The EMBO Journal vol.7 no.6 pp. 1 597 - 1604, 1988

The three-dimensional structure of P2 myelin protein

T.Alwyn Jones, Terese Bergfors, Jan Sedzikand Torsten Unge

Department of Molecular Biology, Biomedical Centre, Box 590,S-751 24 Uppsala, Sweden

Communicated by C.-I.Branden

The three-dimensional structure of P2 protein from peri-pheral nervous system myelin has been determined at 2.7A resolution by X-ray crystallography. The single iso-morphous replacement/anomalous map was interpretedusing skeletonized electron density on a computergraphics system. An atomic model was built using frag-ment fitting. The structure forms a compact 10-strandedup-and-down fl-barrel which encapsulates residual elec-tron density that we interpret as a fatty acid molecule.This $-barrel shows some similarity to, but is differentfrom, the retinol binding protein family of structures.The relationship of the P2 structure to a family ofcytoplasmic, lipid binding proteins is described.Key words: P2/myelin/fatty acid/protein -lipid interaction/transport/retinol binding protein

IntroductionPeripheral nervous system (PNS) myelin contains a numberof proteins (see Lees and Brostoff, 1984, for a review). P2protein is a minor component accounting for 2-15 % of thetotal protein, depending upon the species (Greenfield et al.,1973). Its localization, reviewed by Weise and Brostoff(1985), is mostly consistent with the view that it is presenton the cytoplasmic side of Schwann cells where it behavesas a peripheral membrane protein (Trapp et al., 1984). Atpresent, the function of P2 is unclear. Injection of bovineP2 protein into Lewis rats induces experimental allergicneuritis (EAN) (Kadlubowski and Hughes, 1979), an animalmodel for Guillain -Barre syndrome, a human demyelinatingdisease of the PNS. Shorter neuritogenic peptides from P2have also been described (Uyemura et al. 1982).

Sequences for P2 protein have been determined frombovine (Kitamura et al., 1980), human (Suzuki et al., 1982)and rabbit sources (Ishaque et al., 1982). The protein con-

tains 131 amino acids and is highly conserved (91 % identi-ty) from species to species. On the basis of sequencehomology, P2 has been recognized as a member of a fami-ly of small, cytoplasmic, lipid binding proteins (Takahashiet al., 1982; Sundelin et al., 1985b; Sacchettini et al., 1986).Taken together with the high degree of species conserva-

tion, this suggests that P2 may function as a specific lipidtransport protein in myelinating Schwann cells.

Transportation of specific small molecules is a generalproblem in biology for which there may be several struc-tural solutions. One family of transport proteins with struc-tural similarity to serum retinol binding protein (RBP)

©IRL Press Limited, Oxford, England

(Newcomer et al., 1984) has already been recognized (Papizet al., 1986; Sawyer, 1987). RBP transports retinol fromits site of storage in the liver to the cell surface receptor thattransfers the retinol into the cell. There it is bound to cellularretinol binding protein (cRBP), a member of the P2 familyof proteins (Sundelin et al., 1985b).

Crystals, suitable for structure determination, have beenreported for five members of this family: cRBP (Newcomeret al., 1981) and P2 (Bergfors et al., 1987) from our ownlaboratory, liver fatty acid binding protein (L-FABP)(Paehler et al., 1985), intestinal FABP (Sacchettini et al.,1987a), and apo-cellular retinol binding protein II (Sacchet-tini et al., 1987b). In this paper we present the three-dimensional structure of P2 myelin protein.

Results and discussion

The P2 structureP2 protein consists of a single, flattened globular domain- 35 A in diameter and -20 A thick (Figures 1 and 2).The domain is a 10-stranded up-and-down fl-barrel with(+ 1)9 topology. It therefore has some similarity to but isdifferent from RBP (Newcomer et al., 1984) and its familyof proteins. As viewed in Figure 1, the barrel appears astwo orthogonal fl-sheets (Chothia and Janin, 1982). The topsheet consists of strands 1-4 and the bottom sheet of strands5-10, with the fourth and fifth strands rather separated.Strands 1 and 6 both show a large change in direction atresidues 11 and 81, respectively, so that strand 1 is sharedby both sheets. This arrangement closes off two diagonalcorners of the sheet sandwich while forming possible en-trances to the barrel at the other two diagonal corners.

Eight of the nine loops connecting the strands are short,reverse turns. The loop connecting strands 1 and 2, however,is very different and consists of a helix-turn-helix motif(Figure 2) that effectively closes off one end of the barrel.The first helix is amphiphilic, with four large hydrophobicresidues (F16, Y19, M20 and L23) pointing into the barreland hydrophilic residues pointing outwards (D17, E18 andK2 1). In the second helix, G33 points directly into the bar-rel while A28, T29 and L32 close the gap between the helixand the neighbouring loop at residue F57. Side chain residuesfrom three other loops (the salt link D76-R78, W97 andM 119) close off the underside of the barrel, as viewed inFigure 1. The entrance to the other open end of the barrelis restricted by a ring of side chains from residues F4, I42,151, F64 and L91 (Figure 3).P2 is a basic protein with 10 net basic residues relative

to acidic residues. Approximately half of the 25 lysine/arginine residues have no well defined density beyond theCG/CD atoms. However, basic residues involved in saltlinks, D76-R78, R39-E54-R52-E61, D17-R30 and D69-K81, are well determined. This is similar to the situationoccurring in virus capsids such as satellite tobacco necrosisvirus where the basic residues lining the inside of the cap-

1 597

, 110 110

47

67 , 67

Fig. 1. Cca stereo plot of P2 myelin protein with bound fatty acid. The view is chosen to show the orthogonal $-sheets. The axis of the barrel runsdiagonally from the lower left to the upper right. To enhance the three-dimensional effect, the drawing is made with bonds closer to the viewerdrawn more thickly.

131 131

7 7

45 45

57 57

M26 26

Fig. 2. Ca stereo plot of P2 myelin with bound fatty acid. The view is into one end of the barrel.

Fig. 3. Ca stereo plot of P2 myelin with bound fatty acid. The view is into the other end of the barrel from Figure 2. The side chain atoms drawn,restrict entrance into the barrel.

sid appear disordered while those forming salt links (for ex-

ample between subunits) are ordered (Jones and Liljas,1984b). In virus capsid proteins these disordered basicresidues are thought to interact with nucleic acid phosphategroups. It seems reasonable to assume that in P2 these dis-ordered side chains on the outside surface of the moleculeinteract with phospholipid head-group phosphates of themyelin membrane.Two arginines however, R106 and R126, are totally buried

in the (3-barrel, and form no salt links with acidic residuesof the protein. There exists a strong, continuous, residualelectron density in the centre of the barrel that cannot beassigned to protein residues. We have interpreted this den-

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sity as a fatty acid CH3(CH2)3CH = CH(CH2)7COOH withits carboxyl group placed between the guanido groups ofthe two arginines and close to the hydroxyl group of Y128.The remainder of the binding pocket is made up of residuesV1 15, I104, Y19, D76, F16 and M20 (Figure 4). Becauseof the relatively low resolution of this study and the possibili-ty that we are observing the average conformation of a

number of different fatty acids, the precise nature of theligand is tentative. The position of the cis carbon-carbondouble bond in our model corresponds to that found in oleicacid [CH3(CH2)7CH = CH(CH2)7COOH] which is knownto have high binding affinity to P2 in vitro (Uyemura et al.,1984). We made no attempt to label P2 with any fatty acid

T.A.Jones et al.

The three-dimensional structure of P2 myelin protein

Fig. 4. Fatty acid binding site in P2 myelin protein.

and we are observing an in vivo ligand (or averaged ligands).This observation strengthens the view that the function ofP2 is to transport fatty acids in Schwann cells.

Neuritogenic regionBovine P2 protein induces EAN in the Lewis rat (Kad-lubowski and Hughes, 1979). EAN is an animal model ofthe human demyelinating disease Guillain-Barre syndrome.Uyemura et al. (1982) report that peptide 53-78 shows thesame induction rate of EAN as does intact P2, while theshorter peptide 66-78 shows mild but definite activity. Pep-tide 70-78 however has no activity. The region 53-78 cor-responds in structure to t(3t(3t (Figure 1) while the shorterpeptide corresponds to a t,Bt unit. In this latter region Q68,E69, E71, T73 and N77 are the most exposed residues andare therefore most likely to be involved in the inductionprocess.

Lipid binding family of proteinsProtein sequence analysis has shown that P2 is a memberof a family of homologous cytoplasmic proteins that bindlipid ligands (Takahashi et al., 1982; Sundelin et al., 1985b;Sacchettini et al., 1986). This family includes cRBP (Ongand Chytil, 1978a; Sundelin et al., 1985a) cRBP II (Li etal., 1986), cellular retinoic acid binding protein (cRABP)(Ong and Chytil, 1978b; Sundelin et al., 1985b), intestinalFABP (Alpers et al., 1984), liver FABP (previously calledZ protein, Takahashi et al., 1982), heart FABP (Sacchet-tini et al., 1986), adipocyte P2 protein (previously called422 protein, Bernlohr et al., 1984) and P2 protein (Kitamuraet al., 1980).The sequence alignment of the eight proteins shown in

Table I is an extension of the data published by Sundelinet al. (1985b) with some minor modifications. Only sixresidues are conserved among all members of the family.Three of these residues are glycines (G6, G46 and G67).G46 and G67 occur in turn conformations while G6 is partof the region (FLGTW in P2) showing sequence homologyto serum RBP (Bergfors et al., 1987) and its family of pro-teins (Papiz et al., 1986). The conformation of this regionshows a glycine preference at residue 6 when checked againsta database of refined protein structures (Jones and Thirup,1986). The suggestion of Bergfors et al. (1987) that thisregion would correspond to the start of a barrel structurein P2 is therefore correct. In this region in RBP an arginineresidue (R139) stacks onto a tryptophan (W24). A similarinteraction is conserved in the P2 family between residues

K130 and W8. Similar amino-aromatic interactions havebeen described by Burley and Petsko (1986).Conserved residue N15 is not involved in side chain inter-

actions. This residue links the end of the first strand andthe start of the first helix. Our database shows a strongpreference for Asn or Gly at residue 15 (6 Asn and 10 Glyresidues in the top 20 best fits to residues 12-17). The highdegree of conservation from F16 to A36 is due to the re-quirement of closing off one entrance to the barrel with apair of amphiphilic helices. G24 terminates the first helixin a standard conformation requiring a glycine preference(Schellman, 1980). Residue 33 has a strong preference foran alanine or glycine (although it is in a helix). The sidechain of a larger residue in this position would protrude in-to the barrel and reduce the volume that could be occupiedby a ligand.Of the five residues forming the inner ring which lines

the other open end of the barrel (Figure 3), two are con-served (142 and F64) and the others are conservative substitu-tions. In the well conserved region 69-72 with sequenceEFEE, both F70 and E72 point into the barrel. E72 interactswith the side chains of S82, Q93 and Q95 and possibly withburied waters. For those proteins having a buried E72, thehydrogen bond donor hydroxyl group is conserved in residue82, and residue 95 is always a Gln. In I-FABP and L-FABP,residues 72, 82 and 95 are hydrophobic, showing size com-pensation at 72 and 82.

In the C-terminal half of the molecules there are noresidues conserved over the entire family. The arginineresidues 106 and 126 that most closely interact with our pro-posed fatty acid ligand are conserved except in cRBP, cRBPII (where they are both glutamines) and L-FABP (whereresidue 106 is a threonine). These substitutions may beenough to account for the specificity of cRBP for retinol(Bashor et al., 1973) and cRABP for retinoic acid (Ong andChytil, 1975). However, both cRBP and cRBP II showsubstitutions V- K40 and T- R102 that could introduce newpositive charges into the barrel.

Five insertions/deletions are needed to align the sequences.Four of these are easy to accommodate in our P2 model sincethey occur in turns. In cRABP the end of the helix at 36needs extending by two residues. In cRBP and cRBP II theturn at 76 needs extending by two residues. In cRABP andL-FABP, the turn at 88 must be extended by one residue.The turn at 98 shows the largest variation. In cRABP it isextended by three, in I-FABP by two and in L-FABPshortened by four residues. The fifth insertion concerns only

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T.A.Jones et al.

Table I. The sequence alignment of the P2 family of proteins

cRABP bovinecRBP rat

cRBP tI rat

I-FABP rat

L-FABP ratH-FABP rataP2 murineP2 bovineSecondary structureInside barrelIdentity count

P2 number

--PNFAGTWKMRSSENFDELLKALGVNAMLRKVAVAAASKPHVEIRQDG-P VDF NGYWKML S NE NF E E YL R AL DV NV AL RK IAN L L - -KP DKE I V Q D GMT KDQNGT WE ME S NE NF E GYMK AL DI DF AT RKI AV R L - -T QT K II V Q D G--MAF DGTWKVYRNENYEKFMEKMGI NVVKRKLGAHD--NLKLTI TQEG--MNFSGKYQVQSQENFEPFMKAMGLPEDLI QKGKDI --KGVSEI VHEGT E K N F VGTWKLVDSKNF DDYMKS LGVGF ATRQV AS MT --K P T T I IE K N GMC D A F VGTWKLVSSENF DDYMKEVGVGFATRKVAGMA --K P N MI IS V N G-SNKFLGTWKLVSSENFDEYMKALGVGLATRKLGNLA--KPRVIISKKG- e e e e e e e e e e h h h h h h h h h h h h h h h h h h h e --e e e e e e e e e t

2_ 2 I I I6 8 I 6 I6 3 I2 2 I I i

2 - 2 3 7 2 8 6 7 6 3 3 6 4 7 8 7 4 3 5 6 6 5 5 6 5 3 3 5 4 7 6 3 5 2 2 3 - 6 5 2 2 4 8 3 4 3 8*

10*20

*

30*

40

cRABP bovinecRBP ratcRBP II ratI-FABP ratL-FABP ratH-FABP rat

aP2 murineP2 bovineSecondary structure

Inside barrelIdentity countP2 number

DQF YI KT ST T VRT T E I N -FDH MI I RT LS T F RN Y I MD -FDNF KT KT NS T F RNYDL D -FNKFTVKESSNFRNIDVV-FKKVKLTI T YGS KVI HNE -F

DT I YG KT HS T F KNT E I S N FDL V T I R S E S T FK N T E I S-FDII TI RTESPFKNTEI S-Ft e e e e e e e e t t e e e e e e -e6233 i 2 -I

6 2 3 3 4 4 5 2 6 5 6 4 6 4 4 4 3 -8*50

*

60

KVGEGFEEETV --DGRKCRS LPTWENENKI HQVGKEFEEDLTGIDDRKCMTTVSWDG-DKLQT VG V E F D E HT K G L DG R N V KT L VT WE G -NT L VE L G V D F A Y S LA - -D G T E L T G T L T MEG -N K L VT L GE E CE L E T M- -T GE K V K A V V K ME GD N K MVQLGVE DDE VT A - -DDRKVKS V VT L DG -GKL VKLGVE F DE IT A--DDRKVKS I I T L DG -GAL VKL GQ E FEET T A - -DNR KT KS T VT LA R -GS L Ne t t t e e e e

3 5 8 4 6 6 4 6*70

e e t - -t e e e e

2 6 4 2 -7 4 6 6 4*

80

e e e e e e e t -t e e e

5 4 3 5 6 3 4 6 -4 5 6 5*

90

cRABP bovinecRBP ratcRBP II ratI-FABP ratL-FABP ratH-FABP rat

aP2 murineP2 bovineSecondary structureInside barrelIdentity countP2 number

C T Q T L L E G D G P K T Y WTRELANDELILTFGADDVVCTRIYVRE-CVQKGE ---KEGRGWTQWI EGDELHLEMRAEGVTCKQVFKKVHC V Q KG E ---K E N R G WK Q WV E GD K L Y L E L T C G D Q V C R Q V F K K K -

GKFKRVDN-GKELIAVREISGNELIQTYTYEGVEAKRIFKKE-T T F K G ---- ---I KS VT E F NGDT IT NT MT LGDI VYKR VS KR I -

HVQKVD --- GQETTLTRELS DGKL ILTLTHGNVVS TRTYEKEAQVQKVD --- GKS TTI KRKRDGDKLVVECVMKGVTS TRVYERA -

QVQKWN ---GNE T TI KRKL VDGKMVVE CKMKDVVCT RI YE KV -

e e e e e t ---t e e e e e e e e e e t t e e e e e e e e t t e e e e e e e e e e -

3 5 6 7 3 2 ---5 2 343 3 3 5 4 3 2 5 5 4 6 3 4 4 2 4 2 3 4 6 5 4 4 6 4 4 4 5 3 -*100

*

110*120

*130

The secondary structure assignment is estimated by inspection on the display (e = extended ,B-strand, h = a-helix, t = tum). The character i signifies that the residuepoints into the barrel.

H-FABP, prior to the conserved F64. This may require a,B-bulge in the strand.A number of internal homologies have been reported for

members of this family. Sacchettini et al. (1986) report analignment in H-FABP corresponding to residues 68-98 to99-129 in P2. In H-FABP this gives six identical and 23conservative substitutions. Both regions correspond to f33fmotifs but they are out of register with respect to one another.The homology identified by Sundelin et al. (1985c) betweenresidues 1-53 and 54-106 for both P2 and cRBP only hasstructural similarity for the first strand in each region.Gene sequences have been determined for aP2 (Hunt et

al., 1986), L-FABP (Sweetser et al., 1986), cRBP H (Dem-mer et al., 1987), cRBP (Nilsson et al., 1987) and cRABP(Shubeita et al., 1987). The exon/intron boundaries are iden-tical and occur at residues 24, 81 and 115 (P2 numbering).These exons do not correspond to any obvious structuralunits.

Sundelin et al. (1985a) report that a cyanogen bromidefragment of unreduced cRBP contains a disulphide that may

be the result of the preparation. The three cysteines in cRBPcorrespond to residues 80, 93 and 124 in P2 and cannot forma disulphide bridge in our model. We suggest, therefore,that the observed disulphide is indeed an artefact of thepreparation.Although our model strengthens the notion that members

of this family of proteins will have similar structures, morestructural information is required to clarify the nature ofligand binding and the effect of its removal. We have col-lected a high resolution 2 A native data set on cRBP(Newcomer et al., 1981) and hope to use our P2 model tosolve its structure using molecular replacement methods.Crystals have been reported for three other members of thefamily: L-FABP (Paehler et al., 1985), I-FABP (Sacchet-tini et al., 1987), and apo-cRBP II (Sacchettini et al., 1987).

Similarity to RBPSerum RBP transports retinol from the liver to cells express-ing a specific receptor (see Goodman, 1984, for a recentreview on RBP). The crystal structure ofRBP has been solv-

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The three-dimensional structure of P2 myelin protein

, 147

50 50

Fig. 5. Superposition of P2 with RBP. RBP Cros are drawn with thin lines. The atoms used to make the superposition are given in Table II.

ed by Newcomer et al. (1984) and has been crystallographic-ally refined to 2 A resolution (Jones and Newcomer, inpreparation). The hydrophobic ligand is solubilized by bind-ing in the centre of what was then a unique 3-barrel topology.Shortly afterwards the (-lactoglobulin structure was solvedby Sawyer et al. (1985) and the two structures shown to bevery similar although the sequence homology is weak (Papizet al., 1986). Only three short regions of four or five residuesshow sequence identity. The family has been further expand-ed on the basis of sequence comparison (Sawyer, 1987) andby two independent structure determinations of insect bilinbinding protein from Pieris brassicae (Huber et al., 1987)and Manduca sexta (Holden et al., 1987). In both structures,the biliverdin ligand is bound in an RBP-like (3-barrel.

Figure 5 shows that RBP consists of an N-terminal coil,an eight stranded up-and-down (3-barrel forming a pair oforthogonal (3-sheets, an a-helix packing onto one of the (-sheets and a C-terminal coil. The two families of proteinstherefore seem to use the same kind of barrel topology tobind their ligand. However, in the RBP family the barrelhas eight strands while in the P2 family there are ten. Assuggested by Bergfors et al. (1987) the barrels can be alignedstarting wtih RBP K17 to P2 S1. Their alignment breaksdown at the beginning of the helix in P2, corresponding tothe first turn in the RBP barrel.

Figure 5 shows the superposition of the barrels with theCot alignments of Table H. This alignment totals 75 residuesout of 131 in P2 and uses the first four and last three strandsof each protein. The root mean square fit of this alignmentis 2.1 A. The positions of the ligands are similar, with theretinol slightly closer to the observer in the view shown inFigure 5. The hydrophilic end groups of each ligand arereversed in the two structures and have functional signifi-cance. Many ligands have been shown to bind RBP provid-ed the (3-ionone ring is maintained and changes are madeprincipally at the end of the isoprene tail (for example, Haseet al., 1976; Horwitz and Heller, 1974). In cRBP andcRABP the reverse is true since they bind retinol (Bashoret al., 1973) and retinoic acid (Ong and Chytil, 1975),respectively. This is easily understood if the retinoids bindin a similar manner to the ligand in P2 since the part of theligand most important for binding then fits into the centreof each barrel and will be involved in more interactions.

Because of their architecture, both proteins need to close

Table II. Structurally equivalent residues in P2 and RBP

P2 RBP

l 16 17 3235 46 38 4947 55 51 5957 66 70 79103 111 103 111112 120 114 122121 130 130 139

off the open ends of their barrels. RBP closes one end withloop residues (RBP 33, 66, 96 and 126) while P2 principal-ly uses the two short helices. The other end of the barrelis more tightly closed in RBP due to very efficient side chainpacking of five Phe rings (15, 20, 45, 77 and 86) aroundthe side chain of M53. The less efficient packing observedin P2 (Figure 3) could result in two binding sites and ac-count for the conflicting reports of the number of fatty acidsbinding to liver and heart FABPs (Offner et al., 1986). Asmentioned earlier, at the start of both barrels a highly con-served basic residue packs onto a highly conserved trypto-phan (W8 and K130 in P2, W24 and R139 in RBP).Why do both families of binding proteins use a similar

folding topology? For even a relatively small protein, thistopology gives a (3-barrel with space for a totally encap-sulated, possibly fragile ligand of -20 atoms. Sequencevariation can then result in very specific ligand binding. Sucha barrel also has the advantage of providing a pair of naturalentrances (or exits) for the bound ligand.

Materials and methodsThe protein was purified from bovine intradural spinal roots and crystalliz-ed as described by Bergfors et al. (1987). The crystals have space groupP212121 with a = 91.8 A, b = 99.5 A, c = 56.5 A. Crystal densitymeasurements did not give conclusive estimates for the number of moleculesin the asymmetric unit. All diffraction data was collected on a Nicolet Xen-tronics area detector (Durbin et al., 1986) using the software system describedby Blum et al. (1987). All data handling and crystallographic calculationswere performed with the program PROTEIN (Steigemann, 1974) unlessotherwise stated. Data collection statistics for the native crystals and thetwo derivatives used in this study are given in Table III. The native dataset was overdetermined both deliberately (in order to test the reproducibilityof the new area detector system) and inadvertently, by the collection of

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T.A.Jones et al.

Table III. Data collection statistics

Native Trimethyl Mercuriclead acetate acetate

Number of measurements 85809 22521 12093Number of unique measurements 11665 11211 5145Percentage of total 69% at 2.7 A 69% at 2.7 A 87% at 3.5 AR-merge (all measurements)a 8.52 8.87 7.07R-merge (rejection ratio 0.4) 7.68 7.62 6.38R-merge anomalousb 2.39 7.29 5.48R-merge to standard native crystal 20.95 17.3

N naR-merge = x 100.0

NE n<l>

where N is the number of unique measurements, n is the number of multiple measurements of a particular reflection, I is the measured and <I> isthe mean intensity of a reflection.

NbRmerge = E (<1+> - <I->) x 100.0

NE (<I+> + <I->)

where +/- refer to Friedel pairs.

Table IV. Heavy atom refinement results. All sites had a temperature factor of 20 A2

Derivative Occupancy X Y Z RmsFh/residual CullisR-factor

Trimethyl lead acetate 6.10 0.10728 0.13402 0.17033 2.34 0.4566.15 0.55067 0.61390 0.694135.50 0.13919 0.48154 0.46403

Mercuric acetate 5.50 0.10754 0.13517 0.17743 2.43 0.3665.74 0.55131 0.61360 0.691835.06 0.13856 0.48089 0.46112

Resolution 7.47 5.97 4.97 4.25 3.72 3.30 2.97 2.7 TotalFigure of merit 0.75 0.80 0.77 0.70 0.68 0.57 0.45 0.35 0.64

Fig. 6. Representative electron density corresponding to 1127.

putative derivatives that turned out to be native crystals. The merging R-factor for data from one crystal was -5%.

Testing a wide range of heavy atom compounds resulted in two derivatives,trimethyl lead acetate and mercuric acetate. Difference Pattersons showedthree sites in the asymmetric unit for both derivatives. Unfortunately, thesites were identical for the two compounds. The lead derivative was pro-duced by co-crystallizing the protein in 6 mM tri-methyl lead acetate witha native crystal seed (Thaller et al., 1981). The mercury derivative was

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prepared by soaking a native crystal in 2.5 mM mercury acetate overnight.The derivative data collection statistics are given in Table III. Althoughthe R-factor of each derivative to a standard native crystal is - 18%, theR-factor between derivatives was 12% in spite of the fact that they occupiedidentical sites in the protein. We therefore performed heavy atom least-squares refinement of both derivatives using the program PHASEREF (writ-ten by Dr J.Remington, private communication). Table IV shows the finalrefinement statistics using anomalous data for both derivatives.

The three-dimensional structure of P2 myelin protein

Fourier maps were calculated with best phases (Blow and Crick, 1959)for both enantiomorphs of the heavy atom sites. As a control, a third mapwas calculated without the anomalous contributions. A fourth map wascalculated after two cycles of solvent flattening (Wang, 1985) of the leadSIR map. These electron densities were skeletonized (Jones and Thirup,1986) at a contour level of 1.5 times the standard deviation of the map andthe whole unit cell was inspected on an Evans and Sutherland PS390 withFRODO (Jones, 1978; Jones and Thirup, 1986). One of the anomalous mapswas significantly superior to the other three. This map clearly showedskeleton density for three protein molecules in the asymmetric unit, withone heavy atom site associated with each molecule. The skeleton of oneof these molecules was interactively edited on the display and the struc-ture's fold determined in 2 h. The side chain skeleton atoms were carefullyedited prior to building the complete structure using five amino acid fragmentsfrom a data base of 32 refined protein structures as described by Jones andThirup (1986). The side chains were then fitted manually by dihedral rota-tions and fragment moves. All residues from the N terminus to the C ter-minus at residue 131 could be accounted for in the density. The main chaindensity was of very good quality with no breaks greater than one gridpointat one sigma contouring. Carbonyl groups were rarely visible in the map.All side chains show good fit to the density except the lysine and arginineresidues not involved in salt links. Figure 6 is a representative fit of a sidechain to the density. The heavy atom binding site is close to the disulphidebond between residues 117 and 124 (Kitamura et al., 1980). Such a siteis unusual in non-reducing conditions (Petsko, 1985) and definitive proofof the disulphide bond must await our refinement of the structure.

Initial models for molecules B and C were obtained by manually fittingthe Cca model of molecule A onto the respective skeletons, using FRODOwith the heavy atom sites as further guide points. The coordinates of fiveCca atoms were then used as guide points to transform the whole moleculeonto the skeleton by least squares alignment (Kabsch, 1978). Each moleculewas then real space refined as rigid bodies into their density (Jones andLiljas, 1984a) with further shifts of 1.1 and 2.3 A, respectively. Thismodel had a crystallographic R-factor of 45% to 2.7 A resolution. Fur-ther real space refinement using small constrained rigid groups (Jones andLiljas, 1984a) and minor rebuilding, reduced the R-factor to 40.5%. Thismodel, consisting of all of the protein atoms, has been partially refined us-ing standard reciprocal space least squares methods. Four cycles of COR-ELS (Sussman et al., 1977) and 11 cycles of PROLSQ (Hendrickson andKonnert, 1980) reduced the R-factor to 29% with an overall temperaturefactor of 20 A2.The experimentally phased electron density map shows similar residual

density in the interior of the 13-barrels of all three molecules which is notaccounted for by the protein sequence. Because of its shape and its prox-imity to two buried arginine residues, we have interpreted this density asa fatty acid molecule.Each molecule has two short cx-helices. These are right handed and con-

firm the crystal enantiomorph.At no stage in this study was there a physical contour plot made of the

electron density. All map interpretation was made using electron densityskeletons at the graphics terminal.

AcknowledgementsWe wish to thank Dr M.Weise for an initial gift of material, Ms KerstinFridborg for help with the initial film processing and Dr Carl Bergfors forhis suggestions of heavy atom derivatives. Finally, we wish to thank RonBurns for the installation of the Nicolet area detector and Michael Blumfor teaching us the use of his software. This work has been supported bythe Swedish Natural Science Research Council and equipment grants fromthe Wallenberg Foundation.

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Received on December 24, 1987; revised on March 2, 1988

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