methylmalonyl-CoA decarboxy- lase, malonate decarboxylase, and glutaconyl-CoA decarboxylase [1–3]. These enzymes are found exclu- sively in anaerobic bacteria. They convert the free energy of a specific decarboxylation reaction into an electrochemical gradient of Na + ions which plays a profound role in the energy metabolism of these bac- teria [4]. Oxaloacetate decarboxylase is a membrane-bound enzyme complex composed of subunits a, b and c with molecular masses of approximately 63–65, 40–45, and 9–10 kDa, respectively. The a-subunit is located peri- pheral to the membrane. It contains the carboxyl- transferase domain in the N-terminal part and the biotin-binding domain in the C-terminal part [5]. The b-subunit is an integral membrane protein with nine membrane-spanning a-helices and a fragment inserting into the membrane but not traversing it [6]. The c-sub- unit contacts the b-subunit with its N-terminal a-heli- cal region and the a-subunit with its hydrophilic C-terminal domain. One histidine residue of the histi- dine triplet near the C terminus of c is specifically required for complex formation with the a-subunit. The c-subunit therefore plays an important role in the in the assembly of the a ⁄ b ⁄ c-complex [7,8]. Vibrio cholerae contains two oxaloacetate decarboxy- lase-encoding gene clusters, termed oad-1 and oad-2. The flanking regions of oad-1 do not code for enzymes of a specific metabolic pathway in which the oxaloace- tate decarboxylase could participate. In contrast, the Keywords association domain; flexible linker peptide; oxaloacetate decarboxylase; protein–protein interaction; sodium ion transport decarboxylase Correspondence P. Dimroth, Institute of Microbiology ETH Zu ¨ rich, ETH Ho ¨ nggerberg, Wolfgang-Pauli- Strasse 10, CH-8093 Zu ¨ rich, Switzerland E-mail: dimroth@micro.biol.ethz.ch *Present address Institute of Physiology, University of Zu ¨ rich, Winterthurerstrasse 190, CH-8057 Zu ¨ rich, Switzerland (Received 18 October 2004, revised 1 December 2004, accepted 10 December 2004) doi:10.1111/j.1742-4658.2004.04524.x The oxaloacetate decarboxylase Na + pumps OAD-1 and OAD-2 of Vibrio cholerae are composed of a peripheral a-subunit associated with two integ- ral membrane-bound subunits (b and c). The a-subunit contains the carb- oxyltransferase domain in its N-terminal portion and the biotin-binding domain in its C-terminal portion. The c-subunit plays a profound role in the assembly of the complex. It interacts with the b-subunit through its N-terminal membrane-spanning region and with the a-subunit through its hydrophilic C-terminal domain. The biochemical and structural require- ments for the latter interaction were analysed with OAD-2 expression clones for subunit a-2 and the C-terminal domain of c-2, termed c¢-2. If the two proteins were synthesized together in Escherichia coli they formed a complex that was stable at neutral pH and dissociated at pH<5.0. An internal stretch of 40 amino acids of a-2 was identified by deletion muta- genesis to be essential for the binding with c¢-2. This portion of the a-sub- unit is connected via flexible linker peptides to the carboxyltransferase domain at its N terminus and to the biotin-binding domain at its C termi- nus. Results of site-directed mutagenesis indicated that a conserved tyrosine (491) and threonine 494 of this peptide contributed significantly to the sta- bility of the complex with c¢-2. This peptide therefore represents a newly identified, separate domain of the a-subunit and has been called the ‘asso- ciation domain’. 846 FEBS Journal 272 (2005) 846–855 ª 2005 FEBS oad-2 genes are part of the citrate fermentation operon and accordingly, the oad-2 genes are expressed during anaerobic growth of V. cholerae on citrate (data not shown). The catalytic cycle starts with the transfer of the carboxyl group from oxaloacetate to the prosthetic biotin group. The carboxyltransfer reaction is catalysed at low rates by the a-subunit alone and with about 1000 times higher rates by the a ⁄ c-complex [8]. This rate increase has been attributed to polarizing the car- bonyl oxygen bond of oxaloacetate by the Zn 2+ metal ion on the c-subunit which is therefore part of the carboxyltransferase active site [8]. In the next step the carboxybiotin switches from the carboxyltransferase site to the decarboxylase site on the b-subunit. Two Na + ions pass through the cytoplasmic access channel contributed in part by the highly conserved helix VIII and bind to specific sites in the middle of the membrane [9–12]. According to a mechanistic model, binding of the second Na + ion to the Y229- and S382- including site abstracts the phenolic proton from Y229. The proton is thought to move through the channel to the carboxybiotin where it catalyses the decarboxylation of this acid-labile compound [11]. This event triggers a conformational change by which the cytoplasmic channel closes and the periplasmic channel opens. The two Na + ions then diffuse into the peri- plasmic reservoir and a proton diffuses from the peri- plasm to Y229 where it restores the phenolic hydroxyl group [11,13]. Overall, the decarboxylation of one oxaloacetate leads to the transport of two Na + ions into the periplasm and the consumption of a periplas- mically derived proton [14,15]. This sophisticated machinery requires specific flexi- ble segments for the mechanical movements side by side with segments that guarantee the structural integ- rity of the three-subunit complex. A remarkable region is the extended proline ⁄ alanine linker between the two domains of the a-subunit of oxaloacetate decarboxy- lase from Klebsiella pneumoniae. Such an extended linker peptide is not apparent, however, in the two oxaloacetate decarboxylases (OAD-1 or OAD-2) of V. cholerae, and the corresponding segments of the OADs known so far differ widely in the linker region and the flanking sequences on both sides. Nevertheless, all of these segments contain numerous proline, alan- ine and serine residues that probably contribute the flexibility necessary for catalysis. Interestingly, also the segments of the cytosolic domains of the c-subunits differ widely among species. As a interacts with c sup- posedly via amino acids in its C-terminal part, these variable regions between the carboxyltransferase domain and the biotin-binding domain might consti- tute a specific interacting interface. Here we probed the interacting parts between subunits a and c by dele- tion and site-specific mutagenesis with the OAD-2 of V. cholerae. The binding domain on a was identified as a stretch of 40 amino acids (480–520) that is flanked at its N terminus by the carboxyltransferase domain and at its C terminus by the biotin-binding domain. This portion of the a-subunit has been termed the associ- ation domain. Particularly important amino acids in this domain for complex stability were Y491 and T494. Results Complex formation between the a- and c-subunits and dissociation at acidic pH A detailed analysis of the interaction of the C-terminal domain of the c-subunit with the a-subunit was per- formed using the recombinantly synthesized sub- units ⁄ domains of the OAD-2 from V. cholerae.As shown in Fig. 1 the c-subunits of the OAD-2 and OAD-1 isoforms harbour the Zn 2+ -binding motif pre- viously identified in the c-subunit of the OAD from K. pneumoniae. The histidine triplet near the C terminus of the c-subunit is also conserved in c-2 of V. cholerae. For practical reasons the membrane part of c-2 was substituted by a peptide of 10 histidine residues. The resulting soluble protein (c¢-2) was synthesized together with the a-subunit (a-2) in Escherichia coli. These two Fig. 1. Domain structure and catalytic zinc binding motif of the oxaloacetate decarboxylase c -subunits from K. pneumoniae and V. cholerae. The sequences of the c-subunits of the oxaloacetate decarboxylases from K. pneumoniae and V. cholerae are compared. They have a common domain structure: a short periplasmic seg- ment (amino acids 1–11) is followed by a transmembrane segment (amino acids 12–32, indicated by the rectangular box) to which the cytosolic domain is linked by a flexible linker peptide rich in proline and alanine residues. Characteristic for subunit c-2 from V. cholerae and subunit c from K. pneumoniae is a histidine triplet at the C-ter- minal end. In the Klebsiella c-subunit D62, H77 and H82 are involved in Zn 2+ binding (indicated by arrows) and H78 is essential for binding of the a -subunit [8]. Amino acid residues supposed to be involved in Zn 2+ binding by the Vibrio c-subunits are D62, H77 and H79 of c-1 and E71, H81 and H83 of c-2 (indicated by arrows). P. Dahinden et al. Association domain of oxaloacetate decarboxylase FEBS Journal 272 (2005) 846–855 ª 2005 FEBS 847 proteins assembled within the E. coli cells to a stable a-2 ⁄ c¢-2-complex as both subunits are purified together by Ni–NTA or monomeric avidin–Sepharose chroma- tography which specifically bind the His 10 tag on c¢-2 or the biotin group on a-2, respectively. The stability of the complex was investigated after binding the a-2 ⁄ c¢-2-complex to a monomeric avidin–Sepharose column. The complex was stable during washing with buffer at neutral pH. However, with citrate buffer of pH < 5.0 the complex dissociated and only a-2 was retained on the avidin column (Fig. 2). The separated subunits reassociated at pH > 5.0 to a stable complex that was retained on a Ni–NTA agarose column (Fig. 2). Therefore, amino acid residues which become protonated at pH < 5.0 seem to be involved in the binding of a-2 to c-2. The most likely candidates are the histidine residues or the glutamate residue at the C-terminal end of c-2 (Fig. 1). Effect of point mutations in the C-terminal domain of c¢-2 on complex stability To investigate whether one of the histidine residues of the histidine triplet at the C terminus of c-2 is import- ant for the interaction with a-2, each histidine was mutated individually to alanine. To analyse the complex formation between a-2 and the c¢-2 mutants, the C-terminal part of a-2 (a-2-C) was synthesized together with c¢-2 and mutants thereof in E. coli. The construct of a-2-C covers the 151 C-terminal residues of a-2 and has an N-terminal extension of the four residues MTVD. The construct contains the C-terminal biotin-binding domain and upstream segments of a-2. As expected, the entire a-2-C protein formed a strong complex with the wild-type c¢-2 protein. In the mutants c¢-2-H82A and c¢-2-H83A binding of a-2-C was not affected, as shown by the copurification of the c¢-2 mutant proteins with a-2-C by affinity chromato- graphy on avidin-Sepharose (Fig. 3). The c¢-2-H81A mutant protein, however, was only copurified in sub- stoichiometric amounts with a-2-C. This indicates that the histidine at position 81 of the c-subunit contributes to the stability of the a ⁄ c complex. It was unclear, however, which residues of a-2 participate in the inter- action. To answer this question, the complex stability was analysed with various deletion mutants of a-2. Complex formation between a-2 deletion mutants and c¢-2 To elucidate the binding domain for c¢-2 on a-2, a number of C-terminal deletion mutants of a-2 were generated. The a-2 deletion mutants were then synthes- ized together with c¢-2 in E. coli and cell extracts were subjected to Ni–NTA affinity chromatography to iso- late c¢-2 via its His 10 tag. The competence of a-2 dele- tion mutants for complex formation with c¢ -2 could thus easily be assessed by the copurification of both proteins. Deletions of a-2 with up to 80 amino acid residues from the C terminus were copurified with c¢-2 showing that this part of the protein is not involved in Fig. 2. 10 Dissociation and reassociation of c¢-2 and a-2. The dissoci- ation of the proteins that were coexpressed in E. coli and purified by Ni 2+ –NTA affinity chromatography was achieved by binding the protein to avidin–Sepharose and washing with buffer of pH < 5. The a-2 subunit still bound to the avidin–Sepharose was then eluted with biotin. Reassociation was analysed by combining the dissociated c¢-2 with the eluted a-2 at pH 8.0. Dissociation and reassociation was analysed by SDS ⁄ PAGE. Two micrograms of pro- tein were loaded on each lane and the gel was stained with silver. M, Bio-Rad 11 broad molecular mass standard (Bio-Rad Laboratories AG, Reinach, Switzerland); 1, a-2 ⁄ c¢-2 complex purified by Ni 2+ – NTA; 2, wash fraction pH 6.0 of purified a-2 ⁄ c¢-2 applied to avidin- Sepharose; 3, wash fraction pH 5.0; 4, wash fraction pH 4.0; 5, wash fraction pH 8.0; 6, elution fraction (pH 8.0); 7, flow-through fraction of reassociated a-2 ⁄ c¢-2 applied to Ni 2+ –NTA agarose; 8, wash fraction; 9, elution fraction. Fig. 3. Complex formation of c¢-2 point mutants with a -2-C. The proteins were coexpressed in E. coli and complex formation was analysed by SDS ⁄ PAGE following affinity chromatography on avi- din–Sepharose. Two micrograms of protein were loaded on each lane and the gel was stained with silver. 1, a-2-C ⁄ c¢-H81A; 2, a-2- C ⁄ c¢-H82A; 3, a-2-C ⁄ c¢-H83A; 4, wild-type a-2-C ⁄ c¢-2; M, Bio-Rad broad molecular mass standard. Association domain of oxaloacetate decarboxylase P. Dahinden et al. 848 FEBS Journal 272 (2005) 846–855 ª 2005 FEBS the complex formation. Deletion mutants of a-2 lack- ing 100 or more C-terminal amino acid residues, however, were not copurified with c¢-2 (Fig. 4A), indicating that amino acids 80–100 from the C termi- nus comprise part of the binding domain. This part of the protein is upstream of the putative linker segment connecting the C-terminal biotin-binding domain with the residual parts of a-2 (Fig. 5). For further analyses of the domain of a-2 which is crucial for complex formation with c-2, the C-terminal part of a-2 (a-2-C) was synthesized together with c¢-2 in E. coli. As was shown above, the entire a-2-C protein formed a strong complex with the c¢-2 domain. This situation did not change if up to 20 amino acid residues were deleted from the N terminus of a-2-C. However, the binding affinity between a-2-C and c¢-2 was reduced if 30 amino acids were deleted and was abolished com- pletely after deleting 40 or more amino acid residues (Fig. 4B). From these results we conclude that a domain of approximately 40 amino acids of a-2-C is essential for the binding of the a- to the c-subunit (Fig. 5). In separate experiments it was shown that the isola- ted N- and C-terminal portions of the a-subunit did not associate to form a complex. For this purpose a-2-N (residues 1–453) and a-2-C (residues 449–599) were syn- thesized separately in E. coli and incubated together before applying the mixture to a monomeric avidin– Sepharose column. Only a-2-C was retained and speci- fically eluted with biotin (data not shown). Effect of point mutations in the binding domain of a-2-C on the binding to c¢-2 To elucidate whether single amino acids in the binding domain of a-2-C were particularly important for the formation of a stable complex with c¢-2, a series of conservative point mutations were constructed on the AB Fig. 4. Complex formation of a-2-C deletion mutants with c¢-2. The proteins were coexpressed in E. coli and complex formation was analysed by SDS ⁄ PAGE following affinity chromatography on Ni–NTA agarose. Two micrograms of protein were loaded on each lane and the gel was stained with silver. (A) D20–D120, deletions of 20–120 amino acids from the C terminus of a-2. (B) D90–D140, remaining 90–120 amino acids after deletion of amino acids at the N terminus of a-2-C. M, Bio-Rad broad molecular mass standard. Fig. 5. Alignment of the C-terminal sequences of subunit a from K. pneumoniae and subunit a-2 from V. cholerae. The sequences shown include the biotin-binding domains (light grey) with the biotin-binding lysine residue 35 residues before the C terminus, the newly discovered association domains (dark grey) and upstream sequences. The association domains are highly conserved in the central portion but deviate significantly within their distal parts. The central portion of 20 amino acids of the association domain includes Y491 and T494 which were shown by site-specific mutagenesis to have a major impact on the stability of the a-2 ⁄ c-2 complex. The association domains are flanked on both sides by linker peptides (black bars above and below sequence) containing an accumulation of proline and alanine residues. These linker peptides were predicted by two independent programs, PSIPRED [26,27] and PSA [28,29]. A particularly extended linker peptide is present in the downstream region of the association domain of K. pneumoniae. The point mutations which have been introduced into a-2 of V. cholerae are shown in the lines labeled ‘mutants 1’ and ‘mutants 2’. The deletions introduced are marked by D20–D140. The corresponding numbers indicate deletions from the C terminus of a-2 or the number of remaining amino acids of a-2-C. P. Dahinden et al. Association domain of oxaloacetate decarboxylase FEBS Journal 272 (2005) 846–855 ª 2005 FEBS 849 plasmid synthesizing a-2-C and c¢-2 (see Fig. 5, ‘mutants 1’). The complex stability of mutant proteins was then assessed by avidin–Sepharose affinity chroma- tography. From 16 mutants 13 had no significant effect on complex stability, but in mutants Y491F, T494V and D509N the stability of the complex was affected (Fig. 6A). To further analyse the significance of these residues for complex formation each one was individu- ally exchanged to alanine (see Fig. 5, ‘mutants 2’). In the mutants a-2-C-Y491A and a-2-C-T494A complex formation with c ¢-2 was completely impaired. In contrast, the stability of the complex between the a-2-C- D509A mutant and c¢-2 was similar to that of the wild- type (Fig. 6B). The complex with the Y491F ⁄ D509N double mutation was less stable than that with the Y491F mutation but significantly more stable than that with the Y491A mutation (data not shown). The c¢-2 protein could only be isolated from E. coli expression hosts that also synthesized a-2 or a-2-C indicating that c¢-2 is degraded in the cells if it is not present as a complex with a-2. This observation can conveniently be used to assess complex formation in vivo with some of the mutants described above. No c¢-2 could be detected upon coexpression with the a-2- C-Y491A or a-2-C-T494A mutant confirming the importance of Y491 and T494 for proper binding to c¢-2. In contrast, c¢-2 was not degraded upon coexpres- sion with the a-2-C mutants Y491F, T494V and D509N. These results indicate complex formation in vivo between c¢-2 and the a-2-C mutants in spite of the fact that these complexes were not strong enough to survive washing with buffer on a monomeric avidin affinity column. The c¢-2 that was eluted in the wash- ing step was competent to form a native-like complex with a-2-C, as both proteins were copurified with a Ni–NTA agarose column which specifically binds the His-tag of c¢-2 (Fig. 7A). Complete degradation of c¢-2 was also observed if it was synthesized in E. coli together with the deletion mutant a-2-Del120. How- ever, if wild-type a-2-C was also synthesized by these cells, c¢-2 was not degraded and could be isolated as a stable complex with a-2-C (Fig. 7B). Discussion Upstream of the biotin domain of the a-subunit is a linker peptide which in case of the K. pneumoniae enzyme, with which most of the fundamental biochem- istry has been explored, contains mostly proline and alanine residues. Peptides with this composition are known to be very flexible and such a flexible region seems to be required for moving the carboxybiotin from the carboxyltransferase site on the N-terminal domain of the a-subunit to the decarboxylase site on the b-subunit. Regions of high flexibility within a pro- tein are disadvantageous, however, for structural stud- ies because they may prevent the protein from adopting a uniform conformation which is the pre- requisite for crystallization. In this context we investi- gated the genome sequences of various oxaloacetate decarboxylase containing organisms and found that V. cholerae contains the genes for two different oxalo- acetate decarboxylases, named OAD-1 and OAD-2, which both lack the extended proline ⁄ alanine linker in AB Fig. 7. Complex formation of c¢-2 with a-2-C mutants. c¢-2 was coex- pressed with a-2-C-mutants in E. coli. One of these proteins was subsequently purified via its specific tag and complex formation with the other one analysed by SDS ⁄ PAGE with 2 lg protein and silver staining. (A) The a-2-C-Y491F ⁄ c¢-2-complex was bound to monomer- ic avidin–Sepharose. The majority of c¢-2 was washed off the column, and the fraction eluted with biotin contained mainly a-2-C- Y491F (1). The wash fractions containing c¢-2 were incubated with wild-type a-2-C and subjected to Ni–NTA chromatography, resulting in the purification of a stable a-2-C ⁄ c¢-2-complex (2). (B) If a-2- Del120 ⁄ c¢-2 was expressed in E. coli, c¢-2 was degraded. To demon- strate the expression of c¢-2 wild-type a-2-C was coexpressed together with the other two proteins and the extract chromato- graphed by a Ni–NTA column. a-2-Del120 and wild-type a-2-C were found in the flow-through (FT) and wash (W) fractions and a stable a-2-C ⁄ c¢-2-complex was eluted with imidazole (lane E). M, Marker proteins. A B Fig. 6. Complex formation of c¢-2 point mutants with a-2-C. The proteins were coexpressed in E. coli and complex formation was analysed by SDS ⁄ PAGE following affinity chromatography on avidin– Sepharose. Two micrograms of protein were loaded on each lane and the gel was stained with silver. The c¢-2 subunit bands are shown. (A) The indicated conservative point mutants were loaded. (B) The indicated alanine mutants were loaded. wt, Wild-type protein. Association domain of oxaloacetate decarboxylase P. Dahinden et al. 850 FEBS Journal 272 (2005) 846–855 ª 2005 FEBS the a-subunit. We reasoned therefore that these enzymes might be more suitable for structural studies. Preliminary experiments with OAD-2 from V. cholerae indicated improved stability properties as compared to the OAD from K. pneumoniae, and we therefore deci- ded to perform further studies with this enzyme. Here, we have identified a domain of 40 amino acid residues within the C-terminal portion of 151 amino acids of the a-subunit which is responsible for the for- mation of a stable complex with the c-subunit and thus is essential for the assembly of the a ⁄ b ⁄ c-complex. This assembly domain is located just upstream of the puta- tive linker peptide that forms the connection to the bio- tin-binding domain (Fig. 5). The linker peptide of OAD-2 from V. cholerae contains three proline and five alanine residues within a stretch of 18 amino acid resi- dues (514–531) while the OAD from K. pneumoniae has seven proline and 15 alanine residues within the 27 amino acids forming the linker peptide (502–528). Upstream of the assembly domain of OAD-2 there is a stretch of 25 amino acid residues (455–479) containing five proline and five alanine residues which could serve as another flexible region within the protein. The cor- responding flexible region of the OAD from K. pneu- moniae comprises residues 450–476 and contains five proline and nine alanine residues. The central part of the association domain of the OAD from K. pneumo- niae (residues 489–509, Fig. 5) is reasonably well con- served. Interestingly, this part of the association domain contains all three residues which were shown by site-directed mutagenesis to contribute significantly to the stability of the complex. Two of the residues (Y491 and D509) are conserved in the K. pneumoniae OAD and T494 is exchanged by a glutamate. These results establish a three-domain-structure for the a-sub- unit consisting of the N-terminal carboxyltransferase domain and the C-terminal biotin-binding domain which are connected by the association domain sand- wiched by a flexible linker peptide on both sides. Astonishingly, the mutant a-2-C-D509A affected complex formation with c¢-2 only marginally although the mutant a-2-C-D509N had a major destabilizing impact on complex formation. As the mutation D509N is much more conservative than the mutation D509A the mutation D509A probably causes a conformational change in a-2-C resulting in a rearrangement of the binding surface which in turn allows another residue to take over the role of D509. Different acidic residues are not far from D509 which could alternatively take over its role. The mutation D509N on the other hand is very conservative and therefore supports the assumption that the negative charge of this residue is of importance for the complex formation with c-2. The dissociation and association of the OAD-com- plex from K. pneumoniae was shown previously to be pH dependent following titration curves with inflection points at pH 6.5 which suggested that a histidine plays an important role in the assembly of the enzyme [16]. According to this model, the enzyme could assemble with the crucial histidine in the neutral form and would dissociate if the histidine becomes protonated. In accordance with this hypothesis it was found by mutagenesis of the OAD from K. pneumoniae that H78 of the c-subunit plays a crucial role in the formation of the a ⁄ c-complex. H78 is part of a cluster of four histidine residues near the C terminus of the c-subunit of which H77 and H82 together with D62 were shown to be ligands for Zn 2+ binding [8]. A similar histidine cluster also exists at the C terminus of the c-subunit of the OAD-2 from V. cholerae, suggesting that two of the histidines are Zn 2+ ligands, whereas one may be involved in complex formation (Fig. 3). This role of a histidine is compatible with the observation that the a-2 ⁄ c¢-2-complex is stable at neutral pH but dissociates at pH < 5.0. By mutagenesis of H81 of c¢-2 to alanine but not by mutagenesis of the other histidines of the cluster, the stability of the a-2 ⁄ c¢-2-complex was signi- ficantly affected, indicating that H81 probably is involved in the interaction between a and c. We would like to emphasize that the results presen- ted here not only provide new structural information but may also reveal a dynamic aspect of the enzyme’s function. It is now clear that the a-subunit binds the c-subunit with a distinct association domain which is flanked on both sides with proline- and alanine-rich linker peptides. These linker peptides may allow hinge movements of the association domain against the carb- oxyltransferase and the biotin domain. The dynamics of conformational motions within the catalytic cycle of the enzyme probably also includes the motion of the entire soluble part of the enzyme against the mem- brane anchor of subunits c and b because a pro- line ⁄ alanine linker peptide also connects the membrane segment and the soluble domain of the c-subunit [7]. A concerted action of these hinge movements may be required to move the prosthetic biotin or carboxybio- tin group back and forth between the carboxyltrans- ferase and decarboxylase catalytic sites on subunits a and b, respectively, as the enzyme operates. Experimental procedures Strains and growth conditions For general cloning purposes E. coli DH5a (Bethesda Research Laboratories, Gaithersburg, MD, USA) 1 was used. P. Dahinden et al. Association domain of oxaloacetate decarboxylase FEBS Journal 272 (2005) 846–855 ª 2005 FEBS 851 For overexpression of protein the strain E. coli RNE41(DE3) (a gift from B Miroux 2 , CNRS-CEREMOD, Meudon, France) was used. Strains were routinely grown at 37 °C and 180 r.p.m. in baffled Erlenmeyer flasks containing 200 mL to 2 L Luria–Bertani medium containing 10 gÆL )1 NaCl. The medium was inoculated with 1% of an overnight culture and incubated at 37 °C and 180 r.p.m. At an attenuance 3 at 600 nm (D 600 )of 0.7 the cultures were cooled on ice, and expression was induced by the addition of isopropyl thio-b-d-galactside (100 lm). The cells were harvested after incubation for additional 3–4 h at 30 °C and 180 r.p.m. Recombinant DNA techniques and sequencing Genomic DNA was prepared by the CTAB method according to Ausubel et al. [17]. Extraction of plasmid DNA, restriction enzyme digestions, DNA ligations, and transformation of E. coli with plasmids were carried out by standard methods [17,18]. PCRs were performed with an air thermo-cycler (Idaho Technology 4 , Salt Lake City, UT; model 1605) using Pfu polymerase. Oligonucleotides used for mutagenesis were custom-synthesized by Micro- synth (Balgach, Switzerland). All inserts derived from PCR as well as ligation sites were checked by DNA sequencing according to the dideoxynucleotide chain- termination method [19] by Microsynth. In the case of site-directed mutagenesis by PCR whole plasmids were amplified, but only the sequence of the genes to be over- expressed was verified by sequencing. Construction of expression plasmids The primers used for site-directed mutagenesis are listed in the Supplementary material. Genomic DNA prepared from V. cholerae O395-N1 [20] served as template for the amplifi- cation of the oad-1 and oad-2 genes by PCR [21]. For the expression of oadA-2 with an N- or C-terminal His tag oadA-2 was amplified from pET24-VcoadGAB-2 harbour- ing the oad-2 genes, with the oligonucleotide primers Vco- adA2_for and VcoadA2_rev containing an NdeIoranXhoI site, respectively. The PCR product was ligated directly with pKS vector restricted with EcoRV. Positive clones selected by blue ⁄ white screening were restricted with NdeI and XhoI, and the obtained oadA-2 fragment was ligated with accordingly restricted pET16b or pET24b vector to give pET16-VcoadA-2 or pET24-VcoadA-2, respectively (Fig. 2A). The N-terminal carboxyltransferase domain and the C-terminal biotin-binding domain of oadA-2 were amplified from pET24-VcoadGAB-2 with the oligonucleotide primers VcoadA2-NT_NdeI and VcoadA2-NT_XhoI or VcoadA2- CT_NdeI and VcoadA2-CT_Xho I, respectively. The accord- ingly restricted PCR products were ligated with vector pET16b restricted with the same enzymes to give pET16- VcoadA-2-N or pET16-VcoadA-2-C, respectively, for the expression of the two VcOadA-2 domains with an N-ter- minal His tag (Fig. 2B). To examine complex formation between c-2 and a-2 a construct was made for the coexpression of the C-terminal, cytosolic domain of c-2 (c¢-2, C-terminal 59 amino acids) with a-2 or C-terminal deletion mutants of a-2. PCR prod- ucts comprising the appropriate DNA fragments were obtained by using the oligonucleotide primers summarized in the Supplementary material. The oligonucleotide primers VcoadG(CT)A2_for and VcoadG(CT)A2_rev were used to amplify an oadG¢A-2 fragment from the vector pET24-Vco- adGAB-2. This fragment and the vector pET16b were restricted with NdeI and XhoI and ligated to give the vector pET16-VcoadG¢A-2. From this clone oadG¢A-2-DelNN fragments were amplified with the primer Vco- adG(CT)A2_for and one of the primers with the suffix ‘_DelNN’, where ‘NN’ is substituted with the number of amino acids missing in the corresponding gene product. The obtained fragments and vector pET16b were restricted with NdeI and XhoI and ligated to give the plasmids pET16-VcoadG¢A-2-DelNN (Fig. 2C). To get deletion mutants from the N-terminal end of the C-terminal part of a-2, a construct for the coexpres- sion of c¢-2 and the 151 C-terminal amino acids of a-2 was made. The oligonucleotide primers used to generate appropriate DNA fragments are summarized in the Sup- plementary material. In a first PCR run the primers VcOG¢_to_A-CT_fo and VcOG¢_to_A-CT_mre containing an XagIoraSalI site, respectively, were used to amplify the oadG¢ fragment and the oligonucleotide primers VcOG¢_to_A-CT_mfo and VcOG¢_to_A-CT_re containing a SalIoraHindIII site, respectively, were used to amplify the oadA-2-C fragment. The two PCR products of the first run were used as templates in a second PCR together with the oligonucleotide primers VcOG¢_to_ A-CT_fo and VcOG¢_to_A-CT_re to give the fragment oadG¢A-2-C, which was restricted with XagI and HindIII and ligated with pET16-VcoadG¢A-2 restricted with the same enzymes to give the vector pET16-VcoadG¢A-2-C. From this clone oadG¢A-2-C-DelNN fragments were amplified with the oligonucleotide primer VcOG¢-A-2- CT_re containing one Pvu I site and one of the primers with the suffix ‘_DelNN’, where ‘NN’ is substituted with the number of amino acids missing in the corresponding gene product. The fragments were restricted with SalI and PvuI and ligated with the plasmid pET16-VcoadG¢A- 2-C restricted with the same enzymes to give the plasmids pET16-VcoadG¢A-2-C-DelNN (Fig. 2D). To examine the competition of a-2-C-D120 with a-2 or a-2-C for binding to c¢-2 the oadA-2 and oadA-2-C genes were cloned into the vector pET124b. The vector was con- structed with the p15A instead of the ColE1 origin of repli- cation [22] and was therefore compatible with the vectors pET16b and pET24b. The oadA-2 gene was amplified from pET24-VcoadGAB-2 with the oligonucleotide primers Association domain of oxaloacetate decarboxylase P. Dahinden et al. 852 FEBS Journal 272 (2005) 846–855 ª 2005 FEBS VcoadA-2_for and VcoadG(CT)A2_rev. The PCR product was restricted with NdeI and XhoI and ligated into pET124b restricted with the same enzymes to give pET124- VcoadA-2 for the expression of OadA-2 without tag. To get pET124-VcoadA-2-C for the expression of a-2-C with- out tag the vector pET16-VcoadA-2-C was restricted with NdeI and XhoI and the VcoadA-2-C fragment was ligated with pET124b restricted with the same enzymes. Site directed mutagenesis by PCR Site directed mutagenesis was performed essentially as described by Fisher and Pei [23]. Each reaction contained in 50 lL 20 pmol of one of the complementary oligonu- cleotide primer pairs summarized in the Supplementary material and 30 ng of the plasmid pET16-VcoadG¢A-2-C as template. DNA was amplified by 12 cycles 15 s at 95 °C, 15 s at 56 °C, and 13 min 30 s at 68 °C with Pfu polymerase. Before the first cycle the DNA was dena- tured for 2 min at 95 °C, and after the last cycle the samples were incubated for an additional 8 min at 68 °C. After cooling to 4 °C the PCR products were treated with DpnI for 1 h at 37 °C to cut the parental DNA strand by adding the enzyme directly to the PCR mix- ture. After heat inactivation for 10 min at 65 °C5lLof the digested PCR samples were used to transform E. coli DH5a. Preparation of cytosolic fraction and membranes For the preparation of cell extracts, cells obtained from expression cultures were resuspended in 7 mLÆg )1 of cells (wet weight) of a suitable buffer. After addition of 0.2 mm diisopropylfluorophosphate (final concentration) and approximately 50 lg DNase I, the cells were disrup- ted by three passages through a French pressure cell at 110 MPa. Intact cells and cell debris were removed by centrifugation (30 min at 8000 g), and the cell-free super- natant was subjected to ultracentrifugation (1 h at 200 000 g) to separate the cytosolic fraction and the membrane fraction. Purification VcOadA-2, VcOadA-2-C, and VcOadG¢A-2 and its derivatives by monomeric avidin-Sepharose affinity chromatography The plasmids containing the corresponding genes were transferred into and expressed in E. coli RNE41(DE3). The cells obtained from expression cultures were resuspended in buffer A (50 mm Tris ⁄ HCl pH 8.0, 250 mm NaCl) contain- ing 1 mm MgK 2 EDTA. The cytosolic fraction was pre- pared as described above and applied to a monomeric avidin–Sepharose column, which was washed with 7 bed volumes of buffer A. Biotinylated protein was finally eluted with 1 bed volume of buffer A containing 5 mm (+)-d- biotin. Purification of VcOadG¢A-2 and its derivatives by Ni 2+ –NTA chromatography The plasmids containing the corresponding genes were transferred into and expressed in E. coli RNE41(DE3). The cells obtained from expression cultures were resuspended in HisBind buffer (20 mm Tris ⁄ HCl pH 8.0, 500 mm NaCl) containing 10 mm imidazole, the cytosolic fraction prepared as described above and then applied to a Ni 2+ –NTA- agarose column (2 mL bed volume, Qiagen AG, Basel, Switzerland) 6 , pre-equilibrated with the same buffer. The column was washed with 10 bed volumes HisBind buffer containing 20 mm imidazole and with 8 bed volumes HisBind buffer containing 25 mm imidazole. Finally, the bound protein was eluted with 4 bed volumes of HisBind buffer containing 150 mm imidazole. Dissociation of VcOadG¢A-2 complex The plasmid pET16b-VcOadG¢A-2 was transferred into and expressed in E. coli RNE41(DE3). The a-2 ⁄ c¢-2 complex expressed by these cells was purified by Ni–NTA chroma- tography. The obtained elution fraction was applied to an avidin–Sepharose column which was subsequently washed with two bed volumes citrate buffer pH 6.0 (20 mm Na- citrate, 250 mm NaCl), 2 bed volumes citrate buffer pH 5.0, two bed volumes citrate buffer pH 4.0 and two bed vol- umes Tris buffer pH 8.0 (50 mm Tris ⁄ HCl pH 8.0, 250 mm NaCl). The wash fractions with citrate buffer pH 5.0 and 4.0 and Tris buffer pH 8.0 each were collected in two bed volumes of 250 mm Tris ⁄ HCl pH 8.0, 250 mm NaCl. a-2 was eluted with two bed volumes Tris buffer containing 5mm (+)-d-biotin. The neutralized wash fractions and the elution fraction were combined and incubated overnight at 4 °C. To prevent unspecific binding NaCl and imidazole were added to a final concentration of 500 mm and 10 mm, respectively, before applying the sample to a Ni–NTA– agarose column (2 mL bed volume, Qiagen), pre-equili- brated with buffer A. The column was washed with four bed volumes HisBind buffer (20 mm Tris ⁄ HCl pH 8.0, 500 mm NaCl) containing 20 mm imidazole and with four bed volumes HisBind buffer containing 25 mm imidazole. Finally, the bound protein was eluted with four bed vol- umes of HisBind buffer containing 150 mm imidazole. Protein detection methods Protein concentration was determined by the BCA method (Pierce, Lausanne, Switzerland) 7 using BSA as standard. SDS ⁄ PAGE was performed as described 8 [24]. Gels were stained with Coomassie Brilliant Blue R 250 or with silver [25]. P. Dahinden et al. Association domain of oxaloacetate decarboxylase FEBS Journal 272 (2005) 846–855 ª 2005 FEBS 853 Secondary structure prediction Two different programs were used for the prediction of sec- ondary structure elements such as flexible regions: psipred [26,27] and psa [28,29]. Acknowledgements This work was supported by Swiss National Science Foundation. We thank Dr Miroux for the gift of the strain E. coli RNE41(DE3). 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Primers used for construction of the expres- sion plasmids and for mutagenesis of c and a and figure 12 depicting the constructs obtained. Fig. S1. Construction of expression vectors to examine complex formation between c-2 and a-2. P. Dahinden et al. Association domain of oxaloacetate decarboxylase FEBS Journal 272 (2005) 846–855 ª 2005 FEBS 855 . Identification of a domain in the a- subunit of the oxaloacetate decarboxylase Na + pump that accomplishes complex formation with the c-subunit Pius Dahinden,. of point mutations in the binding domain of a- 2-C on the binding to c¢-2 To elucidate whether single amino acids in the binding domain of a- 2-C were particularly