Proc. Nati. Acad. Sci. USAVol. 84, pp. 8262-8266, December 1987Biochemistry

Structure of L-3-hydroxyacyl-coenzyme A dehydrogenase:Preliminary chain tracing at 2.8-A resolution

(nucleotide binding domain/fatty acid oxidation/NAD structure/B-side dehydrogenase/x-ray diffraction)

JENS J. BIRKTOFT*t, HAZEL M. HOLDEN*t, RONALD HAMLIN§¶, NGUYEN HUU XUONG§,AND LEONARD J. BANASZAK**Department of Biological Chemistry, Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, MO 63110; and§Department of Biology, Chemistry and Physics, University of California at San Diego, La Jolla, CA 92093

Communicated by Stuart Kornfield, August 3, 1987 (received for review May 11, 1987)

ABSTRACT The conformation of L-3-hydroxyacyl-CoAdehydrogenase (EC 1.1.1.35) has been derived from electron-density maps calculated at 2.8-A resolution with phases ob-tained from two heavy-atom derivatives and the bound coen-zyme, NAD. Like other dehydrogenases, 3-hydroxyacyl-CoAdehydrogenase is a double-domain structure, but the bilobalnature of this enzyme is more pronounced than has beenpreviously observed. The amino-terminal domain, which com-prises approximately the first 200 residues, is responsible forbinding the NAD cofactor and displays considerable structuralhomology with the dinucleotide binding domains observed inother NAD-, NADP-, and FAD-dependent enzymes. Thecarboxyl-terminal domain, comprising the remaining 107 res-idues, appears to be all a-helical and bears little homology toother known dehydrogenases. The subunit-subunit interface inthe 3-hydroxyacyl-CoA dehydrogenase dimer is formed almostexclusively by residues in the smaller helical domain. Adifference map between the apo and holo forms of the crystal-line enzyme has been interpreted in terms of the NAD moleculebeing bound in a typically extended conformation. The locationof the coenzyme binding site, along with the structural homol-ogy to other dehydrogenases, makes it possible to speculateabout the location of the binding site for the fatty acyl-CoAsubstrate.

In most organisms, the metabolic breakdown of long-chainfatty acids proceeds through the,B-oxidation pathway. In thisprocess, a thioester is formed between CoA and the fattyacid, and the alkyl chain is then degraded by the sequentialremoval of two-carbon units. The mitochondrial a-oxidationcycle involves four enzymes; one of these is L-3-hydroxy-acyl-CoA dehydrogenase [(S)-3-hydroxyacyl-CoA:NAD+oxidoreductase, EC 1.1.1.35]. This enzyme utilizes NAD asa cofactor and L-3-hydroxyacyl-CoA as substrate in thefollowing reaction, which in vivo proceeds exclusively to theright:

R-CHOH-CH2-CO-S-CoA + NAD+R-CO-CH2-CO-S-CoA + NADH + H+

The specificity of the enzyme is quite broad, as 3-hydroxyfatty acyl derivatives ofCoA containing four or more carbonsare oxidized at about the same rate (1). While the nucleotidemoiety of the CoA was found not to be essential for catalysis,Km is increased and Vmax is reduced using S-acyl pantotheinederivatives as substrates (2). Transfer of a hydride ionbetween substrate and the nicotinamide ring of the cofactorby L-3-hydroxyacyl-CoA dehydrogenase is "B-side"-spe-cific (3). This is the same specificity found for glyceralde-

hyde-3-phosphate dehydrogenase but differs from that ofother enzymes of known structure, such as malate, lactate,and liver alcohol dehydrogenases, which all have "A-side"specificity.

In eukaryotic cells, L-3-hydroxyacyl-CoA dehydrogenaseis encoded by a nuclear gene and is synthesized in thecytoplasm as a precursor form that appears to contain anadditional 30-35 amino acids (4). Although the mature formof the enzyme can be isolated easily from a variety of tissues,the form from pig heart has been particularly well character-ized (2, 3, 5). Porcine heart L-3-hydroxyacyl-CoA dehydroge-nase is a dimeric protein of molecular weight 67,000 com-posed of two identical subunits, each with 307 amino acids ofknown sequence (6). Some heterogeneity at the aminoterminus has been reported, which may be the result ofproteolytic processing accompanying the translocation of theenzyme into the mitochondrial matrix (6).The enzyme has been crystallized in a number of different

forms (7). The orthorhombic form obtained from polyethyl-ene glycol 6000 at apH near 8 proved usable for x-ray analysis(8). Under these conditions, crystals belonging to the spacegroup C2221 are formed and the following unit-cell dimen-sions are observed: a = 227.2 A, b = 82.1 A, and c = 124.7A. There are eight equivalent positions in the unit cell.

Analysis of electron-density maps based on x-ray dif-fraction data extending to 5.25-A resolution indicated that theprotein subunits are packed in an unusdal fashion in the crystallattice. One enzyme dimer is located in a general position andhas a noncrystallographic 2-fold rotation axis. A second dimeris located with its molecular dyad axis coincident with acrystallographic 2-fold axis. The low-resolution x-ray diffrac-tion study showed that each subunit of the enzyme had adistinctly bilobal appearance (8). The cofactorNAD was boundto the larger of the two lobes near the interface with the smalllobe. The x-ray diffraction data have now been extended to2.8-A resolution and electron-density maps have been calcu-lated. The conformation of the polypeptide chain has beenobtained and was used to determine the structural relationshipbetween L-3-hydroxyacyl-CoA dehydrogenase and other en-zymes that utilize NAD as a cofactor. It is now possible todescribe the NAD-enzyme interactions and, in addition, tosuggest possible modes of interactions between fatty acyl-CoAand the enzyme.

MATERIALS AND METHODSThe procedures used for the purification and crystallizationof3-hydroxyacyl-CoA dehydrogenase were identical to those

Abbreviation: MIR, multiple isomorphous replacement.tAuthor to whom reprint requests should be addressed.tPresent address: Department of Biochemistry, University of Ari-zona, Tucson, AZ 85721.Present address: Area Detector System Corporation, 7343 RonsonRoad, San Diego, CA 92111.

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previously described (8, 9). In the low-resolution study, threeheavy-atom derivatives were used for phase determination,and two of these, methylmercury(II) chloride and K2PtCl6were usable for the high-resolution work.

Diffraction data extending to a maximum resolution of 2.7A were collected on the University of California at San DiegoMark II area-detector system equipped with two detectors(10-12). Data processing included Lorentz and polarizationcorrections and an internal scaling procedure that adjusts forradiation damage and absorption (12, 13). One crystal wasused to collect complete data sets for each of the apo and holoforms of the enzyme and for the platinum derivative. Due tocrystal decay, two crystals were used for data collection fromthe mercury derivative. Bijvoet pairs were not merged for thecrystals containing heavy-atom derivatives. A total of 500,000x-ray reflections were measured, leading to 110,000 uniquereflections. The merging R-factors (13) ranged from 0.046 for theholoenzyme data to 0.073 for the platinum derivative.

Positions of heavy atoms, already known from the low-resolution studies, were confirmed using difference Pattersonand cross Fourier methods. Full use was made of theanomalous data. In addition to the two heavy-atom deriva-tives, the bound cofactor, NAD, was used as a "pseudo"-heavy-atom derivative (14) in a manner identical to thatdescribed in the low-resolution studies (8). Positional param-eters as well as occupancies and temperature factors for theheavy-atom substituents including the pseudo heavy atomsrepresenting the bound NAD molecules were first refined byusing the origin-removed difference Patterson method (15).In the final cycles of the phase calculations, only theoccupancies were allowed to vary and the refinement wasbased on the lack of closure error. The correct handedness ofthe phases was determined from an analysis of anomalousdifference Fourier maps (16). Solvent flattening of the mul-tiple isomorphous replacement (MIR) electron-density mapswas done by a local implementation (17) of the procedureaccording to Wang (18). The procedures of Bricogne (19)were used for the skewing and averaging of electron-densitymaps.Map interpretation and model building (a-carbon model)

were done on mini-maps and on an MMS-X interactivegraphics system (27) using the program NEWNIP (28). Thefitting of the polyalanine model was done on a SiliconGraphics (Mountain View, CA) IRIS 3000 using TOM (C. M.Cambillau, Centre National de la Recherche Scientifique,Marseilles, France), a version of FRODO (29) implementedon the IRIS.

RESULTS

X-Ray Data. Preliminary inspection of the diffractionpatterns on the multiwire area detector confirmed what hadpreviously been observed from still and oscillation photo-graphs-namely, that the crystals of L-3-hydroxyacyl-CoAdehydrogenase do not diffract much beyond about 2.7-Aresolution. The two heavy-atom derivatives showed amarked reduction in intensities at resolutions higher than 3.0A, which consequently was used as the limit for these twodata sets. For both the holo and the apo form of the crystals,data extending to 2.7 A were collected.

Analysis of the heavy-atom phasing parameters (Table 1)shows that beyond 3.5-A resolution, the lack ofclosure error forthe two heavy-atom derivatives is relatively large, and beyond3.0 A only the pseudo-heavy-atom derivative was used. Theplatinum derivative had just one binding site per asymmetricunit, and the mercury derivative had six binding sites, threemajor and three minor. The positions of the major sites turnedout to be in identical locations in each of the three uniquesubunits.

Table 1. MIR phase calculations for L-3-hydroxyacyl-CoAdehydrogenase

Mean No. of Figureresolu- observa- rms AF/rms E oftion, A tions* CH3Hg PtCl6 Apo - holo meritt

7.3 4329 1.5 1.3 1.3 0.694.8 3624 1.3 1.0 1.3 0.594.1 4243 1.4 0.76 1.3 0.563.6 4671 1.2 0.53 1.4 0.493.3 5006 1.0 0.41 1.3 0.353.0 5121 0.86 0.33 1.2 0.242.8 3208 ND ND 1.1 0.12

*Total, 30,202.tThe root-mean-square difference in the observed heavy-atom dif-ference amplitudes divided by the root-mean-square lack-of-closureerror in the protein phase determination.tOverall, 0.43.

Electron-Density Maps and Their Interpretation. The centerof the dimer located in the general crystallographic positionis at (0.15, 0.20, 0.25) and this molecule will be called the A-Bdimer. A second dimeric molecule is centered around thecrystallographic 2-fold axis that is parallel to the y-axis andpasses through the point x = 0.5, z = 0.25. This dimer will bereferred to as the C-C dimer and has its molecular center atapproximately (0.50, 0.35, 0.25). The reader should recall thatall monomers are equivalent except for crystal packingconsiderations; hence in this nomenclature A_ B - C.Three different electron-density maps were used for the

model fitting described here: (l) a MIR map based on threederivatives, (ii) the MIR map that had been subjected to eightcycles of density modification, and (iii) the MIR map that hadbeen both density-modified and averaged around the local2-fold axis. In several regions of the MIR map, helicalstructures as well as segments ofextended polypeptide chain,later found to be component strands of a ,8-sheet, could beeasily identified although other parts of the MIR map weremore difficult to interpret. After eight cycles of densitymodification, a noticeable improvement of the electron-density map in those regions associated with the A-B dimercould be discerned, but the electron density associated withthe C-C dimer did not appear to improve significantly. In asecond stage, the electron-density map from the density-modification calculations was interpreted in terms of ana-carbon-model that was both crude and incomplete. Markersfor 140 a-carbon atoms were placed in each of the A and Bsubunits, and the two sets of a-carbon coordinates were usedto derive a transformation matrix relating the A and Bsubunits. The matrix derived in this manner proved to besuperior to those derived from rotation function analysis andfrom analysis ofheavy-atom positions asjudged from inspec-tion of electron-density maps. Note that averaging onlyincluded the map volume encompassing the A-B dimer.

Initially, an a-carbon model accounting for 252 residueswas obtained, utilizing primarily the MMS-X graphics systembut with occasional reference to mini-maps. This initial chaintracing was followed by fitting of a polyalanine model, againusing the electron-density map, which was both density-modified and averaged. Hence the model consists of an"averaged" interpretation. During this fitting, the unaver-aged MIR map was also consulted, and it is clear that somesmall conformational differences probably exist between thetwo subunits of the A-B dimer. Each polypeptide chain ofporcine heart L-3-hydroxyacyl-CoA dehydrogenase is knownto contain 307 residues, and of these, 288 have been account-ed 'for in each of the A-B subunits.

Initially, an attempt was made to interpret the densityassociated with the C-C dimer in the MIR map withoutmaking any reference to the model derived for the A-B

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dimer. Although it was possible to follow short stretches ofcontinuous polypeptide chain, numerous breaks and discon-tinuities in the electron density made it a difficult task toobtain an unambigous and independent chain-tracing. How-ever, those model segments that were fitted to the electrondensity for the C-C dimer correlated well with correspondingelements of the A-B dimer. The crystallographic relationshipbetween the C-C and A-B dimers was also defined by thesimilar appearance of the difference electron densities attrib-utable to the three NAD molecules. A third source ofinformation used to interpret the electron density for the C-Cdimer was provided by the location ofthe binding sites for themethylmercury(II) chloride used as a heavy-atom derivative.Combining this information, the a-carbon model of the A-Bmolecule was positioned in the electron density belonging tothe C-C dimer. Overall, the correlation between the rotatedmodel and the electron density was only'marginal. In orderto further optimize the model-map fit, the coordinates weresystematically rotated around, as well as translated along, thecrystallographic 2-fold axis in the region of the C-C dimer,and a correlation coefficient was calculated. An optimalposition was found. However, the model-map correlationwas not as good as was observed for the A-B dimer. Oneexplanation for the weaker electron density in the region ofthe C-C dimer is some form of conformational variabilitycaused by crystal packing. Different forms of crystallinedisorder have been observed for other proteins, includinginstances where entire domains are disordered (20).Conformation of L-3-Hydroxyacyl-CoA Dehydrogenase.

The stereodiagram in Fig. 1 shows an a-carbon model of theA-B dimer viewed along the molecular dyad. Also includedin this figure is the cofactor, NAD. The existence of signif-icant amino acid sequence homology between the amino-terminal end of L-3-hydroxyacyl-CoA dehydrogenase andother NAD-dependent dehydrogenases has made it possibleto establish a tentative but plausible relationship between theamino acid sequence and the three-dimensional structure.Using the known three-dimensional structures of severalNAD-dependent enzymes and the above-mentioned se-quence homology, the amino acid sequence for the first 50 or

so model residues has been established. Based on thissequence assignment, it is concluded that no electron densitycan be associated with the first two residues, probably due todisorder in this part of the molecule. Analysis of the bindingsites for the methylmercury derivative used in the MIR phasedetermination provides another sequence reference point. Ineach of the three subunits, the major methylmercury bindingsite is closest to model residue 204. The single cysteineresidue in L-3-hydroxyacyl-CoA dehydrogenase has se-quence number 204, suggesting that the 19 missing residuesprobably belong to the last third of the primary sequence.The conformation ofthe enzyme is that ofa double-domain

structure with a bilobal appearance. The amino-terminaldomain, residues 1-200, is the larger domain and is structur-ally homologous with the NAD binding domains observed inother dehydrogenases (21, 22). As can be seen in Fig. 1, theNAD binding site is indeed found in the amino-terminaldomain and consists of an eight-stranded /B-sheet flanked bya-helices. The first six strands are parallel to each other, andthe last two strands run in the opposite direction. Eventhough the amino acid sequence is not incorporated into themodel at this stage, we are quite certain about the connec-tivity in this part of the structure. As is the case with otherNAD-binding enzymes, the binding site for the coenzyme isat the carboxyl terminus of the parallel 3-sheet and at theamino terminus of the a-helices. The smaller lobe constitutesthe remainder of the subunit. So far, residues 201-288 havebeen incorporated into the model. The only type of secondarystructure seen in this domain is helical (Fig. 1). Parts of thecarboxyl-terminal domain of the A-B dimer are in closeproximity to the C-C dimer in the crystal lattice, and some ofthe 19 residues missing in this domain may eventually beaccounted for when a complete independent model is derivedfor the dimer with crystallographic symmetry.NAD Binding Site. The electron densities for the two NAD

binding sites in the A-B dimer were readily interpretable interms of molecular models ofNAD. The initial interpretationwas facilitated by the use ofNAD coordinates obtained fromthe holo structures of other dehydrogenases, particularlyglyceraldehyde 3-phosphate dehydrogenase. The NAD moi-ety can be placed into the difference density in two ways

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130

FIG. 1. Stereodiagram of the L-3-hy-droxyacyl-CoA dehydrogenase dimer. Theview in this a-carbon diagram is down themolecular dyad axis. The A-B dimer de-scribed ip the text is shown with the Asubunit in the upper half, outlined in thethinner lines, and the B subunit in the lowerhalf, shown with the darker lines. Alsoshown are the bound NAD molecules aspositioned from difference Fourier maps.The numbering system for the A subunit isbased on the current molecular model ofL-3-hydroxyacyl-CoA dehydrogenase and

>A does not correspond to the amino acid se-ZNP«Jquence numbering system (6). Residues in\J V the B subunit have not been numbered, to

improve the clarity of stereoviewing.

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FIG. 2. Holo - apo difference electrondensity. The electron-density map belongingto the bound NAD at the A subunit ofL-3-hydroxyacyl-CoA dehydrogenase isshown. Superimposed on the density is amolecular model ofNAD as observed boundto one of the subunits of glyceraldehyde-3-phosphate dehydrogenase (23). The confor-mation of the NAD was not changed duringthe fitting and has the adenine ring in the anticonformation and the nicotinamide ring inthe syn conformation.

because the cofactor has crude internal 2-fold symmetry.Similar to what has been observed in other NAD-enzymecomplexes, one end of the difference density is noticeablyweaker than the other, and this part has always beenassociated with the nicotinamide moiety. Consequently thenicotinamide ring of NAD was placed in the end of thedifference peak with the lower level of electron density. Thenicotinamide ring can assume two orientations around itsglycosidic bond. In the syn conformation as bound to L-3-hydroxyacyl-CoA dehydrogenase, the A side of the nico-tinamide ring faces the solvent and the B side is adjacent tothe protein surface. In the anti conformation the situation isreversed. Since the enzyme is a B-side-specific dehydrogen-ase, the B side of the nicotinamide ring must face the solventregion where the substrate is bound, and therefore thenicotinamide conformation is believed to be syn when NADis bound to the enzyme. The good correlation between thedifference map and the model of NAD is shown for onesubunit of the A-B dimer in Fig. 2. When this conformationis shown in relationship to the entire dimer, as is done in Fig.1, it is very apparent that the active sites in the dimer arewidely separated, by 31 A at the closest approach, and arefully contained within one subunit.

Subunit-Subunit Interactions. The subunit-subunit inter-face for the dimer is provided nearly exclusively by residuesin the smaller domain found at the carboxyl-terminal end ofthe polypeptide chain. The most extensive contacts are foundbetween two a-helices comprising residues 204-222 (Fig. 1).These two helices are within 300 of being antiparallel to each

other, and the molecular dyad is perpendicular to their helicalaxes. A short stretch of P-structure, residues 237-240, alsocontributes to the subunit-subunit interactions. A third set ofinteractions are found between the helical residues 228-236from one subunit and residues 193-197, which are in anextended conformation, from the other subunit.

DISCUSSIONThe dinucleotide binding domain of L-3-hydroxyacyl-CoAdehydrogenase resembles the corresponding domains in mostother oxidoreductases. Common to these enzyme structuresis the presence of a conformational unit consisting of a four-stranded parallel 8-sheet and one a-helix (22). Anotherfeature of interest in L-3-hydroxyacyl-CoA dehydrogenase isthe occurrence of two a-helices in the crossover connectionbetween p-strands, j8B and PC. This arrangement closelyresembles the conformation present in the FAD bindingdomain of glutathione reductase (24). Also, the last twostrands of the P-sheet in L-3-hydroxyacyl-CoA dehydrogen-ase, PG and PH, are found in the opposite orientation fromthe other sheet components; so in effect, the P-sheet super-secondary structure in the nucleotide binding domain is reallya mixed one, although predominantly parallel.The conformation of NAD that best fits the difference

electron-density map between holo and apo forms of L-3-hy-droxyacyl-CoA dehydrogenase resembles the structure ofthe coenzyme when bound to another B-side-specific dehy-drogenase, glyceraldehyde-3-phosphate dehydrogenase. The

FIG. 3. Hypothetical ternary complex.The stereodiagram shows a schematicdrawing of the molecular model of the AsubunitofL-3-hydroxyacyl-CoAdehydroge-nase together with a bound NAD. Thehypothetical binding mode for the fattyacyl-CoA substrate is also shown. NAD andCoA are shown as ball-and-stick models.

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structure of the enzyme and the position of the bound NADmake it possible to speculate about the substrate binding site,although no definitive substrate binding studies have yet beendone. The nicotinamide ring is located in the cleft separatingthe two domains, with the B side of the nicotinamide ringfacing the solvent and the A side facing the enzyme. Fur-thermore, the stereochemistry at the 3-carbon of the fattyacid is the same as at the 2-carbon of malic acid, the substratefor malate dehydrogenases. Therefore, in our modeling oftheenzyme-substrate complex, we used the same stereochem-ical relationship between carbon atoms in the substrate andNAD as has been postulated for cytoplasmic malate dehy-drogenase (25). When a L-3-hydroxy fatty acyl-CoA moleculeis positioned adjacent to the nicotinamide ring of the NADsuch that the hydrogen on the 3-carbon of the fatty acid isnear the 4-carbon of the nicotinamide ring, only two generalorientations for the substrate seem possible. The morefavorable one places the fatty acid moiety in the narrower endof the cleft between the lobes, with the alkyl chain stretchedacross the surface of a helix comprising residues 251-266.The cleft is visible in both Fig. 1 and Fig. 3. In thisarrangement, the hydrocarbon chain of the fatty acid moietyis in contact with protein on all sides except at the further-most end. The CoA moiety is placed in the more open end ofthe cleft, where it makes relatively few contacts with theenzyme. The structure ofthe CoA part ofthe substrate shownin Fig. 3 is similar to that observed in crystalline complexesof citrate synthase (26). However, the 3'-phospho-ADP-pantothenate moiety appears to be quite flexible and it couldbe in contact with many different parts of the dimer. In analternative orientation, the two ends of the substrate areinterchanged. The adenine end is cramped into the narrowerend of the cleft between the lobes and, in fact, would have tochange conformation into a more extended form than thatshown in Fig. 3. The hydrocarbon chain of the fatty acidmakes fewer contacts with protein in this arrangement, andis more exposed to solvent. Finally, the stereochemicalarrangement in the catalytic center is not as satisfactory as inthe first orientation. Although speculative, the preferredorientation is shown in Fig. 3.The model of L-3-hydroxyacyl-CoA dehydrogenase that

has been presented in this report is still incomplete in thatapproximately 20 amino acids are still not accounted for andside chains have not been fitted, but we believe that most ofthe missing residues belong to the carboxyl-terminal domain.The electron density associated with this part of the structurewas more difficult to interpret due to the proximity ofelectron density attributable to other subunits in the crystallattice, particularly the subunits belonging to the C-C dimer.It should be possible to improve the electron-density map,particularly at higher resolution, by using model phases oncethe amino acid sequence has been incorporated into thepresent interpretation.

We thank D. Schuller for performing the density-correlationexperiments and C. Nielson, D. H. Anderson, and Dr. S. L. Ed-wards (University of California at San Diego, La Jolla, CA) forassistance during data collection. We are grateful to Thom Meiningerfor his continuing help in enzyme purification and to Dr. J. Ross, who

aided in the determinations of the steric relationship of the A-B andC-C dimers. We thank Dr. Paul Bethge for his continuing help withour computer graphics systems and Dr. C. M. Cambillau for hisgraphics program TOM. This work was supported by NationalScience Foundation Grant PCM-8208894 (L.J.B.) and NationalInstitutes of Health Grant RR 01644 (N.h.X.).

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