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

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

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 decarboxydecarboxy-lase, 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-subc-sub-unit 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 choleraecontains 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 choleraeare 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’

oad-2genes 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 Zn2+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

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 seralan-ine 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 Zn2+-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 segseg-ment (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).

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 His10 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 His10tag The competence of a-2 dele-tion mutants for complex formadele-tion 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.

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

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.

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

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.

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 Zn2+ 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 Zn2+ 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)

For overexpression of protein the strain E coli RNE41(DE3)

(a gift from B Miroux

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

600 nm (D600) 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

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 NdeI or an XhoI

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

NT_NdeI and NT_XhoI or

VcoadA2-CT_NdeI and VcoadA2-CT_XhoI, 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 XagI or a SalI 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 SalI or a HindIII 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 PvuI 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

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
C 5 lL of

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 MgK2EDTA 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 Ni2+–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 Ni2+ –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

5 mm (+)-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)

SDS⁄ PAGE was performed as described

stained with Coomassie Brilliant Blue R 250 or with silver [25]

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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Supplementary material

The following material is available from http://www blackwellpublishing.com/products/journals/suppmat/ EJB/EJB4524/EJB4524sm.htm

Table S1 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