Three-dimensional structure of horse liver alcohol dehydrogenase at 2.4 Å resolution

J. Mol. Biol.

(1976) 102, 27-59

Three-dimensional Structure of Horse Liver Alcohol Dehydrogenase at 2.4 A Resolution HANS ~~KLCND. GUSTAF

HONORDSTRGM,~ILAZEPPEZA~JEK

S~DERLVTND.

[NGRID

BENGT-OLOFS~DERBERG.ORLANDO

Agricultura.1

OHLSSON.

TORNE BOIWE BRANDI~N

TAPIA,(:ARL-~.VAR

1)epa:rtment of Chemistry Collpg~ of #wedenT? S-750 07 I’ppsala

7. Sweden

AS I) AiKE LAKESOK Laboratory

Karolinska

for Eszyme Research,. Nobel Institute of Biochemistry Irditute. A’ohvtigen 1. 8-104 01 Btockholm 60. Sweden

(Received 5 August

1.975. and iv& twiwl

,fwm

II ilrowmber

1976)

The crystal structure analysis of horse liver alcohol dehydrogenase has been cxt,ended to 2.4 A resolution. From the corresponding electron density map of t,tlra apoenzyme we have determined the positions of the 374 amino acids in th<, polypeptide chain of each subunit. The coenzyme binding domain of the subunit comprises residues 176 to 318. 45!/ of these residues are helical and 32% are in t,he central six-stranded pleated sheet structure. The positions and orientations of the helices with respect to the pleated sheet indicate a possible folding mechanism for this part of the subunit nt,ructure. The coenzyme analogue ADP-ribosr binds t,o this domain in a position and orientation very simila,r to roenzymn binding to lactate dehydrogenase. The adenine part binds in a hydrophobic pocket. the adenosine ribose is hydrogenbonded to the side chain of Asp223, the pyrophosphata is positioned by interaction with Arg47 and thr nicotinamide ribosn is 6.4 away from the cat)alytir zinc atom. Thr catalytic domain is mainly built up from three distinct) antiparalkl pleated-sheet, regions. Residues within this domain provide ligands to the catalytic zinc atom; Cys46, His67 and Cys174. AIL approximate tetrahedral coordination of this zinc is completed by a water molecule or lrydroxyl ion depending on the pH. Residues 95 t,o 113 form a lobe that binds the second zinc atom of tile subunit. This zinc is liganded in a distorted tetrahedral arrangement by four sulphur atoms from the cystaine residues 95. 100. 103 and 111. The lobe forms OIIR side of a significant cleft in the enq-me surface suggesting that, this rrgiorl function. might constitute a second catalytic centre of ~11~1~no~m Thr two domains of the subunit are separat,ed by a crevice tllat contains a wide and deep hydrophobic pocket. The catalytic zinc atom is at the bottom of this pocket, with the zinc-bound water molecule projf,cting out into the pocket. This natcr molecule is hydrogen-bonded to the side c*ttaill of Sear48 which in turn is hydrogen-bonded to His51. The pocket which in all probability is the binding site for the substrate and tJhf: nicotinamide moiety of the coenzyme, is lined almost exclusively with hydrophobic side chains. Roth subunits contribute rcxsidues to each of the two srlbstrate binding pockets of the molecule. The onI5 accessible polar groups in the vicinit,y of the caatalytic rentre are Ser48 and Thrl7R apart from zinc and the zinc-bolmd water molecsulr.

“<

ti. I~:I~I,I-S~I ~7’ .tr,

1. Introduction Horse liver alcohol dehydrogenase (EC’ I .l. 1.1) is an NAD +-dependent enzyme which cat’alvzcs the oxidat’ion of various primary and secondary alcohols to the wrresponding aldehydes (Sund & Theorell. 1963: Brandhn et ccl.. 1975). The actiw cnzymc has a molecular weight, of 80.000 and is a dimer of two idernical subunits. The sequence of t’hc 374 amino acids in the single polypeptide chain of one subunit, has been determined by Jornvall (1970). Each subunit, firmly binds tnw zinc at,oms and has one coenzyme binding sit,e. The enzyme crystallizes in an ort,horhombic space group in the absence of coenzyme (Zeppezaurr of al.. 1967) with one subunit in t,he asymmetric unit. Binar! wmplexes wit,11 inhibitors such as ADY-ribose (Abdallah PI nl.. 1975). salicylate (Einnrsson of al., 1974), 1.lO-phcnanthroline and imidazole (Boise & Brand&, 1976) crysta,Jlize isomorphously wit,11 the apornzyme. The presence of alcohol during crystallization does not induce any major conforma,tionnl change since such cryst’als are isomorphous t,o t’hosr obbained from a highly concentrated cnzym~~ solution in weak phosphate buffer (Brand&r of ab.. 1965). However. complexes containing cocnzyme result in different crystal forms, triclinic or monoclinic, depending on slight variations of cryst~allization conditions (Zeppezauer Pt ~1.. 1967). Both t,hcse wyst’al forms contain t’he whole dimeric molecule in the asymmet8ric unit. The relationship between the symmet’ry and cell dimensions of t8hest crystal forms has been discussed a,nd int,erpreted in terms of a coenzymc-induced conformational change of t’hc enzyme molecule (Brand&n. 1965). Low-resolution studies of bhe orthorhombic apoenzyme crystals in combination with inhibitor studies gave t,he overall dimensions of the molecule and the position of the coenzyme binding site (Brand& it al., 1972). From a preliminary high-resolution study to 2.9 A resolution (Brand&r it aZ., 1973) we obt.ained the general fold of the ma,in chain and t’he positions of the two zinc atoms in the alcJho1 dehydrogenase subunits. Simultaneous comparisons of this strucbure and that of glyceraldehyde-3determined phosphate dehydrogenase (Buehncr et cd.. 1973) with t’he previously sbructure of lactic dehydrogenase (Adams et aZ., 1970) revealed some basic structurefunct~ion principles within the family of dehydrogenases. The subunits of these enzymes are divided into t,wo separate domains, each associated with a particular function. The coenzyme-binding domains exhibit, fundamental similarities in their st,ruct,ures as well as in their mode of coenzymc binding, whereas t’he catalytic domains, which bind different substrat,e molecules have very different’ stru&ures. The st.ructural and functional similarit(ies within these coenzyme-binding domains have been studied in det,ail (Ohlsson et al., 1974) and an evolutionary relationship including flavodoxin and kinases has been established (Rossmann et al., 1974). m’e report here a more complet’e descript’ion of the structure of liver alcohol dehydrogenase as well as crystallographic details of t.he high-resolution work, which has now been extended to 2.4 w resolution. From a knowledge of the primary structure we have been able to position the side chains of the molecule within t’his improved elect,ron density map with a high degree of confidence. In t,he following paper (Eklund et ccl.. 1976) we compare this structure with the sequences of other alcohol dehydrogrnases and predict t,hat t#he general folds of the alcohol dehydrogenase subunits. including the yeast enzyme, are quite similar. A preliminary report of the structure and some of its functional implications has been published (Eklund of ccl.. 1974).

30

H.

EKLUND

ET

ill,.

We thus obtain a substantial improvement in accuracy with a reasonable extension of t,otal counting time for reflexions that have A medium or moderately weak intensity (1). From experience we have found that only marginal improvement is obtained when reflexions are measured in this way whorl S is larger than S,,,. These reflexions are in general very weak and would require much longer counting times to be improved. Thus we do not remeasure those reflexions but accept their low accuracy. Using this method it was possible to measure about 30 to 50 reflexions per h with a satisfactory relat,ive accuracy. In Fig. 1 WC show a histogram tha,t gives the number of reflexions z’ersu8 relative accuracy for the tlative (Iata set.

( I

2500

2000

Cc)

(b)

a)

-----1

t

?

1

5 1500

1

1000

I-

5oc

)I

C )O

4

8

12

16

.20

1 0

I

crl

:_

1

8

12

u F/F

16

>20

I04,TLLn 8

12

16

FIG. 1. Histogram showing the number of X-ray reflexions, S. of nat,ivo ADHas Jesus their relative accuracy, 100 x u(P)/&‘. (a) Data for Bragg spacings cc to 3.7 a (b) Data for Bragg spacings 3.7 to 2.9 A. (c) Data for Bragg spacings 2.9 to 2.4 A. Only the 3166 strongest reflexions were measured in interval C.

20

‘20

crystals

The 8570 independent reflexions to 2.9 f, resolution were divided into 4 different shells as a function of sin 0 with approximately the same number of reflexions in each shell. In general we increased the measuring time for reflexions in shells of high 0 in order to obtain an approximate equal relative accuracy for most reflexions. Indices for all reflexions within each shell were computed in advance and stored on disc. During data collection these indices were read by the computer one at a time and the corresponding reflexions were measured. All reflexions with Bragg spacings between 2.9 and 2,4 .& were treated as a fifth shell containing a total of 6940 possible reflexions. All these reflexions were measured for the native enzyme. However, since many of these were very weak, we only measured derivatives. the 3155 strongest reflexions (Snative less than 0.30) for the heavy-atom 11,725 independent intensities were thus collected for the native protein and 3 of the the mercury complex, data to on15 heavy-atom derivatives. For the fourth derivative, 2.9 A resolution were collected.

HORSE

LIVER

iZLCOHOL

DEHYDROGENASE

3I

The reflexions from each crystal were measured in sets of 100. Five standard reflexions. chosen within the corresponding shell. were measured before each such set in order to follow the time-dependent decrease in intensity. When the mean intensity of the standard on the crystal were stopped and reflexions had decreased about 15 “/b the measurements continued on a fresh crystal. Somewhat larpcr radiat,ion damapt> was accepted for thti mercury derivative.

The intc*nsitias were corrected for t,he Lorrntz factor in the usual way. Polarization corrections were applied according to the formula given by Arndt & Willis (1966) fol ideal mosaic crystals of both the sample and the morrocllromat~or. Empirical absorption corrections were determined and applied according to t’he methods given by Nortall ef al. ( 1968). A linear least-squares fit of normalized F-values Z’~TSIL~ time was calculated for each standard reflexion in order to correct for the tilne-dependent decrease in intensity. The mean values of t,he coefficirnts of thesct crc1unticjrls Tvere theta used to computcl thts time corrections. Dat,a from different crystals of t,he same derivatives \~ere scaled together by comparing the F-values of 30 strong reflexions selected from diffrrcnt’ regions of tho reciprocal lat,ticch. These reflexions were always mcwsured at the hepirlninp of the data collection rontint, for evr+r’y new crystal. By maltirlg an immediate preliminary analysis of thcase scaling rc+iexic)nn we could easily detect crystals with nnonialios it) th(x diffracted intensities. and discard these crystals boforr any more mainly due to low heavy-atom occupancy. wvhfxi a complete clata set, lrad been niraslirrd for a data. \vere collected. Furthormorcl, particular derivative, all crystals werp scaled t,ogetllcr and tlio r.csult)s analysed. If tllrl P’-v;tll~r%s of thrsr scaling reflrxions for a particular clrystal differed by mom than 3O,, from t hc m(san values of all cq,stals \VP discarded all tlatx tllat Ilad been collected on that crystal. As a further test of the corrc&uns for thr t,iInc~-tlt:perldt!lIt decreascl in intrnsit\and on this scaling procedure we collected additional ovorlappin, (7 data from a freslt cryst,al. \j-ci thus remeasured 50 reflexiorls from the beginning and end of t,he dat,a sets collect,ed on c~cll individua,l crystal. From tht: analysis of those overlapping reflexions we found t,hat t,hc seal<’ factors were in general correct but t.hat, additional small but significant. tirnc corrcac.tions wore necessary in scjmc casrs. As a final tchst of tile scaling procedure we (*on1paled corresponding reflexions from raeI Ilcavy-atoln derivative with those from tIlta tlati\-(3 crystals. Mean vallles of tllr scale factors for 100 corrsc~cntivc~ly collcctcd rrfkxiol~r; \vere computed. An error in the scalr fartor of a parti~lllar (arystal cc)llld (&I\ bfs spottrtl I)y inspwtiolr of these mpan \-alrir*s. (d)

Hearty-atoms

ant1 phase

analysis

‘I%(~ lltravy-atom complexes 11s~~l here bind at 4 differelrt positions at the surface of the subunit, each complex having H preference for crrtaitl sit,es. The single binding site of Pt(CN):-. site A, and the maill binding site of Au(CN); , site B, were located (Siiderkrg et al., 1970) and rc&od (BrHndtn et al.. 1972) in tllr low-resolution study. High -rt~solut,ion difference electron drnsity maps revralrcl t,lle presence of a second sit)e with lower occupancy, site C. for tile Au(CN),~ complex. In addition a small number of tnolecules also bind Au(CN); at site A. In tile double derivat.ive containing both Au(CN), and Pt(<‘N): this site is occupictl by Pt(CN):. The Ilt~avy-atom positions of the mercuq dtsri\.wtive were located from difkc?ncc electron density maps using phases derived from the otller 3 derivatives. \Ve found that ethylmercurythiosulicylate binds at 2 sites, C and D, CHC’II with less than half occupancy in most crystals. Site D is very close to the 2-fold rotation axis that relates 2 molecules in the crystal latt,ice. The low occupancy of this site may reflect steric hindrance for the D-sites of adjacent molecules to be simultaneously occupied. Since this derivative was prepared with excess Pt(CN):--, we also found that site A was fully occupied. All major sites were confirmed by inspection of high-resolution difference Patterson maps. Final difference electron density maps gave no indication of additional minor sites. A description of t,hr side rhains in\~olrnd in t,hrse binding sites is given in a later section of this paper.

06 250

200

15c

IOC

50

I C

I

I

I

I I 0 IO Y”e/t

1

I

I

1 020

PIG. 2. Accuracy of phane determination as a t’unctilJn of sm B/h. Th $1~~xplanation of the ewves from the top downward is as follows: CUI’VP m(ordinate at, top right,) is the average figure of derivatives merit of the phase angles. The 4 (upper) curves for the IfI valurs of thr heavy-atom refer to the averagr root-mean-squares of l,fFxl. The E; values (low-w 4 curves) represent the rootmean-square lack of closure errors of thr phase triangles for the 4 drrivativns. / , (~thylmhrcurythiosalicylatt~ I K,l’t(m),; : 1 K,Pt,((‘N), ; KAll((‘N),. . li,lY((‘S), , KAn(C’N),:

3

34

H.

EKL

UN 1) E 1’ d I,.

Final hoa.vy-atom parameters as ~011 as R-values are given in Table 1. Some additional rc+intrment~ criteria useful in e&mating the accuracy of thn phase determination are shown in Fig. 2. (H) Electron density map and model building Elrctron density maps WHIW computed by a programmu kindly provided by G. N. Keekt, using the “best” Fourier synthesis (Blow & Crick, 1959). Sections wore computed perpendicular to the y-axis wit11 computational imervals 0.42 A%in a, 1.00 !I in b and 0.63 A4 in c. Contours wcrc computar printed using different symbols for different~ levels of electron tlensity. Since the scale \+-a~ 2 cm to the A&strom those contours could be directly taraced onto Mb-lar sllccts for IIW it1 an optical comparator (Richards. 1968). The first contour lovcl was cllosen at 0.33 c~,I:%~and t,ltc? following contours at intervals of 0.10 e/.q3. The maximum map dnnsit,ios at the centrcs of the 2 zinc atoms wore 2.9 1 e/AR and 2.42 e/AZ. Some sections t,hrouglr tllc: map are showrl ill Fig. 3 to illust,rate tlrc% quality of the map. In ortlcr to study thtl detailed shapes and positioris of tlrr, zinc ligands wc calculated a special map, p (ctlzymc ~zinr). itr tllc regions around ttrc 2 zinc atoms :

lilt:. 3. Vievv of 5 composite sections of constant y from 0.1729 t,o 0.2261 from t,he horse liver alcohol dehydrogenase electron den&y map to 2.4 d resolution. This se&ion goes through the cent,rel part of the molecule and illustrates part of the pleated-sheet regions of t,ho coonzym~~. binding domain, the catalytic zinc atom and some side chains. where f+,,,,, = calculated structure factor contribution, to reflexion h, from thu 2 observed zinc positrons in the asymmetric unit. The occupancy of the zinc atoms was obtained by trial and error to produce zero electron donsity at the zinc positions. The values were

HORSE

LIVER

ALCOHOL

DEHYDROBENASE


X

=

J.

FIG. 4. Part (a) Electron (b) Electron calculated zinc

of electron density

3i I

density from

density of contribution

the

map

showing

“best”

Fourier.

the smm region WRS wbt,rart,ed.

tho ligands but

computrcl

J

to the act~ivo-site from

*tructure

zinc atom. f’artors

where% thrs

xi

H . E I( I,17 N 1) E:T' d 7,.

found to he 33 and 30 &ctrons, showing t,llat the absolute scale of th(l F-values is approximately correct. The result,ing map around tho catalytic zinc atom is shown in Fig. 4 and c>omparcd to the “hrst” electron density map in the same region. Cloarly the nature of tllo zinc ligands ca,n be identified wit11 A high degree of confidence bb- t,his proaedurc and their positions can, furthermore, be more accurately determined. mirror device and a skeleton Tho electron density map was mounted in a Richards Kondrew model was built, in the usual way. ln order t’o saVe space and at the same time maintain a rapid method for changing sections for display. we have made some modifications in the usual procedure which aro useful for the model-building of big molecules. The sheets of Mylar tape were fixed to and wound around a,luminium tubes with a 2-cm diameter. These tubes were fitted with rolling spring devices so that each sheet could br easily drawn up or down like a roller blind. When the maps are displayed they arc kept at the correct posit,ion by fitt,ing 2 aluminium bars at the bottom edge of the map into corresponding holes on 2 girders at, the bottom of the map area. The girders are oriontrd perpendicular t,o the sheets of tllch map and the distance between the 2 girders is slightly shorter tlhall t,lle dist,ance het\vPt%n t.he ends of tile bars on each sheet. For the measurements of initial approximate at-omit co-ordinat,es we have used a measuring screen, the same size as a section of the map, which can bc translated to any position in the map area. A light point, whicll is movable in 2 perpendicular directions, is fitted to the rear of the screen. The light point can tllus be moved ho coincide with the mirror image of an atom in the Kendrew modf>l and tho co-ordinates of tile light point are recoidod. The co-ordinate sot obtained in this way is now being refined against idealized bond lengths and angles losing a programm(x kindly- provided by R. Diamond (1966).

3. Results (a) Interpretation.

of the electron

density

rnq

Compared to the earlier map (BriindBn et al.. 1973), it is much easier to follow the main chain density unambiguously in this 2.4 A resolution map and to obtain the correct amino acid assignment. Cys174, Phe176 and TyrlSO, at the beginning of the nucleotide-binding domain, are very well defined and were used as starting points for the interpretation. A number of internal well-defined residues within this domain, mainly phenylalanines. methionines. cysteines, leucines, isoleucines. valines and Trp314 were used as markers for the positioning of other less well defined, polar. external side chains. Knowledge of the assignment of residues in the coenzyme-binding domain facilitated the interpretation of the second domain. The quality of the present map is such that almost all carbonyl groups of the peptide bonds are clearly visible and that there is a dimpling of the electron density at the centres of six-membered rings. Using the amino acid sequence determined by Jiirnvall (1970) we could thus position all residues, except the first four, at t’he amino end where the density fades out on t’he outside of the molecule. The amino acid assignment reported here differs from our preliminary assignment, of that map most at the 2.9 A stage (BrBndbn et al., 1973). In the interpretation main chain regions were found in the whole molecule and they were correctly connected in the nucleotide-binding domain. However, in the second domain, the two ends of the chain were not correctly identified and a few connections between main chain regions were in error. In the earlier map, the side chains were in general not’ sufficiently well defined to permit an unambiguous identification. Independent. confirmation of the correctness of the amino acid assignment reported here has been obtained. Sequence analyses have shown that Cys46 can be selectively carboxymethylat,ed by iodoacetate hoth in solution (Harris, 1964; Li & Vallee, 1964;

HORSE

LIVER

Al,(‘OHOl,

DEHYDROGENASE

37

Jornvall. 1970) and in the crystalline state (Zeppezauer et aE., 1975). A difference electron density map of the carboxymethylated enzyme shows one significant positive peak close to the position of the sulphur atom of Cys46 in the present model (Zeppezauer et al., 1975). Furthermore, specific labelling in solution of cysteine residues in the yeast and liver enzymes l)y modified coenzyme analogues (Jiirnvall et al., 1975) shows t,hat, Cys46 and Cys174 mast bc close together in space. They are in fact bot’h ligands to the same zinc atom. In conclusion, knowledge of t,he amino acid sequence facilitated the correct tracing of the main chain and was a prerequisite for the exact identification of most side chain densities, Furthermore, there are no inconsistencies between the reported amino acid sequence and those side chains in our electron density map that can he independently ident,ified hp the shape of t’he density. (h) lkwcription~ of the structure (i) The s&unit Each subunit is divided into two domains which are separated by a cleft containing a deep pocket. This pocket accommodates the substrate and the nicotinamide moiety of bhe coenzyme. One of the domains is responsible for binding the coenzyme and the other provides the groups necessary for substrate binding and specificity. The two domains are unequal in size ; the catalytic domain is larger and comprises 231 residues, whereas the coenzyme binding domain is built up from 143 residues. The two subunits of the dimeric molecule are joined together by homologous interactions (Monod et al.. 1963) within corresponding regions of the coenzyme binding domains. The fold of the polypeptide chain within one subunit is illustrated in Figure 5(a). The whole molecule has an approximate helical content of 297; of the residues while 34% are in pleated-sheet regions. Figure 6 lists the primary structure as determined by Jijrnvall (1970) and the various regions of secondary structure of the subunit. (ii) Z’hp catalytdc domain The catalytic domain comprises residues 1 to 175 and 319 to 374. Both ends of the polypeptide chain are thus within this region. The two zinc atoms of the subunit are bound to ligands from this domain. A st’ereo diagram of the a-carbon positions and th(b zinc atoms of the catalyt’ic domain is shown in Figure 5(b). The corresponding main chain hydrogen-bonding pattern is shown in Figure 7. There are only four helical segment,s in this domain comprising 19”/b of the residues. In contrast, 35% of the residues are in pleated-sheet, regions and 147(, in reverse bends. Thus a large number of residues, 320/o have no regular secondary structure. The polypeptide is folded into a complicat’ed network of /?-structures in this domain. There are three main regions of pleated sheet which are called PI, PII and /?III. The strands of these sheets are mainly anti-parallel. Schematic diagrams of these three regions are shown in Figure 8. Region /?I is composed of six strands, comprising residues 41 to 45, 68 to 71, 88 to 92, 156 to 160, 347 to 352 and 369 to 374, which form a twisted wall through the top half of t,his domain. The strands are antiparallel except the last two. Most’ of the remaining residues in the amino-terminal half of the chain are on one side of the twisted wall formed by the strands of PI. These residues can be divided into three separate regions; the two pleated-sheet structures PII and PI11 and a lobe comprising residues 95 to 113 that binds the non-catalytic zinc atom.

3s

H.

EKLCND

ET

AL.

Sheet /3II is built up by four anti-parallel strands, comprising residues 34 to 40, $2 to 78, 86 to 87 and 148 to 152. One of these. /311:3, is short and provides only two hydrogen bonds to neighbouring strancls. Three of the stranck, flII : 1. /311:2 and PII :3, are direct continuations of strands in ,31 (1. 2 and 3, respectivefy). There is, however,

HORSE

LIVER

ALCOHOL

39

DEHYDROGENASE

Ce) FIG. 5. Stereo diagram of the molecule’s (I,,, atom backbow. (a) One subunit viewed along the direction of the 2-fold axis. (b) The catalytic domain viewed in the same direction as in (a). (c) The coenzymo-binding domain wit,h the bound ADP-ribosr molcculr direction to that in (a). (d) and (R) The complete enzyme molecule with 2 honn~l A [)I’-rihow different, directions.

viewed in the oppo&r molrcules

viewed

in 2

a change in direction of approximately 90” between corresponding strands in the two sheet regions. The strands of these anti-parallel pleated sheets are furthermore twisted in such a way that the strands of /311 in combination with some of the strands of PI enclose an approximate cylinder as shown in Figure 9(a). Inside this cylinder there is a tightly packed core, Kl. of 16 hydrophobic side chains. half of which are valine. These residues are listed in Table 2. The third pleated-sheet region within this domain. /XII, is built up by six strands comprising residues 9 to 14, 22 t’o 29, 62 to 65, 129 t,o 132, 135 to 138 and 145 to 146. These strands are linked in a rather irregular fashion and are twisted so that they form a closed set. in which each strand is hydrogen-bonded to two of its neighbours. A stereo diagram of this arrangement is shown in Figure 9(b). Similar cylinders of pleated-sheet have been found in the serine proteases (Birktoft et al., 1970; Shott,on t Wats.on. 1970) and in thermolysin (Colman et al., 1972). Although the number of strands in these cylinders is usually six, there seem to be no general similarit,ies between these different superstructures, but rather a way of clust,ering residues inbo hydrophobic cores. Side chains in the interior of this cylinder, in alcohol dehydrogenase. form a second core, K2, of hydrophobic residues. These residues are listed in Table 2. Tryptophan 15 is at the base of this cylinder and covers the open end t,owards the solution. One side of the aromatic ring system of this tryptophan is thus well exposed to the solvent, wherea,s t#he other side facts the hydrophobic core.

c

FIG:. 7. The main chain hydrogen-bonding itntl:ilM

scheme in the catalytic

domain

(residues

I to 174

to 374).

There is a clustering of internal serine and threonine side chains from residues 43, 143. 144, 145, 147 and 150 between the two hydrophobic cores Kl and K2. The OH-groups of these side chains are generally hydrogen-bonded to carbonyl oxygen atoms from sequentially quite different, regions of t’he main chain. The resulting network of hydrogen bonds forms a wall in t’he interior of the domain t#hat sepa,ratcs the two hydrophobic cores Kl and K2. The four helices present, in bhis domain compzisr residues 46 to 53. 168 to 175. 324 to 338 and 355 t’o 365. These are all a,ligned at the surface of the molecule outside interior pleated-sheet regions. In general. one side of the helices faces t.he solut’ion and t*he other side provides non-polar residues for either of’ t’wo additional hydrophobic cores. K3 or K4 (see Table 2). These hydrophobic regions form interaction helices and pleated-sheet’ regions. Thtw is an aromat8ic cluster of six areas ~JdXW?ll phenylalanine residues in cow K3. Ttw strucbure determination confirms the prwence of two tirmly twund zinc atoms per subunit, in liver alcohol dehydrogenasc (akeson, 1964). Both t,hese zinc atoms are bound to ligands within the catalytic domain. The co-ordination around the zinc atoms is presented in a later section of this paper. An internal charge interaction is found close to the cntal*vtic zinr atom hdween t,ht side chains of argininc 369 and ylutmlic*

krcitl 68.

FIG. 8. Schematic

diagram

of the

:< differrut,

ple,ztd-sheet

struct~ures

in thu

cittrtlytic

dom&u.

A large proportion of the residues that exhibit no obvious secondary structure are located in the region 93 to 122. This region comprises the lobe that binds the second zinc atom (see Fig. 10) and a stretch of residues that form part of the entrance to the deep pocket between the subunits.

HORSE

LIVER

ALCOHOL

FIG. 9. Stereo diagrams of the strands of pleated-sheet approximate cylinders. (a) Strands from pleated-sheet regions @I and ,!llI. (b) Strands from pleated-sheet region /3TII. TABLE

Residues in the catalytic Cbre Kl Val36 11038 Met40 Ala69 Va173 Val80 Va183 Val89

Pro91 Met123 Thr143 Thrl46 ThrlBO VallSl Vall62 Va1157

in the catalytic

domains that enclose

2

domain which form hydrophobic Core K3

Core K2 Ala11 Va113 Va126 Ile64 Phe130 Ile137

43

DEHYDRGGENASE

Ala1 2 Leu14 PhePl 11045 Thr59 Va163 Ala65 Gly66

Core Kl comprises residues from the pleated-sheet of the cylinder formed by /III, core K3 comprises from fir, a2 and ~3.

cores Core K4

Phe140 Phe146 Phe352 110335 Phe359 Leu362 Thr370 Lou372 Phe374

ala70 IleQO 110160 Pro166 Leu166 Va1169 Lou171 Ile172 Gly173

Phe176 Va1328 Leu331 Va1332 Phe336 Met336 Phe340 Leu342

regions /31 and PII, core K2 is at the interior residues from /?I, PI11 and ~4, and core K4

FIG. 10. St,ereo zinc atotn.

(iii)

diagram

of t,he lobe

The coenzyme binding

ropion

comprising

residues

96 to 1 I4 that.

hind

t,hP second

domai-n.

The coenzyme binding domain comprises residues 176 to 318. A st’ereo diagram of the or-carbon positions of this domain is shown in Figure 5(c). The corresponding main chain hydrogen-bonding pattern is shown in Figure 11. The amount of secondary structure within this domain is considerable: 4574 of the residues are helical. mainly

FIG.

11, The

main

chain

hydrogen-bonding

scheme

in the coenzyme-binding

domain.

HORSE

LIVER

AI~COHOI,

4.7

I)EHYDROGEK.ASE

r-helices, 32% are in the pleatsed-sheet structure and 137; in reverse bends. Thus only about 10:); of the residues have no regular secondary structure. and no continuous stretch of irregular structure is longer than four residues. The basic element of secondary structure in this domain is a central pleated-sheet with a right-handed twist. comprising six parallel strands (/?A t,o BF) flanked by helices (aA, aB. aC. aCD. aE and 31°S) in a regu1a.r pat.tern. The residues involved in these strands and helices are shown in Figure 6. A schematic diagram of t’he fold of the polvpept,idr chain in this domain is shown in Figure 1%.

FIG. 12. Schematic diagram liver alcohol dehyclrogenasP.

of the main chain fold of the coenzyme-binding

domain

of horse

A description of the main chain fold of this domain has been presented earlier (Brand&r et al., 1973). The general similarity of this structure to that of a corresponding coenzyme-binding domain in lactic dehydrogenase (Adams et al., 1970) and malic dehydrogenase (Hill et al.; 1972) was recognised and discussed at the 2.9 A stage (Brand& et al., 1973). The same general polypeptide fold has since been reported for the coenzyme-binding domain of glyceraldehyde-3-phosphate dehydrogenase (Buehner et al., 1973) and for the ATP-binding domain of muscle phosphoglycerate kinase (Blake & Evans, 1974). Most important for these comparisons is that the sequential order of the elements of secondary structure is the same in all these domains. Similar folding pat’tern with slight deviations from t’his basic structure has been

40

H.

F:KL~:sl,

ET’

.4I,.

found in adenyl kinase (Schulz rf ~1.. 1974), in the second domain of yeast phosphoglycerate kinase (Bryant et al., 1974). and has also been recognized in flavodoxin and subtilisin (Rao & Rossmann, 1973). A more dishntly related patter11 has ken reported for phosphoglyceratemutase (Campbell et ccl.. 1974). Topological comparisons of some of these structures have been published (Schulz $ Schirmer. 1974) and evidence for an evolutionary relationship has been presented and dis(;ussd 1)~ Rossmann et al. (1974). We have reported earlier (Ohlsson et al., 1974) a detailed study of the similarities and differences in sequence and structure between the coenzyme binding domains in ADHasei. lactic dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Here we will only report some additional aspects of the structure of this domain in ADHase in particular t,he binding of the coenzyme analogue ADP-ribose. The unique fold of the coenzyme-binding domain creates a specific crevice for the binding of a dinucleotide molecule. The coenzyme is bound in a similar manner. in all the dehydrogenases investigated so far, in the central region of the carboxyl end of the parallel pleated-sheet. Residues from /?A. /IB. /3D, aB, aE and the loops con netting PA to aB and BD to aE are involved in this binding. The middle strand @A is shorter at the carboxyl end than it.s neighbouring strands BB and /3D. Furthermore. the loop which connects PA to /3B turns to the left whereas t,he loop below PA connecting fiD to aE turns to the right. By this arrangement a crevice is formed outside the carboxyl end of fiA below aB and above aE. The coenzyme molecule is bound in this crevice with t,he AMP part in the region above helix aE and the NMN part in the region below aB (see Fig. 5(c)). A pseudosymmetrical arrangement’ of both the coenzyme-binding domain and the bound coenzymr molecule is thus obtained. as was noted by Rao & Rossmann (1973) for lactic dehydrogenase. Experimental details of an X-ray study to 2.9 A resolution of the complex between alcohol dehydrogenase and the coenzgme analogue ADP-ribose as well as a description of the conformation of the bound ADP-ri hose molecule have previously been reported (Abdallah et al., 1975). From that study and the present model of the enzyme molecule. the details of the int,eraction between enzyme and ADP-ribose listed in Table 3 have now been established. The adenine moiety is situated in a hydrophobic pocket,, the adenine N6 atom points out of this pocket towards the solution. This atom is close to the guanidinium group of Arg271, which in turn forms a salt linkage with Asp273. The 2’-hydroxyl of the adenine ribose hydrogen-bonds to one of the oxygen atoms of the carboxyl group of ~~~223. This ribose closely approaches Gly199. The side chain of Lys228, which can be specifically modified (Zoltobrocki et al., 1974). is also found in this region. The electron density map indicates that t,his residue may occupy two different positions. In one of t,hese positions the side chain forms a hydrogen bond to the second oxygen atom of Asp223 and is quite close to 03’ of the adenosine ribose. The pyrophosphate moiety makes a bend over the side chain of Ile269 and forms a salt-linkage with the guafi&nium group of Arg47. The main chain of this residue belongs to the catalytic domain, but the side chain bridges the gap between the two domains. The terminal ribose points towards the catalytic zinc atom but is 6 A away. Possible contacts with this ribose are hydrogen bonds from 02’ and 03’ to main chain carbonyl oxygen atoms of Gly293 and Ile269, respect’ively. t Abbreviation

used:

ADHase,

alcohol

dehydrogenase.

HORSE

LIVER

ALCOHOL

47

DEHYDROGENASE

TARLE 3 &served

Adenosine

interactions

ribose

I)yrophc,splratr

betwee% liver alcohol dehydrogenase and AD P-ribosp

PhelYX Val222 110224 Pro243 Ilt32.50 11~269 ‘Shr2i-I Thrli7 Arg27 I .jsp273

.%I1side chains lining thn hydrophobic adoninebinding pocket, have bsen includwl here, although scmo are not, in actu&l contact with the aIlenim+ xm%xt\-

Asp223 Gly199 Ila269 Asn225 1,,\322X

Hydrogen

Arg47 TlC26R

(‘barge intf~ract,ion

bond to 02’

()Y’ dose to these residues Hands not, established

Gly293 Gly293 110269

The two domains in the subunit of liver ADHase are more separated in space than the corresponding domains in the subunits of the other dehydrogenases of known three-dimensional structure. Thus helix xA belongs t,o the coenzyme-binding domain in ADHase both sequentially and struct,urally. No equivalent sequential helix is present in the other dehydrogenases in this region of t’he structure. However. helix aG in lactic dehydrogenase and molate dehydrogenase as well as the carboxy-terminal helix of glyceraldehyde-3-phosphate dehydrogenase occupy equivalent positions in space wit,h respect to the rest of this domain. Sequentially. t,hese helices belong to the second domain of the subunits in these dehydrogenases. The positions and orientations of the helices with respect, t,o the strands of the pleated-sheet in this domain provide a good example of the observation (Nagano. 1974) that pleated-sheet surfaces are frequently covered by helices. Helices aA, aB and ac’ are on one side of the sheet and helices uCD and IE: on the other side. The loop between PC and /3D that forms aCD in ADHase has an irregular structure in the obher dehydrogenases but an equivalent position in space (Ohlsson et al., 1974) a,F is absent’ in ADHase but the sequentially corresponding loop has a structural position in ADHase equivalent to z,F in lactic dehydrogenase (Ohlsson et al., 1974). Thus, in all dehydrogenases both sides of the twisted sheet, are almost completel) covered by helices or at a few positions loops of irregular structure. This effect is accentuated by the fact that the directions of the helical axes have a similar twist as do the directions of the strands of the pleated-sheet, Thus, the helical axes of aC, aB and aA are parallel to the strands j3C, /3A and /3E. respectively. Furthermore, these helices are stacked in such a way that, each helical axis is a,t the same level as it’s corresponding parallel strand of pleated-sheet,. Similarly, helices &D, & and

48

H.

Ir:KLUND

KY’

.If,.

a,F on the other side of t,he sheet are at tshe same level and parallci t,o st#rands PH. ,!?D and /3F. Only non-polar residues arc found in the interior of t’his domain brt~\lrc~n the helices and the sheet. Two hydrophobic regions. ?il and X?. one on each side of the sheet are thus present in t’his domain. Table 4 lists the residues involved in these regions in ADHase. Equivalent’ residues in lactic dehydrogenase and glpcrraldehydehydrophobic. 3-phosphate dehydrogenase (Ohlsson r/ c/l.. 1974) art. almost inrari~l~ly TABLE

lie&dues

Ala183 Vall86 Ala187 Va1189 Cysl95 Va1197 Leu200 \‘a1203 Core Nl is formed

4

in the coe?Lzylrae-bi,ndin$ domairb

Gly204 Va1207 Ile208

Cys211 Ala216 110219 Gly221 Xla232

Ala237 Phe264 Phe266 Va1268 Va1288 Val290 Val292 .IlaX Ii

which jorm hydrophobic

.\la196 Phe198

llr220 Val”22 1 llP250

by residues from aA, nB, XC! and the skands from uDC. ME and the skantls.

cores

Leu2.54 Va1262 Ala278 (lya281

(‘ys282

of the parallel

pleated-sheet.

Core N2 is formed by r&duos

(iv) The dimet The dimeric molecule is shaped approximately like a prolate ellipsoid with dimensions 45 A x 60 A x 110 8. The molecule is, however, more bulky at the ends of the long axis than in the central region. Stereo diagrams of the 748 a-carbon positions and the four zinc atoms of the molecule as well as two bound ADP-ribose molecules are shown in Figure 5(d) and (e). The two subunits of the dimer are related by a crystallographic Z-fold axis and joined together mainly through interactions within the coenzyme-binding domains. These domains thus form a core in the middle of the molecule. The catalytic domains are at, the two ends of the dumb-bell shaped molecule and the catalytic sites are in the junctions between these domains and t’he core. The two catalytic zinc atoms are rabher deeply buried in the middle of each subunit. From the surface of the molecule they are accessible from two directions one of which is from the front of the molecule as defined in Figure 5(d). The other direction is at a right angle to the first and through a deep pocket, as can be seen in Figure 5(e). The remaining two zinc atoms are situated at the back side of the molecule close to the surface. The two crevices where the coenzyme molecules bind are at the front of the molecule. The subunit arrangement is such that both coenzyme molecules are bound on the same side of the enzyme molecule with the adenine ends closest to the 2-fold axis. The nicotinamide moieties could enter the catalytic sites from the front side of the molecule, as deduced from the study of ADP-ribose binding, and thereby close off the entrance to the catalytic zinc atoms from this direction. The only entrance left to each of the catalytic zinc atoms, after coenzyme binding. is thus through the deep pocket between the coenzyme binding core and the catalytic domain as deduced

HORSE

LIVER

ALCOHOL

4!)

DEHYDROGENASE

from this crystal structure. Sub&rates might thus diffuse into the catalytic centre from a different direction than the nicotinamide moiety, involving interactions with different parts of the structure. Some distances between different groups in the moltcule and between t’hese and bound inhibitors a,re listed in Tahlt: 5. TAHLE~ 1)istance.s between d$fferent groups of the liver alcohol deh,ydroqewasp ~molecule Dist,ance Z%, Z&L Zn,, Zn,, Zk Zn,, Zn,, Zn,, 74, zn,, Zna, .A , A, 1. 1 .\I 4, .A , -4 I Trp314,

A zn,, Zn,,

Znb2 Ai 1 A, - Nl ‘11, TrplB, TrplB, Trp314, Trp314, *-\z N, N2 Trpl5, TrplS, -- Trp314, Trp314, Trp314,

I)istancr

A

“0.6 44.6 42.2 IX 40 5 41 24 (ix 21 24 38 15 38 35 54 18 2.3 6

Subscripts 1 and 2 refer to the 2 subunits of the molecule. Zn, is the catalytic Zn, is the second zinc atom of the subunit. A refers to t.he observed posit.ion of t,he of bouncl ADP-ribose, N refers to an assumed position of t,hv nicotinamide moiety 1974) using the observed position of bound ADP-ribose and the conformation bound to lactic dehydrogenase. Distances are computed to thr\ midpoint of aromatic For xymmet,ry reasons tlint,ance X, IT2 is equal to X, 1’,.

zinc atom and adenine moiety (Ohlsson et (II.. of NAD when ring systems.

The direction of the 2-fold axis in ADHase with respect. t’o t,he structure of bhc coenzyme-binding domain is quite different from the directions of any of bhe three mutually perpendicular 2-fold axes in lactic dehydrognase or glyceraldehyde-3phosphate dehydrogenase (l3uehner et al.. 1974). ln the ilDHasc molecule the parallel pleated-sheet structures of the two coenzyme-binding domains are joined t#oget’her across the 2-fold axis by anti-parallel p-binding between the fiB’-strands. The pleatedsheet, structure thus extends through the whole dimt~r comprising 12 strands arranged in two pairs of six parallel strands joined in an ant’i-parallel fashion. There is a right ha,nded twist of about 200” from the first to t,he last strand. A stereo diagram of this central pleated-sheet core of the molecule is shown in Figure 13. Furthermore, residues 301 to 303 of the extended chain BS are also joined by antiparallel /Lbinding across the 2-fold axis to corresponding residues in the second subunit. These two regions of anti-parallel strands are 20 A a,part and form two edges of a flat subunit interaction area with approximate dimensions 20 A ./ 30 A. The other two edges of this area are symmetry-related and formed by non-polar interactions bet’ween side chains from helix 31°S in one subunit and bhe loop that connects BE and /3S in the other subunit,. In thr imerior of t’his closed interaction 4

Fra.

binding

13. Stereo diagram of the domains of the molecule.

1%stranded

pleated-aheet

structure

through

the

2 oocnzyn~-

area there are only hydrophobic residues mainly from BE, PF, jZ3 and 31°S of both subunits. These subunit interactions are illustrated in Figure 14 and the residues involved are listed in Table 6. The two tryptophans with residue number 314 from both subunits are situated at, the centre of this hydrophobic core close to the 2-fold axis. Tryptophan 314 is thus buried in an extremely hydrophobic environment in contrast to TrplB. The geometrical arrangment of the four tryptophans in the molecule and its implications for t’he non-linear protein fluorescence quenching observed in ADHase has recently been discussed (Br&ndBn et al., 1975). Some additional subunit interactions are formed between residues from the lobe around the second zinc atom in one subunit (ArglOl, Va1102, HislOS, Gly108 and

“U

.

FIG. 14. Stereo 8re differentiated

diagram by open

of the Hubunit interaction tlre~. and solid bonds, respectively.

Rcuidnw

from

the 1’ (lifferent

whunits

HORSE

LIVER

ALCOHOL TABLE

/&ractions lntcractim

51

DEHYDROGENASE ti

between the two subunits oj the alcohol dekydrogenase rnoleculp type

Renidm in snbnnit I

R&clue

in subunit 2

-Main chain hydrogen bonds

Non-polar hntwnen

contact sick chains

0 0 N N s 0 N 0

of of of of of of of of

Arg312 Trp314 Trp314 GIy316 Leu301 Leu301 Met303 Met303

Met275 Ile291 V&1294 Pro296 Leu301 Met303 Pro305 Met306 Leu308 Lsu309 ‘lYp3 14

N of Gly316 N of Trp314 0 of Trp314 0

Ofihg312

0 N 0 s

of of of of

Met303 M*t303 Leu301 Leu:w1

Pro305 Lml308 Pro305, Met306, Leu309 Met306 Met303 LeuYOl, Trp314 Mck275, Va1294 Va1294, Pro296 Ile291, Trp314 Va1294 M&303. Leu308, Trp314

PhellO) and residues in the coenzyme-binding domain in the other subunit (Gln283, Ala286. Tyr286 and Ser310). These interactions are. however. few and do not seem t.0 contribute much to the stability of the dimer. In view of the large hydrophobic interaction area between t,he subunits it is not surprising that, it has been very difficult t’o separate the subunits of this molecule without’ denaturation. It is possible, however. to perform hybridization experiments in 8 Jr-urea between the closely simi1a.r EE and SS isozymes producing ES hybrids (Pietruszko it al.. 1969). (v)

7’hp zin.c atoms and the substrate-bin,disg

pocket

The t.wo domains of the ADHase subunit are separated by a cleft containing a wide and deep pocket. At the bot.tom of this pocket, 20 A from the surface of t,he molecules, is the zinc atom, that has been identified as the catalytic zinc (Brand&r et al.. 1973). This zinc atom is bound to three protein ligands from the catalytic domain. two sulphur atoms from Cgs46 and Cys174 and one nitrogen atom from His67 (see Fig. 15). A water molecule or hydroxyl ion, depending on the pH. completes a tetrahedral co-ordination. This water molecule is involved in a system of hydrogen bonds which include Ser48 and His51 (see Fig. 16). These hydrogen bonds may be important for proton release upon NAD+ binding (Shore et al., 1974) and for substratNe polarization in the catalytic mechanism (Eklund et a2.. 1974). Residues 95 to 113 form a lobe that binds the second zinc atom of the subunit. This lohe projects out from the catalytic domain having few side chain interactions wit#h the remaining parts of the subunit (see Figs 5(a) and 10). This zinc is liganded

Cvs 46

0 0

Hz0

s 0

or OH-

cy597

(a) FIG.

16. Schematic

diagram

(a) Ligands

to the catalytic

(h)

to thr

Ligands

second

(b) showing

the zinc ligands

in ADHaxe.

zinc atom. zinc atom.

in a. distorted tetrahedral arrangement by four sulphur atoms from cysteine residues 97: 100, 103 and 111. The deep pocket between the domains that contain the catalytic zinc atom has been tentatively identified as the substrate-binding pocket from the positions of bound inhibitor molecules, such as l.lO-phenanthroline and imidazole (BrLndBn et al.. 1973; Boiwe & BrlindBn, 1976). This pocket is lined almost exclusively with hydrophobic residues. These are listed in Table 7. Stereo diagrams of this pocket are shown

FIG. 16. Stereo diagram showing the spatial orientation of the zinc-bound water molecule, side chains of Ser48 and His61 and the hydrogen bond system between these groups.

the

Residues lining Subunit 8er48 Leu57 V&158 PheW PhellO Leull6

one active-site pocket

1 SW117 Leu141 Thr178 Pro2!xi Ik318

in Figure 17. There are only two polar residues, tier48 and Thr 178. in t,he part of this pocket where the catalytic reaction occurs. All other residues are non-polar. creating a hydrophobic environment for the nicotinamide moiety. t,he substrate and the outer sphere of the zinc atom. Both subunits contribute residues to each active site pocket. The inner regions of this pocket which binds zinc, the nicotinamide moiety and the reactive part of the substrate are contained entirely within each subunit. The second half of this pocket, closer to the surface of the molecule, is, however, lined with residues from the catalytic domain of one subunit and from the region 31°S of the coenzyme binding domain of the other subunit. This arrangement implies that, large substrate molecules like steroids (Waller et aE., 1965) or w-hydroxy fatty acids (Bjorkhem, 1972) may be bound to both subunits in each of the two possible binding sites. However, details of the interactions between enzyme, substrate and the nicotinamide part of the coenzyme, including possible half-of-the-sites rea’ct,ivity (Seydoux et al., 1974) can only he studied in the triclinic holoenzyme crvst.als. The structure determination of that modification is in progress.

FIG. 17. Stereo diagram showing the residues involved

in the aswmed

substrate-binding

pocket.

54

H.

EKLUND

(c) Heavy-darn

ET’

.AI,

bin.ding sites

Two of the four heavy-atom binding sites are located in the crevice where the coenzyme molecule binds. Site A, which is t’he single binding site for the anion Pt(CN):and a minor site for Au(W);-, corresponds to the position uhere we find the pyrophosphate part of ADP-ribose. This sit,e can be characterized as a shallon pocket lined with the hydrophobic side chain of lle269 and the main chain of residues 201, 202, 268 and 269. There are two positively charged residues. Lys228 and Arg271 at the fringes of this pocket,. The shallow pocket, which is creaeed by residues from the coenzyme-binding domain is situated close to the interface between the two domains of the subunit. The side chain of Arg47 in t,he catalytic domain can bridge the gap between the two domains and provide the closest positfive charge for this site. Electron densit’y maps of the Pt(CN)iderivative (Zeppezauer et al., 1975) also show that this side chain, which is poorly defined in the maps of the native enzyme, is rigidly fixed in the derivative and that the guanidinium group interacts with the Pt(CN)iion. Furthermore, Brg369 in the catalytic domain is also sufficiently close to interact but at a considerably longer distance from the platinum atom. The negative charge of a bulky group like Pt(CN)$- bound to this site is thus compensated for by the positive charge of Arg47 and by partial positive contributions from Arg271. Arg369 and Lys228. Site B which is the major binding site for Au(CN); is in the hydrophobic pocket, where the adenine moiety of the coenzyme binds. A number of hydrophobic residues, listed in Table 4, line this pocket. The only positively charged side chain in the vicinity of this site is the guanidinium group of Arg271 at the surface of this pocket. The occupancies of sites A and B for the double derivative containing both Pt(CN)iand Au(CN), are lower than those of the two single derivatives. The only reasonable explanation is that the affinity of one site for its heavy-atom complex is decrea,sed when the other site is occupied. This can not be due to steric interference since the distance between the centres of the two sites is 115 A. Site C which is one of the binding sites for ethylmercurythiosalicylate, and also a minor site for Au(CN); is close to Cys240 in PC of the coenzyme-binding domain. The sulphur atom of this residue is the only group from the protein that can coordinate with the mercury atom. There is a hydrophobic pocket between /3C and crC where the aromatic part of the mercury complex might bind. This pocket is lined with the side chains of Leu200, Ala232, Phe229 and the hydrophobic part, of the side chain of Lys233. Site D which is the second binding site for the mercury complex is found close to Cys132. This binding site is situated in a polar region close to a 2-fold rotation axis that relates two molecules in the crystal lattice. The side chains of Arg133, Trp15 and Glu24 from bobh molecules are in the vicinity of this site. The positions of the mercury atoms are so close to the 2-fold axis that the complex probably randomly occupies only one of the two symmetry-related positions.

4. Discussion The structure of alcohol dehydrogenase presented here has been correlated (Brand&r et al., 1975) to a number of physico-chemical studies in solution. Here we will only discuss some structural aspect’s of t,his molecule.

HORSE

LIVER

ALCOHOL

DEHYDROGENASE

*56

From the arrangement of helices and strands of pleated-sheet in the coenzymebinding domain it is possible to make some conject,ures about the later stages of the folding process of this domain. It is reasonable t,o assume that this domain folds independently of the rest of the molecule for the following reasons. The domains are fairly well separated in space from each other in the subunit. Furthermore, the folding pattern of the coenzyme-binding domains has been shown to be very similar in different dehydrogenases (Rossmann et al., 1974; Ohlsson et al., 1974) although these domains have completely different interactions with the remaining parts of the dehydrogenase molecules. Not only are the catalytic domains structurally quite different and oriented differentSly with respect to the coenzyme-binding domains in these molecules but the subunit interactions are also completely different in ADHase and involve different parts of the subunits. Although t’he details of the following argument are illustrated by the ADHase structure t,he general conclusions should apply to all dehydrogenases. Rao & Rossmann (1973) recognized that the coenzyme-binding domain in lactic dehydrogenase is built up by two similar halves related by an approximate 2-fold rotabion axis between and roughly parallel to the two middle strands PA and PD. We w-ill now assume that these two halves fold independently into distinct units separated by the loop that connects ,W and /3D. and show some consequences of this assumption for the folding of the complete domain. The assumption implies that there are two separate nucleation centres (Anfinsen, 1973), one in each folding unit. comprising three parallel strands of pleated-sheet, connected in the same sequential order by two helices. These helices are both on the same side of the corresponding sheet. Thus /3A, aB, BB, aC and /3C fold as one unit and /3D, aE, BE, a,F and /3F as a second unit. AR was noted by Rao & Rossmann (1973) these units have very similar folds and roughly superimpose. The assumption t,hat’ these substructures are fairly stable and fold as d.iscrete,.units is reasonable, since by this folding each unit has formed a large part of one of the hydrophobic cores bet,ween the helices and sheet that are present in the completed subunit. Furthermore, the polar residues of the helices that face the solvent in these substructures are also at the surface of the compleDed subunit. The second side of the three-stranded sheet has, however, a surrounding in this hypothetica,l substructure t,hat is quite different from that in the completed domain. The non-polar residues on this side of the sheet face the solvent in the isolated substructures whereas they are buried in the completed domain. Wr will now present structural evidence that the presence of this exposed hydrophobic area in the first folding unit, from PA to /?C? might provide a strong driving force for joining together the two m&s in the correct configuration. In the completed domain these non-polar residues are covered by hyd.rophobic side cha,ins from the loop that connects PC and PD. The following side chains line that side of t,he sheet in ADHase: Thr194, Ala196 and Phe198 from PA; Arg218, Ile22O and \Ta1222 from /3B; and Glu239. Va1241 and Pro243 from PC. In the completed domain Thr194, Arg218, Glu239 and Pro243 face bhe solvent, whereas the remaining residues which are all non-polar are buried in the hydrophobic core N2. They are shielded from the solvent by the side chains of Ile250, Va1253, Leu254, Met257 and the aromatic ring of T.vr246. All these residues belong to the loop, in&&g helix aCD, that joins the two folding units. The remaining residues of this loop are all polar residues that face the solvent. In other words, by bringing that loop in ADHase in cout+lct with t,he exposed side of the t~hre+strancled sheet, in the first folding unit,,

ix

H. EKLUI1‘Ll

f2T

AL

all exposed non-polar residues of the sheet and all non-polar residues of the loop are buried in a hydrophobic core. When the loop has been positioned by these hyd.rophobic interactions t,here are severe restrictions in t,he possible wa,ys to join the two three-stranded sheets together into a six-stranded sheet. The remaining number of residues in this loop is too sma,ll t’o bring the strands together in an anti-parallel fashion. Furthermore, alignment of /3F with /IA or /3D with /?C in a parallel fashion would bring all the helices to t,he same side of the six-stranded sheet with overcrowding as a, result. Thus the units click together into correct relative orientation by the formation of hydrogen bonds between /IA and /3D after having been approximately oriented by the hydrophobic interactions described above. The folding process described so far leaves one side of the sheet, in the second unit, from /ID to /3F exposed t,o the solvent. However, this is precisely the region which, in the completed subunit, is covered by the extra helix mentioned earlier, aA in ADHase, aG in lactic dehydrogenase and t’hc carboxy-terminal helix in glyceraldehyde-3phosphate dehydrogenase. The fact that these structurally equivalent helices are formed from sequentially different regions in the different dehydrogenases provides strong argument that these helices are positioned at a late stage in the folding process and that the sequence of folding events described here is plausible. The catalytic domain is divided in two parts comprising residues 1 to 175 and 319 to 374, respectively. All residues of the carboxy-terminal region are on the outside of the molecule consistent with the notion that this part is folded after the proper conformation of the first region of the catalytic domain has been attained. Helix a3, comprising residues 324 t,o 338, is outside helix a2 which is formed by residues 168 to 180. The remaining residues form the top of the catalytic domain including the last two strands of pleated-sheet in /31. These last residues 347 to 374 form a structure that strongly resembles parts of the folding units wit,hin the coenzymebinding domain. These residues are folded into two parallel strands of pleated-sheet possible evidence for a gene duplication joined by a helix. In order to investigate within the ADHase molecule, based on this three-dimensional homology, we have compared the region 347 to 374 with that of structurally similar regions in the coenzyme-binding domain of ADHase. There are four possible units of two parallel strands of pleated-sheet joined by a helix in that domain, comprising residues 193 to 223,218 to 243.263 to 294 and 287 to 318. The amino acid sequence of the carboxyterminal region were aligned against the sequence of each of these four regions using methods similar to those used in the comparison of the whole coenzyme-binding domains of different dehydrogenases (Ohlsson et al., 1974). No genetic relationship could be found with any of t,hese four regions based on the calculation of various homology criteria (Dayhoff et al., 1972). We can thus conclude that the presence of two parallel strands of pleated-sheet joined by a helix in this domain reflects the structural stability of this arrangement. and no apparent evolutionary relationship to the coenzyme-binding domain. A comparison of the co-ordination of the catalytic zinc in carbonic anhydrase, carboxypeptidase and alcohol dehydrogenase shows an interesting and probably functionally significant correlation with the anion binding capacity of the proteinbound zinc atom. Zinc is co-ordinated to three histidine residues in carbonic anhydrase (Liljas et al., 1972) and to one glutamic acid and two histidine residues in carboxypeptidase (Lipscomb et al., 1970). Anions bind strongly to the zinc atom in carbonic charged protein anhpdrasr (Lindskog it al.. 1971) w h ere there are no negatively

HORSE

LIVEN

ALCOHOL

DEHYDROGEKABE

5;

to balance the positive charge of the zinc ion. In alcohol dehydrogenase, on the other hand, where two of the ligands are negatively charged cysteine residues, anions do not bind to zinc with any appreciable affinity (Norne et al., 1973). Although this enzyme is inhibited by anions like chloride ions, ib has been shown conclusively that these ions generally bind at a different site (for a discussion see BrBnd& et al. 1975). However, when one of the zinc ligands, Cys46. is carboxymethylated, and thus one negatively charged ligand is changed into a neutral thioether ligand, anions ]ikc iodide ions are bound t)o zinc (Zeppezauer et al.. 1975). The zinc atom in carboxypept.idase. which has one negatively charged ligand in the nat’irc state. binds anions. hut presumably with lower affinity than does carbonic anhydrase (Norne et al., 1973). CystSeine ligands to zinc have previously not heen found in enzymes. Spectrophot,ometric evidence has, however, indicated its presence in alcohol dehydrogenases (Drum $ Valle, 1970). Garbett et al., (1972) actually proposed a co-ordination of two cysteine and one histidine residues to t’he catalytic zinc atom of yeasb alcohol dehydrogcnascl. Zinc co-ordination in this enzyme is probably the same as in the liver enzyme since partial sequence studies have shown homology with invariant residues corresponding to a,11zinc ligands including the ligands tSothe second zinc (J6rnvall. I973a : ,JSrnvall rf al.. 1975). TJw co-ordination of t’his second zinc atom in SDHase is similar to the tetrahedral arrangement of four c,ysteine ligands around iron in rubredoxin (,Jensen. 1974). Even more striking is the similarity to the four cysteine ligands around the iron clusters in the bacterial ferredoxins (Adman et al.. 1973). Three of the cysteines in both ferredoxin and ADHase are separated by two obher residues and the fourt,h ligand is some distance away in the sequence. Formally, these ligands thus have sequerlcc~ numbers X, X + 3, X + 6 and X+N (see Figure 10). The similarities exter!d even further. Looking down t’he axis defined by the metal and ligand X+ R’. the sequential arrangement of the other three ligands has the same unique hand in these two structures. These similarities may be the result of an energetically favouritbk structural arrangement or it may have evolutionary implications. Th(b t\\o zinc-binding sites in the ADHase subunit might have different requirements on the ava,ilability of zinc for the proper folding of the polypeptide chain. The residurs that, provide ligands to the catalytic zinc atom belong to quite different structural regions of ehe catalytic domain and are far apart in the sequence. Thus the main part of’ the catal,ytic domain must’ be folded in the final conformation in order to form this zinc-binding site. In contrast. the residues that provide ligands to the second zinc-binding site are close together in sequence and belong to only one st’ructural region. It is thus possible that zinc is required here in order to bring about the final conformation of this lobe region. In the absence of zinc. t’his region might thus 1~1flexible or obtain a different’ conformation from t#he OIle found here in the presence’ of zinc. There is no evidence from the st,ructure that, this would affect the conformation of the remaining part, of the subunit but only of the lobe region involving residues 95 to 113. Tilt, function of t,his extra zinc atom remains unknown. In rat liver ADHase t,hr presence of isozymes has been correlated to disulphide bridges involving the Jigands to this zinc atom (Jiirnvall. 19733). The rat isozymes are active in ethanol oxidation. The lobe region that binds zinc is, thus. in all probability. not essential for the cat,alytic action of alcohol oxidation. 10 has been suggested that t,he extra zinc at,om is tassrntial for the structural stability of the enzyme. There is no rviden(*r in tht.

ligan&

58

H. E:KLUNl)

67’

AL.

structure that this lobe region is necessary e&her for tertiary or quntcrnary structuw st’abilization of the remaining part’ of the molecule. From the structural point, of view-. this region looks much more like a second catalytic centre. The zinc atom is situated on one side of an obvious cleft int,o which the lone pair of ele&rons of the sulphur atom of Cvs97 project. The similarit,ies with rubredoxin and ferredoxin suggest that, this region might be a catalytic centre for a redox process possibly with one or two of the cvsteine ligands as catalytic groups or it may he a molecular fossil for such a centre. Skilful technical assistance from Mrs (:. Lund@& B.-M. Eklund, B. Lindahl and K. Michelson are gratefully acknowledged. We thank Dr K. K. Kannann for helpfld advice and assistance with computing problems. We are grateful for financial support from the Swedish National Research Council (grant no. 2767), the Knut and Alice Wallenberg Foundation, the Tri-Centennial Fund of the Bank of Sweden and the U.S. National Institut,e on Alcohol Ahnsn and Alcoholism (grant, no. AA00323-01).

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