Báo cáo khoa học: Dynamics driving function ) new insights from electron transferring flavoproteins and partner complexes pdf

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REVIEW ARTICLEDynamics driving function ) new insights from electrontransferring flavoproteins and partner complexesHelen S. Toogood, David Leys and Nigel S. ScruttonManchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, UKIntroductionElectron transferring flavoprotein (ETF) is positionedat a key metabolic branch point, and is responsible fortransferring electrons from up to 10 primary dehydro-genases to the membrane-bound respiratory chain, thenature and diversity of which vary between organisms[1]. ETFs are highly dynamic and engage in novelmechanisms of interprotein electron transfer, which isdependent on large-scale conformational sampling toexplore optimal configurations to maximize electroniccoupling. Sampling mechanisms enable efficient com-munication with structurally distinct redox partners[2], but require additional mechanisms for complexassembly to impart specificity in the protein–proteininteraction.ETFs are soluble heterodimeric FAD-containingproteins that are found in all kingdoms of life. Theycontain a second nucleotide-binding site which isusually occupied by an AMP molecule [1]. In bacteriaand eukaryotes, ETFs function primarily as solu-ble one- or two-electron carriers between variousKeywordsacyl-CoA dehydrogenase; conformationalsampling; electron transferring flavoprotein;imprinting; trimethylamine dehydrogenaseCorrespondenceN. Scrutton, Faculty of Life Sciences,University of Manchester, 131 PrincessStreet, Manchester M1 7DN, UKFax: + 44 1613065201Tel: + 44 1613065152E-mail: nigel.scrutton@manchester.ac.ukWebsite: http://www.mib.manchester.ac.uk(Received 10 July 2007, revised 24 August2007, accepted 14 September 2007)doi:10.1111/j.1742-4658.2007.06107.xElectron transferring flavoproteins (ETFs) are soluble heterodimeric FAD-containing proteins that function primarily as soluble electron carriersbetween various flavoprotein dehydrogenases. ETF is positioned at a keymetabolic branch point, responsible for transferring electrons from up to10 primary dehydrogenases to the membrane-bound respiratory chain.Clinical mutations of ETF result in the often fatal disease glutaric aciduriatype II. Structural and biophysical studies of ETF in complex with partnerproteins have shown that ETF partitions the functions of partner bindingand electron transfer between (a) a ‘recognition loop’, which acts as a staticanchor at the ETF–partner interface, and (b) a highly mobile redox-activeFAD domain. Together, this enables the FAD domain of ETF to sample arange of conformations, some compatible with fast interprotein electrontransfer. This ‘conformational sampling’ enables ETF to recognize structur-ally distinct partners, whilst also maintaining a degree of specificity. Com-plex formation triggers mobility of the FAD domain, an ‘induced disorder’mechanism contrasting with the more generally accepted models of pro-tein–protein interaction by induced fit mechanisms. We discuss the implica-tions of the highly dynamic nature of ETFs in biological interproteinelectron transfer. ETF complexes point to mechanisms of electron transferin which ‘dynamics drive function’, a feature that is probably widespreadin biology given the modular assembly and flexible nature of biologicalelectron transfer systems.AbbreviationsACAD, acyl-CoA dehydrogenase; DMButA, n-butyldimethylamine; ETF, electron transferring flavoprotein; ETFQO, electron transferringflavoprotein ubiquinone oxidoreductase; Fc+, ferricenium ion (oxidized); GAII, glutaric acidaemia ⁄ aciduria type II; MCAD, medium-chain acyl-CoA dehydrogenase; SAXS, small-angle X-ray solution scattering; TMA, trimethylamine; TMADH, trimethylamine dehydrogenase.FEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS 5481flavoprotein-containing dehydrogenases. Electrons areaccepted or donated to ETF via the formation oftransient complexes with their partners [3]. Almost allETFs are mobile carriers containing a flexible domainessential for function [4]. ETFs need to balance pro-miscuity with specificity in their interactions with pro-tein donors and acceptors, in keeping with theirfunction in respiratory pathways. In this review, wediscuss new aspects of the structure and mechanismof ‘typical’ ETFs, and explore the diversity in func-tion and structure of ETFs across kingdoms. Finally,we analyse, in the context of new structural informa-tion, the role of clinical mutations in human ETFsand their partner proteins that give rise to severemetabolic diseases.ETF familiesETFs across kingdoms interact with a variety of elec-tron donors ⁄ acceptors that are involved in diverse met-abolic pathways. ETFs belong to the same familiesof a ⁄ b-heterodimeric FAD-containing proteins [5–7].Members of these families can be divided roughly intothree groups based on sequence homology and func-tional types.Group I ETFs are a well-studied group of electroncarriers, typically found in mammals and a few bacte-ria. Mammalian ETFs are physiological electronacceptors for at least nine mitochondrial matrix flavo-protein dehydrogenases [4,8]. These dehydrogenasesinclude the chain length-specific acyl-CoA dehydrogen-ases (e.g. medium-chain acyl-CoA dehydrogenase,MCAD) involved in fatty acid b-oxidation, isovaleryl-CoA dehydrogenase, 2-methyl branched-chain acyl-CoA dehydrogenase, glutaryl-CoA dehydrogenaseinvolved in amino acid oxidation, as well as dimethyl-glycine and sarcosine dehydrogenases involved in cho-line metabolism [4,8]. Electrons are passed from theseprimary dehydrogenases through ETF to membrane-bound ETF ubiquinone oxidoreductase (ETFQO)[9,10].Another well-studied group I ETF is from the bacte-rium Paracoccus denitrificans [11–13]. It is capable ofaccepting electrons from P. denitrificans glutaryl-CoAdehydrogenase, in addition to the butyryl-CoA andoctanoyl-CoA dehydrogenases from pig liver. Thephysiological electron acceptor for ETF has beenfound to be ETFQO [12].Group II ETFs are homologous to the proteinsFixB and FixA, equivalent to a-ETF and b-ETF,respectively, which are found in nitrogen-fixing anddiazotrophic bacteria [14]. These ETFs are oftenelectron donors to enzymes such as butyryl-CoAdehydrogenase, and may also accept electrons fromdonors such as ferredoxin and NADH [15]. No ETF-dependent activity has been observed with the mem-brane-bound respiratory enzymes in nitrogen-fixingbacteria, and so it is thought that the electron transferpathway from ETF to dinitrogen is via the enzymesETF:ferredoxin oxidoreductase, ferredoxin, nitrogenasereductase and nitrogenase [14].A well-studied group II ETF is from the bacteriumMethylophilus methylotrophus strain W3A1, which con-tains only one known dehydrogenase partner, namelytrimethylamine dehydrogenase (TMADH) [3,16]. FixB ⁄FixA proteins have been characterized from the micro-aerobic Azorhizobium caulinodans, which is known toaccept electrons from pyruvate dehydrogenase underaerobic conditions [14]. The nitrogen-fixing organismBradyrhizobium japonicum contains two sets of ETF-like genes: one with high homology to group I ETFs(etfSL), and the other very similar to group II FixB ⁄FixA proteins [17]. Under aerobic conditions, only theetfSL genes are expressed, whereas the reverse is truefor anaerobic growth, as nitrogen fixation only occursanaerobically [17].One ETF from the anaerobe Megasphaera elsdenii(formerly Peptostreptococcus elsdenii) is unusual, as itcontains two FAD-binding sites per ETF molecule,and so does not bind AMP [6,15,18,19]. This ETFserves as an electron donor to butyryl-CoA dehydro-genase via its NADH dehydrogenase activity [6], andis an electron acceptor for d-lactate dehydrogenase[15]. It has also been shown to contain a low percent-age of the modified flavins 6-OH-FAD and 8-OH-FAD [6].Group III ETFs include a pair of putative proteins,YaaQ and YaaR, located adjacent to the cai operon,which encodes carnitine-inducible proteins in Escheri-chia coli [7]. Group III members will not be discussedfurther in this review.An examination of the databases of genomicsequences shows organisms containing multiple ETF-like genes as well as ETFs fused with other proteins(Pedant; http://pedant.gsf.de). The genome of theeubacterium Fusobacterium nucleatum ssp. nucleatum(ATCC 25586) suggests the presence of two completeETF molecules, each positioned upstream of an acyl-CoA dehydrogenase. The genome also contains a largeORF (GI:19704756; Pedant; http://pedant.gsf.de) con-taining a fusion of three proteins comprising an N-ter-minal short-chain acyl-CoA dehydrogenase, followedby the a-subunit only of ETF and a C-terminal rubre-doxin (Fig. 1). As no functional studies of this enzymehave been published, it is presumed that the absence ofthe b-ETF subunit is a result of its role as a ‘fixed’ETF and partners – structure, function and dynamics H. S. Toogood et al.5482 FEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBSelectron carrier, although flexibility within the multi-domain complex may be possible.Another example of an organism with multiple ETFcontent is the iron-reducing, nitrogen-fixing bacteriumGeobacter metallireducens (Pedant; http://pedant.gsf.de). At least three of the sets of ETF genes areunusual (e.g. ORF4) as the N-terminal portion of thea-ETF subunit contains the gene sequence encoding a[4Fe)4S]2+ ⁄ +ferredoxin domain (Fig. 1). These ETFsare found upstream of genes such as putative Fe–Soxidoreductases (Pedant; http://pedant.gsf.de). At leastnine other putative [4Fe)4S]2+ ⁄ +ferredoxin-contain-ing ETFs have been identified (NCBI blast; http://www.ncbi.nlm.nih.gov/BLAST).Many archaea contain ETF- or FixB⁄ A-likesequences, such as Archaeoglobus fulgidus DSM 4304,Pyrobaculum aerophilum st. IM2, Aeropyrum pernixand Thermoplasma volcanium st. GSS1, but these areabsent in methanogens (Pedant; http://pedant.gsf.de).Several genera, such as Thermoplasma and Sulfolobus,contain multiple ETF genes, including a fusion proteinof the two subunits, with the b-subunit at the N-termi-nus (ba-ETF). In Sulfolobus solfataricus, ba-ETF isfound in an operon-like cluster of genes containing theprimary dehydrogenase 2-oxoacid ferredoxin oxido-reductase, a putative ferredoxin-like protein and aFixC-like protein, homologous to the membrane-bound ETF ferredoxin oxidoreductase in nitrogen-fixing organisms [14].A blast search of the structurally equivalent N-ter-minal (non-FAD-binding) a-ETF and b-ETFsequences against known ORFs showed homologywith a variety of adenosine nucleotide-binding enzymes(NCBI blast; http://www.ncbi.nlm.nih.gov). Suchenzymes include members of the adenosine nucleotidea-hydrolase superfamily from Oryza sativa, which con-tains an ATP-binding fold [20]. The thiamine bio-synthesis-like protein from three Leishmania speciescontains b-ETF and aminotransferase components atthe N- and C-termini, respectively [21]. This class ofenzyme is known to bind ATP. Other ATP-bindingenzymes with homology to b-ETF in the database(NCBI blast; http://www.ncbi.nlm.nih.gov) includeadenylyl-sulfate kinase from Anaeromyxobacter sp.Fw109-5 (GI:121539501), the predicted glutamate-dependent NAD(+) synthase from Strongylocentrotuspurpuratus (GI:115971088) and the asparagine synthasefrom Desulfovibrio vulgaris ssp. vulgaris DP9(GI:120564303). As b-ETF typically binds AMP,homology to domains of other enzymes known to bindadenosine nucleotides is not surprising.Sequence homology of ETFsAn alignment of a- and b-ETFs from all kingdoms oflife (Fig. 2) shows that, within the a-ETF family, theoverall sequence homology is low, although highsequence homology is found in the C-terminal region.By contrast, in the b-ETF family, there is a similardegree of sequence similarity throughout the length ofthe protein. Group I ETFs align better than group IIETFs, although both groups contain significantsequence similarity in conserved regions.The C-terminal portion of a-ETF contains a highlyconserved region, known as the b1ab2region of FADenzymes, which binds the adenosine pyrophosphorylmoiety of FAD [22]. Within this region is the a-ETFconsensus sequence of PX[L,I,V]Y[L,I,V]AXGIS-GX[L,I,V]QHX2G [7], similar to the consensussequence for FAD-binding dehydrogenases ofGXGXXGX15[E ⁄ D] [22]. The b-ETF family contains aconserved signature sequence of VXRX2[E,D]-X3[E,Q]X[L,I,V]X3LP[C,A][L,I,V]2which is used toidentify members of the b-ETF family [7]. Adjacent tothis signature sequence, group I b-ETFs also showthe highly conserved region of DLRLNEPR-YA[S ⁄ T]LPNIMKAKKK (residues 184–204; humannumbering), containing the recognition loop and thehighly conserved L195 necessary for partner binding inFusobacterium nucleatumButyryl-CoAdehydrogenaseα-ETFRubredoxinβ-ETFFusion proteinProbable Fe-S oxidoreductaseGeobacter metallireducensFerredoxinα-ETFRubredoxinoxidoreductaseFusion proteinFig. 1. Schematic diagram of the ‘operon-like’ arrangement ofgenes and fusion proteins from Fusobacterium nucleatum ssp.nucleatum (ATCC 25586) and Geobacter metallireducens (ORF4;Pedant; http://pedant.gsf.de).H. S. Toogood et al. ETF and partners – structure, function and dynamicsFEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS 5483humans [23]. The group II b-ETF from M. methylotro-phus also contains a recognition loop and the highlyconserved L193 partner binding to TMADH [3]. Othergroup II members appear not to contain a significantgroup I-like recognition loop, suggesting a differentmode of partner binding.ETF and partners – structure, function and dynamics H. S. Toogood et al.5484 FEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBSStructure of ETFDomains of ETFThe three-dimensional structures of group I ETFs havebeen solved from humans (Fig. 3A) [1] and P. denitrifi-cans [13], and group II ETF from M. methylotrophus(W3A1; Fig. 3B) [3]. The structure of the P. denitrifi-cans ETF is nearly identical to human ETF, with themajor difference being a random loop between residuesb90–96 which is an a-helix in humans [13]. All threestructures can be divided into three distinct domains.Domain I is composed of mostly the a-subunit,whereas domain III is made up entirely of the b-sub-unit [1]. These domains share nearly identical polypep-tide folds related by a pseudo-twofold axis, in spite ofa lack of sequence similarity. Both domains I and IIIare composed of a core of a seven-stranded parallelb-sheet, flanked by solvent-exposed a-helices. Thesedomains also contain a three-stranded antiparallelb-sheet with a fourth strand coming from the oppositedomain. Together these two domains form a shallowbowl shape, and make up the ‘rigid’ or more staticpart of the molecule upon which domain II rests.Domain III contains a deeply buried AMP moleculewhich plays a purely structural role [1].Domain II is the FAD-binding domain, and isattached to domains I and III by flexible linker regions(Fig. 3) [1]. Domain II can be subdivided into twodomains, II a and IIb, which are composed of theC-terminal portions of the a- and b-subunits, respec-tively. Domain IIa is the larger of the two, folds in amanner similar to bacterial flavodoxins [24] and formsmost of the region that binds FAD. This is the regionof high sequence similarity within the a-subunit. Thisfold consists of a core of a five-stranded parallelb-sheet surrounded by alternating a-helices [1]. A sixthstrand of the b-sheet is provided by the b-subunit.FAD is bound in an orientation in which the isoallox-azine ring is situated in a crevice between domains IIand III, with the xylene portion pointed towards theb-subunit. By contrast, domain IIb does not interactwith FAD, but instead wraps around the lower portionof domain IIa near domains I and III [1].Despite the low sequence similarity between thetwo groups of ETF, the overall folding of the struc-tures is very similar, with the exception of the orien-tation of the flavin-binding domain. Domain II ofW3A1 ETF is rotated by about 40° relative to thehuman and P. denitrificans flavin domains, withVa190 and Pb235 (W3A1 numbering) serving ashinge points [3]. In human ETF, the conservedEb165 of domain III interacts with Na259, which islocated near the conserved Ra249 (Ra237 in W3A1)and FAD (Fig. 4A). There are also hydrophobicinteractions between the C7- and C8-methyl groupsII IIFADABFADIII I IIIIHuman W3A1Fig. 3. Overall structures of the ETFs fromhumans (A) and Methylophilus methylotro-phus W3A1 (B). PDB codes: human, 1EFV[1]; W3A1, 1O96 [3]. a- and b-ETF chainsare shown as magenta and blue cartoons.FAD and AMP are shown as yellow andorange sticks, respectively. ConservedLeub195 ⁄ 194 for human and W3A1 ETFs,respectively, are shown as red spheres.Fig. 2. Alignment of a-ETFs (A) and b-ETFs (B) across kingdoms. Organisms: BRADI, Bradyrhizobium japonicum etfSL genes(P53573 ⁄ P53575); BRADII, Bradyrhizobium japonicum FixB ⁄ A genes (P10449 ⁄ P53577); HUMAN, mature human sequence(P13804 ⁄ P38117); METH, Methylophilus methylotrophus (P53571 ⁄ P53570); PARA, Paracoccus denitrificans (P38974 ⁄ P38975); SULF, Sulfol-obus solfataricus (Q97V72 ⁄ Q97V71). Sequences were obtained from the Swiss-Prot database (http://www.expasy.org) with accession num-bers in parentheses. The numbering for W3A1 and P. denitrificans a-ETF residues in the text are for the cloned forms of the protein inwhich a methionine (in bold typeface) has been inserted at the beginning of each gene. Residue colours: orange, FAD binding; blue, AMPbinding; red, interaction with partners; green, interaction between domain III and flexible domain II; violet, b-ETF signature sequence; yellow,hinge points. The dotted red line refers to the recognition loop.H. S. Toogood et al. ETF and partners – structure, function and dynamicsFEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS 5485of the isoalloxazine ring of FAD and residues Fb41and Yb16, respectively, of domain III [1]. Theseinteractions are likely to transiently stabilize the fla-vin domain in this position [25]. Sequence alignmentsshow that Eb165 (human numbering, Fig. 1) ishighly conserved amongst mostly group I ETFs,including P. denitrificans ETF (Eb162), which alsocontains the flavin domain in the same position ashumans. This suggests that this may be a commonorientation of the flavin domain amongst group Imembers.As a result of the change in orientation of the flavindomain in W3A1 ETF, Eb163 (equivalent to humanEb165) interacts instead with the conserved Ra237 viaa bifurcated salt bridge (Fig. 4B) [3]. This arginine resi-due also forms a single salt bridge with Da241 ofdomain II. A second interaction between these twodomains is seen in the low-resolution W3A1 ETFstructure [3], between residues Ra211 and Eb37. Inhumans, the equivalent arginine residue, Ra223, inter-acts directly with the flavin and is over 8 A˚fromdomain III [3].R 211E37L184D241W38R237FADE163F 41FADR249E 165N259ABCD3 structures Multiple positions of the flavin domain Low resolution solutionstructureIIIIIII IIIIIFig. 4. Interactions between domains II and III in human (A) and Methylophilus methylotrophus W3A1 (B) ETFs. PDB codes: human,1EFV [1]; W3A1, 1O96 [3]. a- and b-ETF chains are shown as magenta and blue cartoons and sticks. FAD is shown as yellow sticks and awater molecule is shown as a red sphere. Hydrogen bonds and hydrophobic interactions are shown as dotted and broken lines, respectively.(C) Small-angle X-ray scattering solvent envelope of W3A1 ETF, with a superimposition of the crystal structures of free ETF within it [4].a- and b-ETF chains are shown as blue and magenta cartoons, respectively. Domains are labelled with Roman numerals. Adapted from [3].(D) Superimposition of three free ETF structures showing the two positions of the flavin domain. Adapted from [4]. a- and b-ETF chains areshown as green and red cartoons, respectively. Domains are labelled with Roman numerals.ETF and partners – structure, function and dynamics H. S. Toogood et al.5486 FEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBSSolution structure of free ETFSmall-angle X-ray solution scattering (SAXS) studiescarried out on human, P. denitrificans and W3A1ETFs have shown that the solvent envelopes of eachETF are almost identical, in spite of the different con-formations of domain II [4]. A superimposition of thesolvent envelope of W3A1 ETF onto the structure ofits free ETF shows that, although domains I and III fitwell, the envelope around domain II shows the exis-tence of multiple conformations in solution (Fig. 4C)[3]. These conformations appear to arise from domainII rotating about 30–50° with respect to domains I andIII via two flexible hinge regions. This corresponds toa shift in position of domain II from the W3A1 posi-tion to the human ⁄ P. denitrificans position. The lackof an appropriate shoulder in the intermediate anglerange, which can be associated with the static lobeddomain structures, suggests that all three ETFs possesssimilar domain arrangements in solution, with the fla-vin domain sampling a range of conformational states.These states are likely to include multiple discrete, buttransient states. A superimposition of W3A1 ETFswith different flavin domain positions, modelled byweighted masses molecular dynamics, has shown thatthese conformations are consistent with the solventenvelope of ETF [3]. The solvent envelopes of bothoxidized and reduced W3A1 ETF are essentially identi-cal, suggesting that no large conformational changeoccurs as a result of changing the redox state [4]. Theconformations seen crystallographically may havearisen from the trapping of a particular discrete stateas a result of crystal packing constraints, but may alsoreflect differences in the proportions of the discretestates between the different ETFs [25].Cofactor bindingThe isoalloxazine rings of FAD from human andW3A1 ETFs are sandwiched between several conservedresidues that make distinct, but structurally equivalent,interactions (Fig. 5A) [1,3]. A key characteristic ofETF FAD-binding domains is the ‘bent’ conformationof the ribityl chain of FAD as a result of 4¢OH hydro-gen bonding with N1 of the isoalloxazine ring [1]. It isthought that the 4¢OH group helps to stabilize thesemiquinone ⁄ dihydroquinone couple, and may beinvolved in electron transfer to ETFQO. Another char-acteristic feature is the absence of aromatic residuesthat stack parallel to the ring. One or two aromaticresidues (Yb16 and Fb41 in humans) are within hydro-phobic interaction distance, but the rings are not ori-ented towards FAD. In its place the guanidiniumportion of the side chain of the conserved Ra249 isperpendicular to the xylene portion of the isoalloxazinering, which may function by stabilizing the anionicreduced FAD [13], and also by conferring a kineticblock on full reduction to the dihydroquinone [3].Other key interactions include the N1 residue ofHa268 with O2 of the isoalloxazine ring, which mayalso function in stabilizing the anionic semiquinone [1].The hydroxyl group of Ta266 interacts with N5 ofFAD, which may aid in modulating the redox poten-tial. The ADP moiety of FAD is solvent exposed,more so in W3A1 ETF [3]. Stabilization of the nega-tive charge imposed by the phosphates is achievedthrough interactions with residues such as Sa248 andSa281 [1].ABFig. 5. (A) Schematic representation of the FAD-binding region ofhuman ETF. PDB code, 1EFV [1]. FAD residues and water areshown as atom-coloured sticks and red circles, respectively.(B) AMP-binding region of human ETF. Residues and FAD areshown as atom-coloured sticks and water molecules are shown asred spheres. Potential interactions are shown as dotted lines.H. S. Toogood et al. ETF and partners – structure, function and dynamicsFEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS 5487The AMP-binding sites of all three ETF structuresare very similar, both in terms of the position andtypes of interaction between AMP and b-ETF. AMPis buried deeply within domain III and is thought toplay a purely structural role (Fig. 5B) [1]. These inter-actions are mostly backbone interactions; thus,although there is a high degree of conservation of posi-tion of the interacting residues, there is often a lowsequence conservation (Fig. 2; blue residues). Thephosphate moiety of AMP from humans forms hydro-gen bonds with the residues Ab126, Db29, Nb32,Qb33 and Tb34, as well as a water molecule. A fewhydrogen bonds are found to anchor the rest of theAMP molecule, including backbone interactions withCb66 and Ab9 and two water molecules [1]. It isthought that AMP binding may be a structural rem-nant of a NADP-binding site, which is a known elec-tron donor of the group II ETF from Megasphaeraelsdenii, which does not bind AMP [6].Structure of ETF–partner complexesMethylophilus methylotrophus TMADH:ETFThe first structure of an ETF in complex with its part-ner protein was solved between TMADH and ETFfrom M. methylotrophus W3A1 [3]. The structure ofthe free TMADH dimer had been solved previously,and was shown to contain the redox-active cofactors6-S-cysteinyl FMN and [4Fe)4S]2+ ⁄ +(electron donorto ETF), as well as a purely structural ADP molecule(Fig. 6A) [26,27]. Two crystal forms were obtained forthe wild-type complexes, which were found to be virtu-ally identical, suggesting that the structure is largelyindependent of crystal packing contacts. The total bur-ied interfacial surface visible in the structures was elon-gated in shape and covered 1750 A˚2, with 10% and8% of the surface contributed by ETF and TMADH,respectively [3]. Surprisingly, there was a completeabsence of density for the mobile flavin domainof ETF, in spite of SDS-PAGE analysis of theTMADH:ETF crystals showing its presence [3].The structures showed that there was an interactionsite between the two proteins, which was distinct fromthe predicted location of the flavin-binding domain ofETF [3]. This consists of a hydrophobic interactionbetween a surface patch in the ADP-binding domainof TMADH and a loop in ETF domain III (residuesPb189–Ib197), termed the ‘recognition loop’ (Fig. 6B).This loop consists of the N-terminal portion of ana-helix and part of the preceding loop. A residue keyto this interaction is the ETF residue Lb194 (redsphere in Fig. 3), which is buried within this hydro-phobic patch of TMADH. Other hydrophobic residuesof ETF interacting with TMADH are Yb191, Ib197and Sb193, the latter of which stabilizes the initial turnof the a-helix in the recognition loop. These residuesare highly conserved, in particular within group IETFs (Fig. 1). Several residues preceding Yb191 whichdo not contact TMADH are also conserved, includingLb186, Nb187, P b189 and Rb190. The recognitionloop is stabilized by both the close packing of theseresidues and a bifurcating salt bridge between Rb190and residues Eb44 and Eb51. Several other residuesinvolved in complex formation include a salt bridgebetween the N-terminus of TMADH and Db16 ofETF, and a number of direct or water-mediated hydro-gen bonds. This relatively small number of interactionshelps to explain why the dissociation constant($ 5 lm) of TMADH:ETF is weak [3,28].In free ETF, the recognition loop is more flexibleand is oriented slightly differently, with Pb189 andPb204 serving as hinge points [3]. Limited trypsin pro-teolysis, which removed the recognition loop, producedan ETF whose structure and redox capabilities withdithionite were virtually identical to native ETF, yet ithad lost its ability to accept electrons from TMADH.This shows the pivotal role of the recognition loop incomplex formation, and serves as an ‘anchor’ distantto the redox centres [3]. This anchor may serve as ameans of recognizing specific redox partners, as allthat would be required would be a suitably placedhydrophobic patch to interact with the recognitionloop [3].The absence of density for the flavin domain of ETFoccurs after residues Va190 and Pb235, which serve ashinge points [3]. This total lack of density was initiallysurprising, as the free ETF structure showed clear den-sity for the flavin domain, in spite of the known flexi-bility of the molecule in solution from SAXS studies[4]. This suggests that either the flavin domain has anincreased mobility within the complex, or packing con-straints with the free ETF structure lock the domain inone position. This mobility of the flavin domain withinthe complex lends support to the transient nature ofthe electron transfer-competent state, as predicted fromkinetics and other studies [4,25].Several mutant TMADH:ETF complexes weredesigned which altered the interactions between theflavin domain and domain III of ETF, as well as itsinteraction with TMADH (see ‘Human MCAD:ETF’section below). At least two of each of the mutant com-plex structures were determined, TMADH WT:ETFEb37Q and TMADH Y442F:ETF WT, includingtwo structures in a new space group (H. S. Toogood,D. Leys & N. S. Scrutton, unpublished results). AllETF and partners – structure, function and dynamics H. S. Toogood et al.5488 FEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBSstructures were virtually identical to the wild-typecomplex, including the absence of the flavin domain,highlighting the rapid mobility of this domain.Modelling studies in which the flavin domain ofETF was docked into the TMADH:ETF complex,based on its position in free ETF, showed that theflavin domain had to undergo a significant conforma-tional change to prevent clashes with TMADH [3,4].This is supported by the detection of structuralchanges on complex formation by observing spectralchanges during difference spectroscopy studies ofTMADH:ETF [29]. Shifting the domain into a human-like conformation would allow the domain to fit withinthe allowable space. The ‘empty volume’ observedgpFMN9[4Fe-4S]2+/+R37L 194Reco nition looY442AMP6-S-cysteinylL14ADPABCTMADH(monomer)ETFFAD2Y442V344FADG479A480S391L393T414Q462H416Y478A464R 195S 193A192Y 191Fig. 6. (A) Structure of the TMADH:ETF complex. Only one TMADH and ETF are shown for clarity. PDB code for all, 1O94 [3]. a- and b-ETFchains and TMADH are shown as magenta, blue and green cartoons, respectively. The TMADH cofactor 6-S-cysteinyl FMN is shown as yel-low sticks, and the [4Fe)4S]2+ ⁄ +centre is shown as red and yellow spheres. TMADH ADP and ETF AMP are shown as orange sticks. Resi-dues Y442 and V344 are shown as blue sticks. The recognition loop of ETF is shown as a red cartoon with the conserved Lb194 residueshown as red sticks. The dotted circle refers to the approximate position of the missing flavin domain. (B) Structure of the recognition loopin TMADH:ETF. Residues are shown as atom-coloured sticks with green and blue carbons for TMADH and ETF, respectively. (C) Model ofETF domain II in the TMADH:ETF complex. a-ETF and TMADH are shown as magenta and green cartoons, respectively. The two FAD mole-cules are shown as yellow sticks. Highlighted residues are shown as atom-coloured sticks with green and magenta carbons for TMADH andETF, respectively.H. S. Toogood et al. ETF and partners – structure, function and dynamicsFEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS 5489between TMADH and ETF is of sufficient size andshape to allow the flavin domain of ETF to undergo a‘ball-in-socket’ type of motion [3], suggesting that mul-tiple (> 2) conformations are possible. This suggestsan ‘induced fit’ model for partner association, withelectron transfer likely to be possible from an ensembleof thermodynamically metastable complexes ratherthan one discrete species [3].Kinetics studies have shown that, in the electrontransfer-competent state, the flavin of ETF is likely tobe close to a surface groove of TMADH close to resi-dues V344 and Y442 [30]. Molecular dynamics calcula-tions were performed on the flavin domain of free ETFsuperimposed onto the complex to determine potentialelectron transfer-competent states [3]. A model of oneof the putative ‘active’ conformations between the[4Fe)4S]2+ ⁄ +centre of TMADH and the flavindomain of ETF gives an intercofactor distance of lessthan 14 A˚(Fig. 6C) [3]. In this state, the guanidiniumion of the conserved Ra237 is located close to the aro-matic ring and hydroxyl group of Y442 of TMADH.Cross-linking studies using bismaleimidohexane withTMADH Y442C and ETF Ra237C mutants led to therapid formation of a cross-linked complex, establishingthe close contact of these residues in the complex. Also,difference spectroscopy studies with TMADH and theETF mutant Ra237A showed that electron transferwas severely compromised as a result of a change in therate of rearrangement of ETF to form the electrontransfer-competent state, rather than a change in theintrinsic rate of electron transfer [29]. However, anyinteractions between TMADH and the flavin domainof ETF are likely to be fleeting, and simply increase thehalf-life of the electron transfer-competent states toallow fast electron transfer [3].Human MCAD:ETFTo investigate the way in which ETF can interact withits structurally distinct partners, the structure ofhuman ETF with its partner MCAD was determined[23]. The structure of free MCAD had been solved pre-viously, and was shown to be a homotetramer of43 kDa monomers (dimer of dimers) containing oneFAD per monomer [31]. The first structure of the com-plex between MCAD and ETF was found to contain atetramer of MCAD with one ETF molecule [23]. Thetotal buried interfacial surface visible in the structures(excluding the ETF flavin domain) was elongated inshape and covered 536 A˚2, with 3.2% and 4.3% of thesurface contributed by ETF and MCAD, respectively.In this structure, the flavin domain of ETF was barelyvisible in the density [23].Four mutant MCAD:ETF complexes were designedwhich altered the interactions between the flavindomain and domain III of ETF (MCAD:ETFEb165A), as well as its interaction withMCAD (MCA D:ETF Ra249A; MCAD E212A:ETF;MCAD E359A:ETF) [25]. The aim was to alter theratio of the different conformational states sufficientlyto trap discrete flavin domain positions. Kinetic studiesof these complexes showed a reduction in electrontransfer rates [when using 2,6-dichloroindophenol asthe terminal electron acceptor], except for the MCAD:ETF Eb165A complex, which showed both a dramaticincrease in rate and decrease in the apparent Kmvalue.Crystal structures of all four mutant complexes wereobtained (Fig. 7A; last three: H. Toogood, A. vanThiel, D. Leys & N. S. Scrutton, unpublished work),which showed an increase in density for the flavindomain to about 70% occupancy (except for MCAD:ETF Ra249A), with the flavin domain in the sameposition as in the wild-type structure. In these struc-tures, ETF is interacting with a dimer of MCAD [25].As with the TMADH:ETF structures, human ETFcontains a recognition loop (Pb190–Ib198), includingthe highly conserved residue Lb195, which interactswith a hydrophobic pocket on MCAD (Fig. 7B) [23].The recognition loop interacts with the MCAD surfacein such a way that causes an extension of a-helix C ofMCAD [31], with a nearly perfect alignment of theaxes and corresponding dipoles of both helices [23].The side chain of Lb195 is buried within a hydropho-bic pocket formed by a-helices A, C and D of MCAD,and is lined by residues such as F23, L61, L73 andI83. ETF residues which also interact with this pocketinclude Yb192, Pb197, Ib198 and Mb199 [23].A comparison of the free and complex crystal struc-tures reveals that, although MCAD adopts a nearlyidentical conformation in both structures, ETF adopts aslightly different backbone conformation with moreextensive side chain rearrangements, including Lb195[23]. The structure of the free ETF mutant Lb195A doesnot show any significant rearrangements of the recogni-tion loop, yet kinetic studies with both MCAD, isovale-ryl-CoA dehydrogenase and the structurally distinctpartner dimethylglycine dehydrogenase show a severedecrease in electron transfer rates (A. van Thiel,H. Toogood, H. L. Messiha, D. Leys & N. S. Scrutton,unpublished work). Mutations of MCAD, such asL61M, L73W and L75Y, which were designed to ‘fill in’the binding pocket, were all severely impaired in elec-tron transfer rates with ETF [25]. Microelectrosprayionization mass spectrometry and surface plasma reso-nance studies showed competitive binding of ETF toacyl-CoA dehydrogenases and dimethylglycine dehydro-ETF and partners – structure, function and dynamics H. S. Toogood et al.5490 FEBS Journal 274 (2007) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS[...]... existence of two possible routes of electron transfer from the [4Fe)4S]2+ ⁄ + centre to an external electron acceptor The shortest pathway extends from C345, a ligand on the [4Fe)4S]2+ ⁄ + ETF and partners – structure, function and dynamics centre, to V344, which is located at the bottom of a small groove on the surface of TMADH The second pathway extends from C345 to E439 and finally to Y442, the latter... dehydrogenase In Flavins and Flavoproteins (Edmondson DE & McCormick DB, eds), pp 687–690 Walter de Gruyter, Berlin Jones M, Talfournier F, Bobrov A, Grossmann JG, Vekshin N, Sutcliffe MJ & Scrutton NS (200 2) Electron transfer and conformational change in complexes of trimethylamine dehydrogenase and electron transferring flavoprotein J Biol Chem 277, 8457–8465 Thorpe C (199 1) Electron- transferring flavoproteins. .. 1977–1989 Scott JD & Ludwig RA (200 4) Azorhizobium caulinodans electron- transferring flavoprotein N electrochemi- ETF and partners – structure, function and dynamics 15 16 17 18 19 20 21 22 23 24 25 26 27 cally couples pyruvate dehydrogenase complex activity to N2 fixation Microbiology 150, 117–126 Pace CP & Stankovich MT (198 7) Redox properties of electron- transferring flavoprotein from Megasphaera elsdenii Biochim... [3] and small-scale conformational changes in the formation of electron transfer-competent state(s) A simplified kinetic scheme for such a system, where A is one -electron- reduced TMADH (4Fe)4S +) and B is oxidized ETF, is shown in Scheme 1 [30] In this scheme, kr (and k–r) refer to the reversible rate of reor- A branching kinetic steady-state scheme has been proposed for intra- and interprotein electron. .. MJ (200 0) Trimethylamine dehydrogenase and electron transferring flavoprotein Sub-Cell Biochem 35, 145–181 Scrutton NS (200 4) Chemical aspects of amine oxidation by flavoprotein enzymes Nat Product Rep 21, 722–730 Marcus RA & Sutin N (198 5) Electron transfers in chemistry and biology Biochim Biophys Acta 811, 265– 316 Page CC, Moser CC & Dutton PC (200 3) Mechanism for electron transfer within and between... because, although the substrate can donate two electrons at a 5494 FEBS Journal 274 (200 7) 5481–5504 ª 2007 The Authors Journal compilation ª 2007 FEBS H S Toogood et al ETF and partners – structure, function and dynamics ETFsq 11 FMN.S (CH 3)3 N 4Fe-4Sox ETFsq + HCHO 4Fe-4Sred 2 FMN 1 (CH 3)2 NH 3 4Fe-4Sox FMNH2 + HCHO (CH 3)3 N 4Fe-4Sox ETFox 4 6 FMNsq.S 7 9 ETFsq (CH 3)3 N 5 FMNsq 10 4Fe-4Sox FMNsq 4Fe-4Sred 4Fe-4Sred... Acta 1433, 139–152 ETF and partners – structure, function and dynamics 66 Dwyer TM, Mortl S, Kemter K, Bacher A, Fauq A & Frerman FE (199 9) The intraflavin hydrogen bond in human electron transfer flavoprotein modulates redox potentials and may participate in electron transfer Biochemistry 38, 9735–9745 67 Olsen RK, Andresen BS, Christensen E, Bross P, Skovby F & Gregersen N (200 3) Clear relationship between... Mayhew SG (199 5) Cloning of electron- transferring flavoprotein from Megasphaera elsdenii Biochem Soc Trans 23, 379S Sato K, Nishina Y & Shiga K (200 3) Purification of electron- transferring flavoprotein from Megasphaera elsdenii and binding of additional FAD with an unusual absorption spectrum J Biochem 134, 719–729 Yu J, Wang J, Lin W, Li S, Li H, Zhou J, Ni P, Dong W, Hu S, Zeng C, et al (200 5) The genomes... which are compatible with fast electron transfer Transient stabilization of electron transfer-competent states is achieved through interactions between the two partners, including interactions with the conserved Ra237 (W3A1 numbering) [23] This separation of the partner recognition site (recognition loop) from the electron transfer site (flavin domain) is critical in understanding how ETF can interact... transfer flavoprotein families ETF-alpha and ETF-beta Res Microbiol 146, 397–404 Frerman FE (198 8) Acyl-CoA dehydrogenases, electron transfer flavoprotein and electron transfer flavoprotein dehydrogenase Biochem Soc Trans 16, 416–418 Beckmann JD & Frerman FE (198 5) Electron- transfer flavoprotein-ubiquinone oxidoreductase from pig liver: purification and molecular, redox, and catalytic properties Biochemistry . REVIEW ARTICLE Dynamics driving function ) new insights from electron transferring flavoproteins and partner complexes Helen S. Toogood, David Leys and Nigel. green and magenta carbons for TMADH and ETF, respectively.H. S. Toogood et al. ETF and partners – structure, function and dynamics FEBS Journal 274 (2007)
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