©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Binding of the Pyruvate Dehydrogenase Kinase to Recombinant Constructs Containing the Inner Lipoyl Domain of the Dihydrolipoyl Acetyltransferase Component (*)

(Received for publication, July 28, 1994; and in revised form, October 19, 1994)

Shengjiang Liu (§) Jason C. Baker Thomas E. Roche (¶)

From the Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The dihydrolipoyl acetyltransferase (E2) component of the mammalian pyruvate dehydrogenase complex forms a 60-subunit core in which E2's inner domain forms a dodecahedron shaped structure surrounded by its globular outer domains that are connected to each other and the inner domain by 2-3-kDa mobile hinge regions. Two of the outer domains are 10 kDa lipoyl domains, an NH(2)-terminal one, E2, and, after the first hinge region a second one, E2. The pyruvate dehydrogenase kinase binds tightly to the lipoyl domain region of the oligomeric E2 core and phosphorylates and inactivates the pyruvate dehydrogenase (E1) component. We wished to determine whether lipoyl domain constructs prepared by recombinant techniques from a cDNA for human E2 could bind the bovine E1 kinase and, that being the case, to pursue which lipoyl domain the kinase binds. We also wished to gain insights into how a molecule of kinase tightly bound to the E2 core can rapidly phosphorylate 20-30 molecules of the pyruvate dehydrogenase (E1) component which are also bound to an outer domain of the E2 core. We prepared recombinant constructs consisting of the entire lipoyl domain region or the individual lipoyl domains with or without the intervening hinge region. Constructs were made and used both as free lipoyl domains and fused to glutathione S-transferase (GST). Using GSH-Sepharose to selectively bind GST constructs, tightly bound kinase was shown to rapidly transfer in a highly preferential way from intact E2 core to GST constructs containing the E2 domain rather than to ones containing only the E2 domain. GSTbulletE2-kinase complexes could be eluted from GSH-Sepharose with glutathione. Delipoylation of E2 by treatment with lipoamidase eliminated kinase binding supporting a direct role of the lipoyl prosthetic group in this association. Transfer to and selective binding of the kinase by E2 but not E2 was also demonstrated with free constructs using a sucrose gradient procedure to separate the large E2 core from the various lipoyl domain constructs. E2 but not E2 increased the activity of resolved kinase by up to 43%. We conclude that the kinase selectively binds to the inner lipoyl domain of E2 subunits and that this association involves its lipoyl prosthetic group. We further suggest that transfer of tightly bound kinase between E2 domains occurs by a direct interchange mechanism without formation of free kinase (model presented). Such interdomain movement would explain how a kinase molecule can rapidly phosphorylate a large complement of pyruvate dehydrogenase tetramers which are bound throughout the surface of the E2 oligomer.


INTRODUCTION

The mammalian pyruvate dehydrogenase complex (PDC) (^1)is a large assembly composed of six components with nine distinct subunits. Four of these components execute the overall reaction through a series of steps linked by cofactor-mediated active site coupling: E1, the pyruvate dehydrogenase component; E2, the dihydrolipoyl acetyltransferase component; E3, the dihydrolipoyl dehydrogenase component; and protein X, the E3 binding component(1, 2, 3) . Like E2 subunits, protein X contains a lipoyl domain, and its prosthetic group participates in the middle 3 of the 5 step reaction series catalyzed by PDC. Control of the PDC reaction is critically important for regulating cellular fuel utilization, and PDC activity is controlled primarily by a highly regulated phosphorylation-dephosphorylation cycle. This regulatory cycle is carried out by dedicated kinase and phosphatase components that operate to reduce and increase the activity of the complex through phosphorylating and dephosphorylating, respectively, the E1 component.

The E2 component has a central role both in the organization of the complex and in supporting the function of the kinase and the phosphatase(1, 4) . Mammalian PDCbulletE2 subunits are highly segmented structures with seven structural regions consisting of four globular domains connected by three extended linker or hinge regions (1, 2, 3) . Sixty of E2's COOH-terminal domain assemble to form a dodecahedron shaped inner core structure that is surrounded by the remaining three globular domains flexibly connected by the three hinge regions. Along with other components and considerations, the domain structure of E2 is modeled in Fig. 5and Fig. 7. At the NH(2)-terminal end of E2, there are two 10-kDa lipoyl domains, E2 and E2 connected by a 30 amino acid linker or hinge region (H1). In all alpha-keto acid dehydrogenase complexes, one or more lipoyl domain of the E2 component functions in ferrying the intermediate forms of their pendant lipoyl group between active sites. The mobile hinge regions allow a single lipoyl domain to visit not only different active sites but to be available to several equivalent active sites at least in the case of the E1 and E2 components(3) . The remaining globular domain, in the exterior region of the mammalian E2 oligomer, functions in the binding of the E1 component (5, 6) and is designated the E2(B) domain. This small (46 amino acid) domain is flanked on its NH(2)-terminal side by a relatively large, 36 amino acid, hinge region (H2), that connects it to the E2 domain and on its COOH-terminal side by a 20 amino acid hinge region that connects it and the entire flexible, exterior structure to the oligomer-forming inner domain of E2. The inner domain of E2 also catalyzes the acetyltransferase reaction.


Figure 5: Model of the domains of an E2 subunit binding an alphabeta unit of the E1 component by its B domain and a kinase subunit by its L2 domain. In the upper section the model depicts hinge connected L1 and L2 domains with attached lipoyl groups. A major portion of L2 and the inner of its lipoyl-lysine side chain are positioned under (dashed lines) and interacting with a kinase subunit (K). The beta subunit of E1 interacts with the B domain of E2 and the alpha subunit of E1 is positioned to serve as a substrate for the kinase subunit. To emphasize the oligomer forming role of the inner (I) domain of E2 a second I domain is included but without outer domains.




Figure 7: Model of movement of the kinase along the surface of the E2 core. The reaction steps show the reversible association of a kinase dimer (labeled K for PK1 or PK2) with one L2 domain (step 1, equilibrium constant K(1)) and then with a second L2-lipoyl domain (step 2, interaction constant, K(2)). ``Hand over hand'' movement of the kinase is achieved by repeated dissociation and association in the K(2) step only leading to movement by interchange of L2 domains binding the kinase. The model incorporates a conformational change in the kinase dimer with the binding of lipoyl domains that results in negative cooperativity (i.e. a rapid exchange by a weaker K(2) interaction while being held by a tighter K(1) interaction). Although negative cooperativity is included and favored, it is not absolutely required (cf. the text and Footnote 8 for the requirements). The domains of E2 are labeled: oligomer forming inner domain, I; E1 binding domain, B; and the inner, L2, and N-terminal lipoyl domain, L1. The model shows only five E2(I) domains out of sixty in a dodecahedron structure and E1 is not included for clarity but would be bound to the E2(B) domain as shown in Fig. 5but as an alpha(2)beta(2) structure interacting with two B domains.



In typical preparations of bovine PDC, 20-30 alpha(2)beta(2)E1 tetramers and as few as one molecule of kinase are bound to the E2 core. Though very tightly bound to E2 the kinase can efficiently inactivate the complex (k = 0.5 s). Indeed, the phosphorylation of free E1 by free kinase is severalfold slower than when these components are bound to the E2 core. We wish to identify the specific domain of E2 that functions in the binding of the kinase and to gain insights into how this organized state of the complex greatly enhances the activity of the E1a kinase. The dissociation of E1 from the E2 core is slow(7, 8) , and the kinase has an even tighter affinity for the E2 core(9) . In early work to address this question of how an E2-bound kinase molecule rapidly phosphorylates many E2-bound E1 tetramers, studies were conducted with very dilute complex which led to a major portion of the E1 but little kinase dissociating from the E2 core. It was found that the few bound E1 were rapidly phosphorylated (and PDC activity lost) and, moreover, that phosphorylation of free E1 tetramers occurred at a rate that corresponded to that estimated for the rate of association of free E1 with the E2 core(10) . Only with the knowledge of E2's structure and characterization of the location and nature of kinase binding to the E2 can the underlying mechanisms be evaluated.

The E1(a) kinase has been shown to bind to the lipoyl domain region of E2 in bovine PDC(6, 9, 11) . Selective removal of the lipoyl prosthetic group was shown to lead to dissociation of the kinase demonstrating an essential role of this cofactor in tight binding of the kinase(12) . Although the kinase binds very tightly to the oligomeric E2 core, it was found to efficiently transfer to a fragment of bovine E2 that contained both lipoyl domains(9) . We have suggested that transfer involves a direct interchange of the kinase (i.e. without any free kinase) between the exterior lipoyl domain regions of the E2 core(9) . Such movement at the surface of the assembled E2 core would explain how a kinase molecule is able to both bind tightly to E2 core and still phosphorylate 20-30 E1 tetramers that are also bound to the E2(B) domain at the surface of the E2 core.

The kinase is composed of two subunits. One has been identified as a catalytic subunit(13) , and we have designated it as K(c); the other subunit was considered to be a noncatalytic subunit(13, 14) and has been designated K(b) by this laboratory. (^2)The amino acid sequence of the catalytic subunit of the rat heart pyruvate dehydrogenase kinase was recently determined from its full-length cDNA(15) . An exciting conclusion was that the K(c) subunit is a relative of the bacterial histidine kinase family. Recently, Popov et al.(16) have cloned and expressed the second kinase subunit of rat heart PDC. They demonstrated that it has a distinct subunit but related sequence to the K(c) subunit and that it was catalytically active although with a lower specific activity than that of the K(c) subunit. The latter observation needs to be reconciled with not losing kinase activity upon cleaving this second subunit(11, 13) . Nevertheless, we will now change our designation of the kinase subunits and refer to K(c) as pyruvate dehydrogenase kinase 1 (PDK1) and to the other kinase subunit as pyruvate dehydrogenase kinase 2 (PDK2). In the case of complexes and subcomplexes containing both, we will reduce this to K1 and K2 for the sake of clarity, e.g.E2-X-K1K2 subcomplex. PDK2 is present in bovine preparations of bovine complex at variable levels and often decreases in resolution processes that first leave the kinase bound to E2 core and then involve removal of the kinase from the E2 core (e.g. 9, 17). The severalfold activation of the kinase occurs upon its binding to the assembled E2 core, and this only requires PDK1(17) . No known functions of the kinase are lost when PDK2 is proteolized (11, 13, 18) or at very low levels in kinase preparations.

Recently, we have prepared a variety of constructs of the lipoyl domain region of the human PDC-E2. (^3)These were expressed in Escherichia coli as fusion proteins attached to glutathione S-transferase (GST) and as free lipoyl domains selectively released from GST by thombin treatment. When expression was performed with lipoate supplementation, the lipoyl domains were fully lipoylated. We have made the individual domains E2 and E2 and those domains with the connecting H1 hinge region attached, E2 and E2, and also made the bilipoyl domain structure, E2. Several E2 subunits of bacterial PDCs contain two or three lipoyl domains; however, no roles have been described that are performed in a specific or even highly preferential manner by one of the lipoyl domains in an E2. We have established that the bovine E1 kinase binds to human lipoyl domains with the L2 domain serving as a highly preferential binding site. We have demonstrated efficient movement of the kinase between the oligomeric E2 core and lipoyl domain constructs containing the L2 domain. Additionally, we have characterized the effects of the individual and bilipoyl domain constructs on kinase activity.


EXPERIMENTAL PROCEDURES

Materials

The E2-X-K1K2 subcomplex and resolved E1 component were prepared from purified bovine kidney pyruvate dehydrogenase complex(19, 20) . (Protein X functions as the dihydrolipoyl dehydrogenase (E3) binding component and is also referred to as E3-BP but X is a clearer designation for presentation of a series of components in a subcomplex.) In some cases the resolved E1 was prepared essentially free of kinase and in other cases it contained up to 0.35 nmol min mg kinase activity. This activity was measured without E2 activation of kinase activity and indicates about 0.4% contamination by the kinase. The latter was used as a source of kinase that is completely free of E2 in some studies because its kinase activity was more stable than that of resolved and purified kinase. Resolved, homogeneous kinase was prepared from E2-X-K1K2 subcomplex by lipoamidase treatment followed by sucrose gradient separation and DEAE chomatography. (^4)Lipoamidase was prepared from Enterococcus faecalis by modification of the procedure of Suzuki and Reed(21) . (^5)The lipoyl domain constructs were prepared and characterized as described elsewhere.^3 With the amino acid sequence shown in parentheses, the following constructs were used in their GST-linked and free forms: E2(1-98), E2(1-128), E2(120-233), E2(98-233), and E2(1-233). The numbers in parentheses designate the portion of the amino acid sequence of mature human PDC-E2 that is expressed in these structures. GSHSepharose 4B (containing S-linked glutathione) was purchased from Pharmacia Biotech Inc. Human glutathione S-transferase was purchased from Sigma and [-P]ATP was purchased from DuPont NEN. The polyol block polymer Pluronic F68 was from BASF Corp. E. coli lipoyl protein ligase was a gift from John Guest, University of Sheffield.

Kinase Binding and Transfer to GST-Lipoyl Domain Constructs

Binding and transfer studies were conducted in 50 mM sodium phosphate, pH 7.4, 0.2 mM EDTA, 0.5 mM dithiotheitol, 0.1% Pluronic F68, 0.1% Triton X-100 (transfer buffer). Microcolumns were made in standard 200-µl polypropylene pipette tips by inserting and compressing 2.5 mm diameter pieces of polyethylene mesh. Equivalent aliquots of GSH-Sepharose were added as a slurry of 50% packed gel in 50 µl total volume of transfer buffer to the microcolumns followed by washing and removal of excess buffer with 5 µl of buffer restored on top of the column. Transfer of kinase from the E2-X-K1K2 subcomplex to GST-lipoyl domain constructs was evaluated by two procedures. In transfer procedure 1, GST-lipoyl domain or GST and the E2-X-K1K2 subcomplex were mixed for a specific time (typically 25 s) in 20 µl of transfer buffer at 30 °C. This mixture was then added and rapidly mixed with 25 µl of packed GSH-Sepharose at 23 °C using the needle of the microsyringe with which the mixture was added to do the mixing (completed in 5 s). After 30 s, 25 µl of the mixture in the column was passed though by applying mild pressure with a FASTEPPER pipette (Denville Scientific, Inc.) connected to the microcolumn. The column was washed twice with 25 µl of transfer buffer by a sequence of addition, rapid mixing, and immediate elution. The filtrates and wash fractions were pooled together (75 µl of filtrate) and placed on ice prior to determination of kinase activity, E2 activity, and GST activity. The GST construct bound to the GSH-Sepharose was eluted with three 25-µl washes of buffer containing 10 mM GSH. The activities of the kinase, E2, and GST were determined in the combined 75 µl of eluate. GST activity was determined by standard procedures (22) using 4-chloro-2,4-dinitrobenzene as substrate. Other assays are described below.

In Transfer Procedure 2, GST-lipoyl domain constructs were pre-loaded onto GSH-Sepharose columns. In some experiments this was preceded by a procedure to delipoylate a portion of the constructs. In that case, 750-pmol samples of GST constructs were incubated with or without 0.35 µg of lipoamidase for 8 h at 30 °C. Parallel studies demonstrated the lipoamidase treatment completely removed the capacity of the GST constructs to undergo reductive acetylation, but this could be restored, after phenylmethylsulfonyl fluoride inactivation of lipoamidase, by treatment with E. coli lipoyl protein ligase to reintroduce lipoyl groups (data not shown). To each tube, 30 µl of GSH-Sepharose pre-equilibrated in transfer buffer was added and incubated 10 min at 23 °C followed by quantitative transfer to a microcolumn and washing with 30 bed volumes of transfer buffer and removal all but 5 µl of excess buffer. Then 4 µg of E2-X-K1K2 subcomplex in 20 µl was added for the indicated times (typically 30 or 60 s) followed by elution steps and analyses as described above.

Kinase Transfer to Free Lipoyl Domains

Transfer of the kinase from E2-X-K1K2 subcomplex to free lipoyl domain constructs (i.e. free of GST) was evaluated using the sucrose gradient procedure as described by Ono et al.(9) to separate the subcomplex from the free lipoyl domains. The buffer for all steps was 50 mM potassium phosphate, pH 7.0, 2 mM dithiotheitol, 0.5 mM EDTA, and 1 mM MgCl(2). The sucrose step gradients were prepared in 5 times 20-mm ultraclear tubes (Beckman) placed in a custom designed Teflon holder at the bottom of buckets of a Sorvall HA629 swinging bucket rotor. The gradient steps consisted of 50 µl each of 7.5, 10, and 15% sucrose (w/v). A 50-µl sample, containing 3 nmol of free lipoyl domain construct, 25 µg of E2-X-K1K2 (366 pmol of E2 subunits, 12 pmol of kinase subunits), and 50 µg of bovine serum albumin, was incubated at 23 °C for 30 min and loaded onto the top of the gradient followed by centrifugation a 27,000 rpm, at 20 °C for 180 min. After centrifugation, a top fraction of 120 µl, a middle 40 µl, and bottom 40 µl were removed. The pellet was resuspended and dissolved in 40 µl of buffer. Fractions were analyzed for kinase and E2 activity and by SDS-PAGE (23) followed by silver staining(24) .

Enzyme Assays

Kinase activity was determined in 20 mM potassium phosphate, pH 7.1, 1 mM MgCl(2), 2 mM dithiotheitol, 0.1% Pluronic F68, and 0.1% Triton times 100 with other conditions as described previously (9, 24) . When the effects of lipoyl domain constructs on the activity of resolved or E2-bound kinase were evaluated, preincubation conditions were as described in the figure legends. Each assay contained 25-30 µg of E1 component and the indicated kinase source. In standard assays reaction mixtures were incubated for 2 min at 30 °C and then kinase activity initiated by the addition of [-P]ATP (100-300 cpm/pmol) to a final concentration of 0.1 mM. Reactions were terminated after 60 s by spotting 40 µl onto dry trichloroacetic acid containing Whatman 3MM paper disc. Discs were then washed as described previously (25) and [P]phosphate incorporation into E1 determined by liquid scintillation counting.

The transacetylase activity of E2 was measured by monitoring the increase in acetyl-dihydrolipoamide production by recording the increase in absorbance at 232 nm(9) . Reaction mixtures contained in 1-ml cuvette at 30 °C: 30 mM Tris-HCl, pH 7.5, 1.0 mM dihydrolipoamide, 2 mM acetylphosphate, 5 µM CoA, 50 µM cysteine, and 2 units of phosphotransacetylase. After the 232 absorbance became constant (by 10 s) due to conversion of CoA to acetyl-CoA by the phosphotransacetylase reaction, the E2 source was added and the increase in absorbance with time recorded.


RESULTS AND DISCUSSION

Capacity of Human Constructs to Bind Bovine Pyruvate Dehydrogenase Kinase

Initial testing was performed with the bilipoyl domain structure, E2(1-233) to determine whether, like the bilipoyl domain fragment, E2(L), derived from bovine E2 subunits, this structure could serve as an acceptor in the transfer of the kinase from the bovine E2-X-K1K2 subcomplex. We also wished to obtain an estimate of the concentration dependence for GST-E2 as an acceptor (using GST as a control) and to evaluate E2 recovery and GST removal in the pass-through filtrate. As shown in Table 1, the recovery of kinase activity did not vary significantly with different GST levels. However, when 197 or 295 pmol of GST-E2 was incubated with the subcomplex (43.9 pmol of E2 subunits), the kinase activity recovered with the subcomplex was reduced by 46 or 69%, respectively. Nearly all the GST activity was removed by the initial rapid passage through GSH-Sepharose which also allowed, within experimental error, full recovery of E2 activity. Thus, these results supported a rapid transfer of the kinase from the intact protein to the bilipoyl domain structure. Indeed, longer times of incubation of the subcomplex with the GST-E2 did not appreciably increase the loss of kinase activity from the subcomplex (below). The results also indicate that competitive transfer of about half the kinase activity from the intact E2 core to the human construct occurs at an even lower ratio of subunit of the GST-bilipoyl domain structure to core E2 subunit (5:1) than the ratio when the bovine E2(L) fragment was used as the acceptor (12:1(9) ). (A possible explanation of this difference, based on the dimeric nature of GST-constructs, is considered later.) The bovine fragment contains the full H2-hinge region following the E2 domain (i.e. after the COOH terminus of this domain in the E2 subunit structure). This suggests that the H2 hinge region does not contribute to kinase binding.



We evaluated whether the bilipoyl domain structure separated from GST was effective in binding the kinase. Increasing concentrations of homogeneous E2 (released from and free of GST) were incubated with the E2-X-K1K2 subcomplex and followed by each undergoing fractionation in a micro-sucrose gradient as described under ``Experimental Procedures.'' The subcomplex was recovered in a pellet and lower supernatant fraction whereas the E2 was recovered in the upper supernatant fractions. The portion of kinase activity found in the upper supernatant fractions increased from 9.3 to 28% as the molar ratio of the bilipoyl domain structure to E2 subunits was increased from 1.3 to 7.8. At the same time, there was a decrease in the kinase activity associated with the pellet fraction. Thus, transfer of the kinase to the free bilipoyl domain structure was supported but was somewhat less efficient than transfer to the GST-bilipoyl domain construct. These initial experiments did not distinguish whether one or both the lipoyl domains bound the kinase but did indicate the human construct was effective in binding the kinase.

Transfer of the Kinase to Various GST-Lipoyl Domain Constructs

The capacity of individual lipoyl domains to serve as an acceptor of kinase being donated by the E2-X-K1K2 subcomplex was evaluated with GST fusion protein forms of these domains. Each GST-linked domains (14.8 µM) was combined with subcomplex (2.2 µM in E2 subunits, 0.037 µM subcomplex). The 20 µl mixture was incubated for 25 s at 23 °C, added and mixed in a microcolumns and then eluted and washed as described under ``Experimental Procedures.'' In all cases, >90% of GST activity was captured on the GSH-Sepharose 4B matrix; >97% of the E2 activity passed through the microcolumn (filtrate fraction); and essentially all the kinase activity eluted with the subcomplex in a control in which GST was used in place of a GST construct. The GST constructs were then specifically eluted with 10 mM GSH. Fig. 1shows the kinase eluted with the subcomplex (cross-hatched bars) and with the various GST constructs (solid bars). There was both significantly less kinase activity associated with the subcomplex and markedly higher kinase activity associated with the GSH-eluted constructs, when the construct contained the inner lipoyl domain, E2. Thus, there appears to be preferential transfer of the kinase to E2, but these data do not eliminate weaker or slower binding to E2. Although the recovery of kinase activity cannot be fully evaluated without knowledge of the relative effect of the lipoyl domain construct on kinase activity (below), the recovery was nearly as high as in the case of the GST control in which the kinase remains bound to E2. Under the conditions of the kinase assays, the intact E2-X subcomplex increases kinase activity about 5-fold whereas the bovine E2(L) fragment gives about a 40% increase in kinase activity(9) . A very small portion of (<5%) GST or GST constructs were not eluted by the 75 µl of GSH wash. SDS-PAGE analysis of the GSH eluates showed a definite PDK1 band with GST-E2 and GST-E2 but failed to reveal any kinase in the GST-E2 eluate (Fig. 2). Although the kinase activity detected with GST-E2 was higher than with GST-E2, the silver stained pattern showed a higher level of the PDK1 band in the GST-E2 eluate than in GST-E2 eluate. This may be at least partially explained by effects of the E2 domain on kinase activity that still occur in the bilipoyl domain structure (below).


Figure 1: Kinase transfer from E2-X-K1K2 to GST-linked E2, E2, and E2. Three µg of E2-X-K1K2 was combined with 284 pmol GST-linked E2(1-98), E2(120-233), and E2(1-233) in a final volume of 20 µl. In accordance with the steps in Transfer Procedure 1 (see ``Experimental Procedures''), the components were combined and incubated for 25s, mixed with GSH-Sepharose (5 s), and after 30 s, a 75 µl filtrate fraction was collected, followed by 75 µl GSH-eluate. Each experiment was conducted in triplicate and kinase activities for all filtrates and GST eluates were measured in duplicate. The transacetylation activity and GST activity of all fractions were measured as described in Experimental Procedures to establish the distribution of E2 and GST described under ``Results.'' Bars show the range of observed values and bar heights the average values.




Figure 2: SDS-PAGE analysis of GSH eluates. Samples (5 µl) from 75 µl GSH eluates (Fig. 1) were mixed with 10 µl of SDS-sample buffer and separation conducted with a 10% polyacrylamide gel with bands when visualized by silver staining. Lane 1 contained 1.5 µg subcomplex and lanes 2-4 show the patterns for eluates of GST-linked E2(1-98), E2(120-233), and E2(1-233), respectively, and lane 5 for the GSH eluate of GST control. The labels on the right side identify the position of the GST-construct.



Further studies were done to evaluate kinase binding using constructs that contained the full H1 hinge region connecting E2 and E2 and an approach to test binding to GST constructs already bound to the gel matrix (transfer procedure 2). The GST constructs E2(1-128), E2(98-233), and E2(1-233) were preloaded in duplicate at equivalent, 750 pM levels, onto 25 µl of GSH-Sepharose. E2-X-K1K2 (4 µg in 25 µl) was added and mixed with the loaded gel matrix and incubated for 1 min, and then this volume was passed through the column, rapidly followed by two 25-µl washes as described under ``Experimental Procedures.'' There was no contamination of the initial pass-through fraction with GST activity using this procedure. The individual GST constructs were then eluted with GSH as described above. The kinase activity in the GSH eluates was greater than 33 pmol of P incorporated min for the 98-233 structure and the 1-233 structure but only 4.5 ± 3.13 for the 1-128 structure which was only slightly above the experimental error for the background. When GST was used, kinase activity was entirely found in the initial pass-through and none (0 ± 3.1) in GSH eluate. Thus, we found the same pattern of much greater transfer of the kinase specifically to E2 constructs but not to the construct containing only the E2 domain when these H1-containing structures were preloaded onto GSH-Sepharose. Similar results were found with these H1-containing constructs in the case of the nonlipoamidase (-LPA) samples in the transfer experiments shown in Fig. 4, below. We would note that the kinase cannot be observed by SDS-PAGE analysis of the GSH eluate when GST-E2 or GST-E2 were used because these polypeptides comigrated with kinase bands.


Figure 4: Transfer of the kinase from the E2-X-K1K2 subcomplex to lipoylated and delipoylated GST constructs. Following incubation with (+LPA) or without (-LPA) lipoamidase as described under ``Experimental Procedures,'' 750 pmol of the indicated GST constructs were bound to 25 µl GSH-Sepharose. Using Transfer Procedure 2, 4 µg E2-X-K1K2 was mixed for 60 s with the indicated gel-bound GST-construct (or GST) followed by collecting 75 µl of E2-containing filtrate and then 75 µl of GST-containing GSH eluate. Each experiment was performed in duplicate and the kinase activity of each fraction was measured in duplicate. More than 99.5% of the GST activity was removed from the initial (pass-through) filtrate and >95% of the E2 activity was present in that filtrate.



Binding to Free Lipoyl Domain Constructs

The sucrose gradient approach, used above to evaluate the concentration dependence for transfer of kinase from E2-X-K1K2 to various levels of the bilipoyl domain(1-233) structure, was employed to compare binding by all the lipoyl domain constructs prepared free of GST. This approach eliminates interference by GST which might be more pronounced in the case of E2 due to the lack of an intervening hinge region between GST and the lipoyl domain. To minimize loss of material, 50 µg of BSA was included in the mixtures. Fig. 3shows the silver-stained SDS-PAGE pattern for the distribution of the kinase, E2, X, and lipoyl domain constructs after gradient separation and fractionation into three supernatant fractions, S1, S2, and S3 from the top to the bottom of the gradient and a pellet fraction, P. BSA gives a major band just below the E2 band (cf. comments Fig. 3, legend). The kinase activity and the E2 activity of all fractions were also analyzed. Under the conditions used, most of the E2-X was in the S3 fraction with some in the P fraction. Virtually all of the free lipoyl domain constructs were in the S1 fraction and with a small amount in the S2 fraction. The gel electrophoresis patterns revealed the kinase in the S1 fraction of the 98-233, 120-233, and 1-233 constructs which contain the inner lipoyl domain but not in the S1 fractions of 1-98 and 1-128 constructs that exclusively contained the NH(2)-terminal lipoyl domain. Because this gel pattern did not separate the PDK1 and PDK2 subunits well, a second gel was run in which electrophoresis was performed for a longer time. This separated these kinase subunits and revealed the K1 and K2 subunits were present at a similar ratio (3-fold higher K1 than K2) in the E2-X (S3) fractions and in the S1 fractions containing E2 constructs (data not shown).


Figure 3: Transfer of the kinase from E2-X-K1K2 to GST-free lipoyl domain constructs. On top of each microsucrose gradient, 50 µl mixture containing 3 nmol (60 µM) of the indicated lipoyl domain construct [E2(1-98), E2(1-128), E2(98-233), E2(120-233), and E2(1-233)], 25 µg E2-X-K1K2 subcomplex, and 50 µg bovine serum albumin (BSA). Centrifugation was conducted at 27,000 rpm in Sorvall HA629 rotor for 180 min at 20 °C. From the top to the bottom of the gradient, the fractions collected (cf. ``Experimental Procedures'') are labeled S1 to S3 and each redissolved pellet fraction is labeled with a P. One-eighth the volume of each fraction was analyzed by SDS-PAGE. in gels containing 15% acrylamide and bands were visualized by silver staining. Each fraction was assayed for kinase activity and E2 activity. The S1 and S2 fractions contained BSA that migrated slightly below E2. The position of one major and a minor bands from BSA are best observed in S1 lane of 1-98 experiment since the control S1 lane was at the edge of the gel. A band for E3 does come between the two bands for BSA. A small portion of the 1-233 construct underwent degradation; the S1 lanes contain about 0.25 nmol of each construct, this is about 7 µg of the 1-233 construct and silver staining readily detects 0.05 µg.



Analysis of kinase activity in the various fractions of the sucrose gradients also supported binding to E2 and not to E2. However, in contrast to the gel electrophoresis pattern, higher kinase activity was found in E2(120-233) construct supernatant versus that of the E2(98-233) or E2(1-2-233) constructs. This discrepancy of higher activity but lower band density suggests the presence of the H1 hinge region may decrease the observed kinase activity, but further studies are needed to confirm this result. (Inhibition by E2, not present in 98-233 construct, was observed (below)). The kinase activities in all the S1 fractions of all the E2-containing constructs were stimulated about 2.5-fold when assayed in the presence of 10 µg of kinase-depleted E2-X subcomplex.

Rate of Transfer of the Kinase

The speed of movement of the kinase from the E2-X-K1K2 subcomplex to GST-E2 (or GST as a control) was evaluated by mixing 4 µg of subcomplex with GSH-Sepharose preloaded with a 7-fold higher level of construct (22.5 µg) than E2 subunits of the subcomplex. An equivalent amount of gel was distributed into columns from the same initial mixture of gel and construct. The subcomplex was added and 75 µl of filtrate collected at various times (10-300 s). The shortest time for completion of these steps was 30 s. Then a GSH eluate was collected. A near maximal gain of 38 pmol of P incorporated min in the GSH eluate for kinase activity was achieved when the subcomplex was eluted by 30 s with only a 10% additional increase in activity occurring after a 5-min mixing of the subcomplex with the preloaded gel. Thus, as was observed with the bovine E2(L) fragment(9) , transfer of the very tightly bound kinase to the recombinant lipoyl domain is very efficient. Considering the tight binding of the kinase to E2 core, we have argued (9) that transfer is occurring more rapidly than the kinase can dissociate from the E2 core. Procedures are under development for achieving a more rapid separation of subcomplex (or GST-free lipoyl domain constructs) from GST constructs to evaluated the rate of kinase transfer.

Lipoyl Prosthetic Group Requirement

Previous studies (12) indicated that the lipoyl prosthetic group contributes to binding by the kinase. GST constructs were delipoylated by treatment with E. faecalis lipoamidase (+LPA) or incubated in the absence of lipoamidase (-LPA) for 8 h at 30 °C. Lipoamidase-treated GST constructs completely lost the capacity to undergo E1-catalyzed reductive acetylation.^3Fig. 4shows the effects on kinase transfer from E2-X-K1K2 subcomplex to GSH-Sepharose-bound constructs after a 60-s incubation. Lipoamidase treatment greatly reduced or eliminated binding to the L2 domain-containing constructs. However, there was little kinase transfer to the GST-E2(1-128) and, within experimental error, no effect of lipoamidase treatment. Thus, the highly preferential binding of the kinase to the E2 domain requires the lipoyl prosthetic group.

We have suggested that the inner hydrophobic portion of the lipoyl-lysine prosthetic group of E2 directly contributes to binding of the kinase for two reasons(12) . First, unlike removal of lipoate, modification of the reactive dithiolane ring portion of the lipoate did not prevent kinase binding and secondly suggesting that this reactive portion of the prosthetic group was not an essential component of the kinase-binding site. Second, the resolved kinase has a tendency to stick to hydrophobic surfaces suggesting that it has an exposed site designed to interacts with a hydrophobic structure. We cannot eliminate the prospect that delipoylation causes a change in the structure of the L2 domain which prevents its binding of the kinase. This is not expected due to the role of the lipoyl-lysine as a swinging arm. Regardless of how removal of the lipoate prevents kinase binding, unique structural interactions of the kinase with novel features in the tertiary structure of the L2 domain seem required to explain binding by this domain but not by the L1 domain which has a highly related sequence (26, 27) with only the COOH-terminal regions showing substantial sequence and size variation.^3 This COOH-terminal portion of L1 and L2 extends beyond the region that can be aligned with the sequences of lipoyl domains of prokaryotic PDC-E2s or the E2s of other alpha-keto acid dehydrogenase complexes. We have suggested that this unique part of the L2 structure may have a specialized role in interacting with the kinase and/or phosphatase components of mammalian PDC.^3Fig. 5models the interaction of a kinase subunit specifically with the L2 domain of E2 and incorporates interaction with the inner part of the lipoyl-lysine prosthetic group as part of the binding site. The model also shows one alphabeta unit of an E1 tetramer binding via its beta subunit to the B domain of E2 (5) with its alpha subunit positioned to interact with the kinase.

Effects of Lipoyl Domain Constructs on Kinase Activity

Fig. 6shows the effects of free lipoyl domain constructs on resolved kinase activity. Consistent with the evidence above for specific binding of the kinase to the L2 domain, the E2(120-233) and the E2(98-233) constructs enhanced the activity of the resolved kinase. Although the effects were not large, the enhancement increased with the level of free lipoyl domains which were included at the indicated levels. In marked contrast, E2-constructs actually inhibited kinase activity with the inhibition increasing with the level of the domain. Further studies will be needed to determine the basis of this inhibition. The E2(1-233) construct gave a small enhancement of kinase activity at lower levels of the construct but this disappeared at the highest level. That pattern is consistent with competing effects of E2 and E2 in influencing kinase activity.


Figure 6: Effects of lipoyl domain constructs on the activity of resolved (E2 free) kinase. Kinase assays were constructed in 50 µl reaction mixtures with 28.5 µg E1 containing about 31 µunits kinase. (Units are for E2-activated kinase activity; 1 unit = 1 nmol P-phosphate incorporated min mg). The indicated GST-free lipoyl domain constructs were added at the listed concentrations for 60 s just prior to initiation of activity. Assays were conducted in duplicate with a reaction time of 60 s. Other conditions were as described under ``Experimental Procedures.''



Besides the possibility of inhibition by E2 being due to a specific interaction with the kinase, inhibition might result from interaction of the lipoyl domain with E1 making E1 a poorer substrate for the kinase. Both lipoyl domains are substrates of E1 in the second reaction step in the PDC reaction. However, steady state kinetic studies (^6)gave a K(m) of E1 for E2(1-98) of 69.4 µM and, even at the highest level, E2(1-98) was present at only 25 µM in the above study. Thus, it would not be expected that interaction of E2 with E1 caused the observed inhibition.

Final Considerations

As indicated in the Introduction, the kinase subunits appear to be relatives of bacterial histidine kinases (15, 16) . In bacteria these are sensor kinases that form histidine-phosphate by self-phosphorylation on the kinase and then transfer the phosphate usually to Asp residues on response regulatory proteins(28) . In the few cases in which these sensor kinases have been well characterized, they have been shown to be dimers (e.g. 29, 30). While the bovine kinase was suggested to have a structure of a heterodimer containing one PDK1 and one PDK2 subunit(13) , there is very limited evidence for that conclusion(19) . We have found that PDK1, alone, binds to the E2 oligomer and exhibits all known functions of the kinase(9, 17, 18) . We have recently obtained patterns in native gel electrophoresis that are consistent with the following dimer forms for two kinase subunits - [K1](2), [K1-K2], [K2](2). (^7)Such a dimer state would help explain the capacity of the kinase to move between lipoyl domains without dissociation since it makes the prospect likely that the kinase has two lipoyl domain binding sites. Thus, a dimer of kinase subunits could move at the surface of the E2 core by a ``hand over hand'' mechanism involving a continuous process of partial dissociation by one subunit followed by interchange to another lipoyl domain on the E2 core. Fig. 7models such a mechanism for kinase translocation across the surface of the E2 core. Movement proceeds by a repeated series of exchanges via the K(2) process only with different L2 domains in the same local region at the surface of the E2 core participating in that step. The key requirements are that movement is limited by the dissociation of the bi-lipoyl domain held kinase dimer and that the rate of intramolecular association to again have the kinase interact with two lipoyl domain must be much faster than the dissociation step in the K(1) process. Additionally the model includes the likely (but not absolutely essential) (^8)prospect that the association of L2 domains by the K(2) step is weaker than the association by the K(1) step, i.e. negative cooperativity is invoked. As presented, the model suggests symmetry between kinase subunits once two L2 are bound which would allow the K(2) step to occur with either kinase subunit.

The data in Fig. 6indicate the free E2 enhances kinase activity much less than the oligomeric E2 core (1.4 fold versus 3.5 fold by intact E2 core under these assay conditions). Moreover, a much higher concentration of this free lipoyl domain is required to maximally activate the kinase than would be expected if binding to this free domain to the kinase was as tight as to oligomeric E2(9) . The proportion of kinase transferred from E2 to GST-E2 in experiments shown in Fig. 1and Fig. 4is substantial as indicated by the relative bar heights of the hatched (passed though with E2) versus the solid bars (eluted with the GST-E2). The data suggest not only a high level of kinase is transferred but a higher recovery of activity than would be expected if GST-E2 gave the same enhancement of kinase activity as free E2. Thus, the high transfer and good recovery of kinase activity indicate that there is tight binding to the GST-E2 structures and suggest the possibility of a greater enhancement of kinase activity by GST-E2 constructs than free lipoyl domains. Since the GST is a dimer, (^9)one interesting reason that there might be tighter binding of the kinase is the potential capacity to achieve bifunctional binding by an intramolecular reaction of dimeric kinase molecules. This would be particularly true in the case of the GST-E2 and GST-E2 constructs in which the E2 domain is well separated by mobile connecting regions from the GST. Further studies are underway to establish the quaternary structure of the kinase, to estimate the rate of transfer of the kinase, and to evaluate the influence of changes in the oligomeric state of various E2-containing structures on kinase binding and activity enhancement.

Our data demonstrate the effectiveness of the human E2-lipoyl domain constructs that we have prepared in determining the specific domain and specific requirements for efficient accepting of kinase tightly bound to a large E2 oligomer. Our results demonstrate the great versatility in using GST-linked constructs as acceptors. Our results indicate that a combination of the lipoyl prosthetic group and a specific portion of the structure of E2 creates the kinase binding site. Some structure in E2 must distinguish it from E2 and it is unlikely that this simply involves the presentation of the lipoyl prosthetic group since these are attached to highly conserved regions. Both E2 and E2 are larger than the lipoyl domains from bacterial PDC-E2 or the E2 components of other alpha-keto acid dehydrogenase complexes. Additional structure is located at the COOH termini of the human E2 and E2 and these parts of the two lipoyl domains have the greatest differences between them in both sequence and size.^3 We suggest that the COOH-terminal end of E2 (residues 208-229) is the most likely region contributing to kinase binding. A weak interaction with E2 may occur since it inhibits kinase activity (Fig. 6) and the SDS-PAGE analyses suggested (in contrast to kinase assays) somewhat greater binding to the bilipoyl domain construct. The model in Fig. 5shows what we have established whereas the model in Fig. 7is both a reasonable and exciting explanation of our results and will be evaluated further.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK18320, by a grant from the Kansas Affiliate of the American Heart Association, and by Agriculture Experiment Station Contribution 95-6-J. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biochemistry, School of Medicine, Stanford University Medical Center, Stanford, CA 94305-5307.

To whom correspondence should be addressed.

(^1)
The abbreviations used are: PDC, pyruvate dehydrogenase complex; E1, pyruvate dehydrogenase component; E2, dihydrolipoyl acetyltransferase component; E2, NH(2)-lipoyl domain of E2; E2, interior lipoyl domain of E2; E2, hinge region connecting E2 and E2; E2(B), E1 binding domain; E2, hinge region connecting E2 to E2(B); PDK1 or just K1, major catalytic subunit, formerly designated by K(c); PDK2 or K2, minor catalytic subunit, formerly designated K(b); E3, dihydrolipoyl dehydrogenase; E3-BP or X, E3-binding protein or protein X; GST, glutathione S-transferase; GSH, glutathione; PAGE, polyacrylamide gel electrophoresis.

(^2)
This subunit was very difficult to detect following two-dimensional gel electrophoresis (isoelectric focussing followed by SDS-PAGE(21) . However, in a heavy overloading of resolved kinase, we detected a smeared band that was more basic than other components at the position this subunit moved in SDS-PAGE. This was the basis of the b designation. This subunit's sequence indicates it is not particularly basic(16) . The basis for this discrepancy remains to be determined.

(^3)
Liu, S., Baker, J. C., Andrews, P. C., and Roche, T. E.(1994) Arch. Biochem. Biophys., in press.

(^4)
J. C. Baker, K. Ono, and T. E. Roche, manuscript in preparation.

(^5)
S. Ravindran, and T. E. Roche, manuscript in preparation.

(^6)
S. Liu, and T. E. Roche, manuscript in preparation.

(^7)
J. C. Baker, and T. E. Roche, unpublished observation.

(^8)
Considering the estimated concentration of lipoyl domains at the surface of the E2 core of geq 1 mM, it seems unlikely that the k step would be less than 10^4 s = 1 mM effective concentration times k(a) for the tethered L2 domain of at least 10^7M s. (Note K(2) = k/k is unitless.) The rapid transfer to GST-E2 structures ( Fig. 1and Fig. 4) or to two free E2 also only need involve a series of K(2) steps (no K(1) step). But the initial association of mono-held kinase with GST-L2 structure (or for two L2 in the case of free E2) involves a k step with typical M s units for the formation of E2-kinase-L2-GST complex. An analysis of limiting conditions that consider the overall affinity of the kinase with E2 core (K(d) leq 3 times 10M,(9) ), the minimal requirements for how fast the kinase moves from E2 core to GST-lipoyl domain structures, and requirements for intramolecular movement to allow phosphorylation of 30 E1/ min within the complex, leaves some range available for the various rate constants but at the same time makes specific predictions. The high affinity of the kinase can be achieved with tight K(1) binding (near 10M) and very weak K(2) binding or weaker K(1) binding (near 10M) and a stronger K(2) interaction. Using a definition that negative cooperativity is involved if k > k (or when free L2 domains are involved that K(2) > K(1) when expressed as dissociation constants), acceptable solutions do not necessarily involve negative cooperativity, as modeled in Fig. 7, only when feasible K(1) and K(2) constants predict <1% of the bound kinase is in the mono-held form. An interesting feature under those conditions is that k values approach 1s which would make intracore movement of the kinase near-rate-limiting for kinase activity. We do not think that is likely based on our observations to date.

(^9)
Constructs are linked to the COOH terimini of dimeric GST derived from Schistosoma japonicum; the three-dimensional structure of the related pig lung GST has been determined(31) . The latter has COOH termini in opposite corners but on the same side in its dimeric structure.


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