Binding of the Pyruvate Dehydrogenase Kinase to Recombinant Constructs Containing the Inner Lipoyl Domain of the Dihydrolipoyl Acetyltransferase Component

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 NH2-terminal one, E2L1 , and, after the first hinge region a second one, E2L2 . 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 E2L2 domain rather than to ones containing only the E2L1 domain. GST•E2L2 -kinase complexes could be eluted from GSH-Sepharose with glutathione. Delipoylation of E2L2 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 E2L2 but not E2L1 was also demonstrated with free constructs using a sucrose gradient procedure to separate the large E2 core from the various lipoyl domain constructs. E2L2 but not E2L1 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 E2L2 domains occurs by a direct interchange mechanism without of free kinase how a kinase molecule can rapidly phosphorylate a large of pyruvate dehydrogenase which are bound the of the E2 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 NH2-terminal one, E2L1 , and, after the first hinge region a second one, E2L2 . 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 E2L2 domain rather than to ones containing only the E2L1 domain. GST•E2L2 -kinase complexes could be eluted from GSH-Sepharose with glutathione. Delipoylation of E2L2 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 E2L2 but not E2L1 was also demonstrated with free constructs using a sucrose gradient procedure to separate the large E2 core from the various lipoyl domain constructs. E2L2 but not E2L1 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 E2L2 domains occurs by a direct interchange mechanism without of free kinase how a kinase molecule can rapidly phosphorylate a large of pyruvate dehydrogenase which are bound the of the E2 mammalian pyruvate dehydrogenase complex used dehydrogenase dehydrogenase acetyltransferase domain of lipoyl domain of region E2L1 and binding region E2L2 to or by or or or a large of with of the a of by the pyruvate dehydrogenase the dihydrolipoyl acetyltransferase the dihydrolipoyl dehydrogenase and the binding E2 a lipoyl and its prosthetic group in the of the by of the for and activity by a highly by kinase and that to and the activity of the complex and the E1 E2 component a role both in the of the complex and in supporting the of the kinase and the subunits are highly with consisting of globular domains connected by or hinge of E2′s domain to a dodecahedron shaped inner core structure that surrounded by the globular domains connected by the hinge regions. with other and the domain structure of E2 in the NH2-terminal of are ~ lipoyl domains, E2L1 and E2L2 connected by a ~ or hinge region dehydrogenase or lipoyl domain of the E2 component in the forms of lipoyl group between The mobile hinge a lipoyl domain to not only but to be to in the of the E1 and E2 The globular in the region of the mammalian E2 in the binding of the E1 component and the domain. ~ domain its NH2-terminal by a ~ hinge region that to the E2L2 domain and its by a ~ hinge region that and the entire structure to the inner domain of The inner domain of E2 also the acetyltransferase of the domains of an E2 binding an of the E1 component by its domain and a kinase by its domain. the the hinge connected and domains with lipoyl of and the inner of its are and with a kinase The of E1 with the domain of E2 and the of E1 to as a for the kinase the role of the inner domain of E2 a second domain but without outer of bovine 20-30 and as as molecule of kinase are bound to the core. tightly bound to E2 the kinase can the complex the of free E1 by free kinase than are bound to the E2 core. We to the domain of E2 that in the binding of the kinase and to gain insights into how this of the complex the activity of the The of E1 from the E2 core and the kinase an for the E2 to this of how an kinase molecule rapidly phosphorylates E1 were with complex which to a of the E1 but kinase from the E2 core. was that the bound E1 were rapidly activity and, that of free E1 a that to that for the of association of free E1 with the E2 with the of E2′s structure and of the and of kinase binding to the E2 can the be kinase shown to bind to the lipoyl domain region of E2 in bovine of the lipoyl prosthetic group was shown to to of the kinase an role of this in binding of the the kinase binds tightly to the oligomeric E2 was to transfer to a of bovine E2 that both lipoyl We that transfer involves a direct interchange of the kinase without free between the lipoyl domain of the E2 the of the E2 core how a kinase molecule to both bind tightly to E2 core and phosphorylate 20-30 E1 that are also bound to the domain the of the E2 kinase of as a and as the other was to be a and by this was to by in a of resolved a that was than other the this in was the of the not The for this to be The of the of the pyruvate dehydrogenase kinase was from its was that the a of the kinase and the second kinase of demonstrated that a but to the and that was with a activity than that of the The to be with not kinase activity this second of the kinase subunits and to as pyruvate dehydrogenase kinase and to the other kinase as pyruvate dehydrogenase kinase the of complexes and containing this to and for the of in bovine of bovine complex and in that first the kinase bound to E2 core and of the kinase from the E2 core The of the kinase occurs its binding to the E2 and this only of the kinase are or in kinase prepared a of constructs of the lipoyl domain region of the human and in were in as to glutathione S-transferase and as free lipoyl domains selectively from GST by was with the lipoyl domains were We made the individual domains E2L1 and E2L2 and domains with the hinge region and , and also made the domain . E2 subunits of or lipoyl that are in a or highly preferential by of the lipoyl domains in an We that the bovine E1 kinase binds to human lipoyl domains with the domain as a highly preferential binding We demonstrated of the kinase between the oligomeric E2 core and lipoyl domain constructs containing the domain. the of the individual and domain constructs kinase and resolved E1 component were prepared from bovine pyruvate dehydrogenase as the dihydrolipoyl dehydrogenase binding component and also to as but a for of a of in a the resolved E1 was prepared free of kinase and in other up to kinase activity was without E2 of kinase activity and by the The was used as a of kinase that free of E2 in its kinase activity was than that of resolved and kinase was prepared from by lipoamidase treatment by sucrose gradient and and in was prepared from by of the procedure of and and in The lipoyl domain constructs were prepared and as the shown in the constructs were used in and free E2L1 E2L2 and The in the of the of human that in was from glutathione S-transferase was from and was from The was from lipoyl was a from of and Transfer to and transfer were in were made in by and ~ of of GSH-Sepharose were as a of in of transfer to the by and of with of of the Transfer of kinase from the to domain constructs was by transfer procedure domain or GST and the were for a in of transfer was and rapidly with of GSH-Sepharose using the of the with which the was to the in of the in the was by with a connected to the The was with of transfer by a of and The and were of and to of kinase E2 and GST The GST bound to the GSH-Sepharose was eluted with of containing 10 The of the and GST were in the of GST activity was by using as are Transfer domain constructs were GSH-Sepharose this was by a procedure to a of the constructs. that case, of GST constructs were with or without of lipoamidase for demonstrated the lipoamidase treatment the of the GST constructs to but this could be after of by treatment with lipoyl to lipoyl not each of GSH-Sepharose in transfer was and 10 by transfer to a and with of transfer and but of of in was for the or by and as Transfer to of the kinase from to free lipoyl domain constructs free of was using the sucrose gradient procedure as by to separate the from the free lipoyl The for was and The sucrose were prepared in in a the of of a The gradient of each of and sucrose containing of free lipoyl domain of of E2 ~ of kinase and of bovine was for and the of the gradient by a for a of a and were The was and in of were for kinase and E2 activity and by by activity was in and with other as the of lipoyl domain constructs the activity of resolved or kinase were were as in the of E1 component and the kinase were for and kinase activity by the of to a of were after by containing were as and into E1 by activity of E2 was by the in by the in in and of the 10 to of to by the the E2 was and the in with of Constructs to was with the domain to determine the domain from bovine E2 this structure could as an in the transfer of the kinase from the bovine We also wished to an of the for as an GST as a and to E2 and GST in the shown in the of kinase activity not with GST or of was with the of E2 the kinase activity with the was by or the GST activity was by the GSH-Sepharose which also of E2 a transfer of the kinase from the intact to the domain of of the with the not the of kinase activity from the The also that transfer of the kinase activity from the intact E2 core to the human occurs an of of the domain structure to core E2 ~ than the the bovine was used as the ~ of this the of The bovine the region the E2L2 domain after the of this domain in the E2 that the hinge region not to kinase whether the domain structure from GST was in binding the of from and free of were with the and by each in a gradient as The was in a and the was in the The of kinase activity in the increased from to as the of the domain structure to E2 subunits was increased from to the was a in the kinase activity with the transfer of the kinase to the free domain structure was but was than transfer to the domain not whether or both the lipoyl domains bound the kinase but the human was in binding the of the to of individual lipoyl domains to as an of kinase being by the was with GST forms of domains was with in E2 The was for and in a and eluted and as of GST activity was the GSH-Sepharose of the E2 activity the and the kinase activity eluted with the in a in which GST was used in of a GST The GST constructs were eluted with 10 the kinase eluted with the and with the various GST constructs was both kinase activity with the and kinase activity with the constructs, the the inner lipoyl E2L2 . to be preferential transfer of the kinase to E2L2 , but not or binding to E2L1 . the of kinase activity be without of the of the lipoyl domain kinase activity the was as as in the of the GST in which the kinase bound to the of the kinase the intact kinase activity the bovine a in kinase of GST or GST constructs were not eluted by the of of the a with and but to kinase in the the kinase activity with was than with , the a of the in the than in be by of the E2L1 domain kinase activity that in the domain structure transfer from to E2L1 , E2L2 , and . of was with E2L1 E2L2 and in a of with the in Transfer the were and for with GSH-Sepharose and after a was by was in and kinase for and GST were in The activity and GST activity of were as in to the of E2 and GST the of and the of from were with 10 of and with a with by and the for of E2L1 E2L2 and and for the of GST The the the of the were to kinase binding using constructs that the hinge region E2L1 and E2L2 and an to binding to GST constructs bound to the procedure The GST constructs and were in of in was and with the and for and this was the rapidly by as was of the with GST activity using this The individual GST constructs were eluted with as The kinase activity in the was than of for the structure and the structure but only for the structure which was only the for the GST was kinase activity was in the and in the of transfer of the kinase to E2L2 constructs but not to the containing only the E2L1 domain were were with constructs in the of the in the transfer shown in We that the kinase be by of the or were used with kinase of the kinase from the to and GST constructs. with or without lipoamidase as of the GST constructs were bound to Using Transfer was for with the by of and of was in and the kinase activity of each was in than of the GST activity was from the and of the E2 activity was in that to sucrose gradient used to the for transfer of kinase from to various of the was to binding by the lipoyl domain constructs prepared free of by GST which be in the of E2L1 to the of an intervening hinge region between GST and the lipoyl domain. of of was in the the for the of the and lipoyl domain constructs after gradient and into and from the to the of the gradient and a a the E2 The kinase activity and the E2 activity of were also the of the was in the with in the of the free lipoyl domain constructs were in the and with a in the The the kinase in the of the and constructs which the inner lipoyl domain but not in the of and constructs that the NH2-terminal lipoyl domain. this not separate the and subunits a second was in which was for a kinase subunits and the and subunits were a ~ than in the and in the containing E2L2 constructs not of the kinase from to lipoyl domain constructs. of each containing of the lipoyl domain E2L2 and and bovine was in for the to the of the the are to and each with a the of each was by in containing and were by was for kinase activity and E2 The and that The of and a from are in of the was the of the for between the for of the the of each this of the and of kinase activity in the various of the sucrose also binding to E2L2 and not to E2L1 . in to the kinase activity was in E2L2 that of the or constructs. of activity but the of the hinge region the kinase but further are to this by E2L1 , not in was The kinase in the of the E2L2 constructs were in the of 10 of of Transfer of the of of the kinase from the to GST as a was by of with GSH-Sepharose with a of than E2 subunits of the of was into from the of and The was and of various The for of was a was gain of of in the for kinase activity was the was eluted by with only a in activity after a of the with the as was with the bovine transfer of the tightly bound kinase to the recombinant lipoyl domain the binding of the kinase to E2 that transfer rapidly than the kinase can from the E2 core. are for a of lipoyl domain from GST constructs to the of kinase that the lipoyl prosthetic group to binding by the GST constructs were by treatment with lipoamidase or in the of lipoamidase for GST constructs the to the kinase transfer from to constructs after a treatment or eliminated binding to the constructs. was kinase transfer to the and, of lipoamidase the highly preferential binding of the kinase to the E2L2 domain the lipoyl prosthetic that the inner of the prosthetic group of E2L2 to binding of the kinase for of of the of the not kinase binding and that this of the prosthetic group was not an component of the the resolved kinase a to to that an to with a We the that a in the structure of the domain which its binding of the not to the role of the as a of how of the kinase of the kinase with in the structure of the domain to binding by this domain but not by the domain which a highly with only the and of and the region that can be with the of lipoyl domains of or the of other dehydrogenase We that this of the structure a role in with the kinase of mammalian the of a kinase with the domain of E2 and with the inner of the prosthetic group as of the binding The also of an E1 binding its to the domain of E2 with its to with the of Constructs the of free lipoyl domain constructs resolved kinase with the for binding of the kinase to the the E2L2 and the constructs the activity of the resolved the were not the increased with the of free lipoyl domains which were the E2L1 kinase activity with the with the of the domain. be to determine the of this The a of kinase activity of the but this the with of E2L2 and E2L1 in kinase of lipoyl domain constructs the activity of resolved were in with E1 containing are for kinase The lipoyl domain constructs were the for to of were in with a of were as the of by E2L1 being to a with the from of the lipoyl domain with E1 E1 a for the lipoyl domains are of E1 in the second in the and in a of E1 for E2L1 of and, the E2L1 was only in the not be that of E2L1 with E1 the in the the kinase subunits to be of are that by the kinase and transfer the to the in which shown to be the bovine kinase was to a structure of a containing and for that We that binds to the E2 and of the We in that are with the forms for kinase subunits and a the of the kinase to between lipoyl domains without the that the kinase lipoyl domain binding a of kinase subunits could the of the E2 core by a mechanism a of by by interchange to lipoyl domain the E2 core. a mechanism for kinase the of the E2 core. by a of the only with domains in the region the of the E2 core in that The are that by the of the domain kinase and that the of association to the kinase with lipoyl domain be than the in the the the not the of lipoyl domains the of the E2 core of that the be than for the domain of . The transfer to or to free E2L2 also only a of the association of kinase with structure for in the of free E2L2 involves a with for the of of that the of the kinase with E2 core the for how the kinase from E2 core to domain and for to of the for the various but the The of the kinase can be with binding and binding or binding and a Using a that free domains are that as not as in only and of the bound kinase in the that which of the kinase for kinase We not that to that the association of domains by the than the association by the the between kinase subunits are bound which the to with kinase in the free E2L2 kinase activity than the oligomeric E2 core ~ by intact E2 core a of this free lipoyl domain to the kinase than be binding to this free domain to the kinase was as as to oligomeric The of kinase from E2 to in shown in as by the of the with the with the The suggest not only a of kinase but a of activity than be the of kinase activity as free E2L2 . the transfer and of kinase activity that binding to the and suggest the of a of kinase activity by constructs than free lipoyl the GST a are to the of GST from the structure of the GST The in but the in its that be binding of the kinase the to binding by an of kinase be in the of the and constructs in which the E2L2 domain by mobile from the are to the structure of the to the of transfer of the and to the of in the oligomeric of various E2L2 kinase binding and activity the of the human domain constructs that prepared in the domain and for of kinase tightly bound to a large E2 the in using constructs as that a of the lipoyl prosthetic group and a of the structure of E2L2 the kinase binding structure in E2L2 from E2L1 and that this involves the of the lipoyl prosthetic group are to highly regions. E2L1 and E2L2 are than the lipoyl domains from or the E2 of other dehydrogenase structure the of the human E2L1 and E2L2 and of the lipoyl domains the between in both and We suggest that the of E2L2 the region to kinase with E2L1 kinase activity and the to kinase binding to the domain The in the in both a and of and be mammalian pyruvate dehydrogenase complex used dehydrogenase dehydrogenase acetyltransferase domain of lipoyl domain of region E2L1 and binding region E2L2 to or by or or or a large of with of the a of by the pyruvate dehydrogenase the dihydrolipoyl acetyltransferase the dihydrolipoyl dehydrogenase and the binding E2 a lipoyl and its prosthetic group in the of the by of the for and activity by a highly by kinase and that to and the activity of the complex and the E1 E2 component a role both in the of the complex and in supporting the of the kinase and the subunits are highly with consisting of globular domains connected by or hinge of E2′s domain to a dodecahedron shaped inner core structure that surrounded by the globular domains connected by the hinge regions. with other and the domain structure of E2 in the NH2-terminal of are ~ lipoyl domains, E2L1 and E2L2 connected by a ~ or hinge region dehydrogenase or lipoyl domain of the E2 component in the forms of lipoyl group between The mobile hinge a lipoyl domain to not only but to be to in the of the E1 and E2 The globular in the region of the mammalian E2 in the binding of the E1 component and the domain. ~ domain its NH2-terminal by a ~ hinge region that to the E2L2 domain and its by a ~ hinge region that and the entire structure to the inner domain of The inner domain of E2 also the acetyltransferase of bovine 20-30 and as as molecule of kinase are bound to the core. tightly bound to E2 the kinase can the complex the of free E1 by free kinase than are bound to the E2 core. We to the domain of E2 that in the binding of the kinase and to gain insights into how this of the complex the activity of the The of E1 from the E2 core and the kinase an for the E2 to this of how an kinase molecule rapidly phosphorylates E1 were with complex which to a of the E1 but kinase from the E2 core. was that the bound E1 were rapidly activity and, that of free E1 a that to that for the of association of free E1 with the E2 with the of E2′s structure and of the and of kinase binding to the E2 can the be kinase shown to bind to the lipoyl domain region of E2 in bovine of the lipoyl prosthetic group was shown to to of the kinase an role of this in binding of the the kinase binds tightly to the oligomeric E2 was to transfer to a of bovine E2 that both lipoyl We that transfer involves a direct interchange of the kinase without free between the lipoyl domain of the E2 the of the E2 core how a kinase molecule to both bind tightly to E2 core and phosphorylate 20-30 E1 that are also bound to the domain the of the E2 kinase of as a and as the other was to be a and by this was to by in a of resolved a that was than other the this in was the of the not The for this to be The of the of the pyruvate dehydrogenase kinase was from its was that the a of the kinase and the second kinase of demonstrated that a but to the and that was with a activity than that of the The to be with not kinase activity this second of the kinase subunits and to as pyruvate dehydrogenase kinase and to the other kinase as pyruvate dehydrogenase kinase the of complexes and containing this to and for the of in bovine of bovine complex and in that first the kinase bound to E2 core and of the kinase from the E2 core The of the kinase occurs its binding to the E2 and this only of the kinase are or in kinase prepared a of constructs of the lipoyl domain region of the human and in were in as to glutathione S-transferase and as free lipoyl domains selectively from GST by was with the lipoyl domains were We made the individual domains E2L1 and E2L2 and domains with the hinge region and , and also made the domain . E2 subunits of or lipoyl that are in a or highly preferential by of the lipoyl domains in an We that the bovine E1 kinase binds to human lipoyl domains with the domain as a highly preferential binding We demonstrated of the kinase between the oligomeric E2 core and lipoyl domain constructs containing the domain. the of the individual and domain constructs kinase