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Protein Science (1993), 2, 1057-1059. Cambr idge University Press. Printed in the USA.Copyr igh t 0 993 The Pr otein Society.~~ . .

FOR THE RECORD

Topological chirality of proteins IEIBORYEU MA 0Upjoh n Research Laboratories, Kalamazoo, Michigan 49001( R E C E I V E D M a r c h, 1993; REV ISED MANUSCRIPT RECEIVED M arch 23,993)

Topologicalstereochemistryofproteins was first dis-cussed for the tructure of a mammal-active scorpion neu-rotoxin (variant 3) from Centruroides sculpturatus Ewingand related proteins in other scorpion species. For sometime, colipase was thought tobe only the econd proteinfor which the covalent structures represented by a non-planar graph (Klapper & Klapper, 1980; Mao, 1989) andfor which thereare opologicalstereoisomericforms(Mao, 1989). The recent publication of the structure e-termination of colipase in a lipase-procolipase complex(van Tilbeurgh et al., 1992) showed that, like almost allother proteins, thedisulfide bonding pattern of colipasebelongs in the planarcategory, and themolecule is in facttopologically achiral. However, a survey of recent liter-ature anda search of he Brookhaven Protein Data ankshowed that a second topologically chiral protein does ex-ist, namely the light chain of quinoprotein methylaminedehydrogenase (Vellieux et al., 1989; Chen et al., 1992).Analysis of the covalent structure of methylamineehy-drogenase light chain indicated that, like the scorpiontoxin, the structure is topologically simple (i.e., withoutknots or links) and that it belongs to the same topologi-cal chirality as the scorpion variant 3 toxin.In topological stereochemistry, he molecular topologyis defined by covalent linkages in a molecule which, foranalytical purposes, are considered o be infinitely flexi-ble conceptually and mathematically but never breakable.Given a sufficient number of branching intramolecularlinkages, the arrangement and onnectivity of these link-ages could be such that there aresomeric forms, namelytopological stereoisomers for the molecule, that are nottopologically interchangeable. Topological stereochemis-try has beenobserved in syntheticorganicmolecules(Walba, 1985) as well as in nucleic acids (White & Coz-zarelli, 1984).For proteins, however, topological stereochemistry hasrarely been an issue in structural studies, in spite of the

~~ ~~ ~ ..Reprint requests to: Boryeu Mao, Upjoh n Research Lab oratories, 301Henriet ta St reet , Kalamazo o, Michigan 49001.

richness of motifs and folding patterns found n proteinstructures (e.g., Richardson et al., 1992). In polypeptideand protein molecules, branching intramolecular linkages(the necessary structural requirement for topological ste-reochemistry) are generally provided by disulfide bonds.Although disulfide linkages are common n proteins, andtheir general geometric properties have een investigated(e.g., Thornton, 1981; Kikuchi et al., 1989), until recentlythere has been only one protein that could have topo-logical isomeric forms (Mao, 1989), namely the scorpionvariant 3 toxin from C. sculpturatus and similar toxinsfrom related species (Almassy et al., 1983; Fontecilla-Camps et al., 1988). That thepolypeptide chain ofa pro-tein hasa well-defined and unique folding conformationdictates that thenative structure of that proteine in oneunique topological isomeric form; for the scorpion eu-rotoxin, the topological structures thatare allowed for itscovalent connectivity were found to fall into two chiralityclasses (D and L) , and the native variant 3 toxin structurebelongs in chirality class D (Fig. 1B; Kinemage 1). Theclassification scheme was based on the observation (andhence a simplifying assumption) that structures of glob-ular proteins are topologically simple and free of topo-logically complicated constructs such as knots and linksand other types of entanglements. Physically, this is sug-gested by the fact that polypeptide chains n globular pro-teins are made f finite numbers of amino acid residues andhave physically determined mechanical properties (e.g.,length, tensile and torsional flexibilities), and that int ra-molecular interactions exist among aminoacid side chainsduring intermediate and final stages of protein foldingsuch that only topologically simple forms are realized.

In addition to scorpion neurotoxin, colipase containsmultiple intramolecular disulfide linkages (Fig. 2) that forsome time were thought tobe potentially represented bya nonplanar graph also Klapper & Klapper, 1980; Mao,1989), and thus the proteinwas thought to be a topolog-ically chiral molecule. If colipase were topologically chi-ral, it would have been an interesting case for checkingthe hypothesis that proteins couldhave only topologically

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1058A A

B. Ma0

B @ C fFig. 1. A: Covalent structure of variant 3 neurotoxin from the scorpionCentruroides sculpturalus. The thick line represents the polypeptidebackbone from theN-terminus to theC-terminus. T he eight cysteines(residues num bered 12, 16,25,29,41,46,48, an d 65, respectively) ar elabeled alphabetically, and the disulfide bond s are schematically rep-resented by thin lines. Th e crossing of cf an d dg edges cannot be avoidedin the plane of the paper.B Graph representation of the covalent struc-ture. Th e positioning of the dg edge over the cf edge specifies the chi-ra l D topology, whereas a reversal of the relative spatial disposition ofthe two bonds gives the L topology. See Mao (1989) for details.

simple structures, and forchecking whether he other to-pological chirality class, L, could be adopted in proteinstructures. The recent determination of the colipase struc-ture in a lipase-procolipase complex (van Tilbeurgh etl.,1992) showed instead that the disulfide linkages in factgive a planar covalent structure and thatcolipase is there-fore a topologically achiral protein (Fig. 2B).A surveyof recent literature nonetheless found a secondtopologically chiral protein in the ight chain of methyl-amine dehydrogenase (Vellieux et al., 1989; Chen et al.,

A

B

Fig. 2. A: Covalent structure of colipase based on sequence analyses(see Mao, 1989); cysteine residue numbers are 17,23,27,28,38,48,59,61,67, an d 85. The two disulfide bonds drawnbelow the polypeptidechain were not explicitly resolved in the seq uence determination; in thisfigure, the structurewould be topologically chiral, whereas in the otherpossible arrangement, in which the cf an d dg edges are replaced by cgan d df edges, the structure would be planar (Mao, 1989). B Disulfidelinkages shown in the colipase structure determined by X-ray diffrac-tion. Misplaced disulfide bonds based o n sequence analyses shown ingray in A ar e now corrected (edgesad an d g h ) . Note the earrangementof the be edge that now allows all disulfide bo nds to be drawn in theplane of the paper without any bon d rossing; thus, the structure s to-pologically achiral.

1992). (This is confirmed by an examination of all pro-teins in the Brookhaven Protein Data Bank [Mao, un-publ.].) The C a tracing of the polypeptide chain of themolecule is shown in Figure 3A and Kinemage 2. Theschematic drawing ofhe molecular structure in Figure 3Bis obtained by topological rearrangements of polypeptidesegments in the three-dimensional structure, most nota-bly the shortening and relocation of he segment from cys-teine 88 to the C-terminus (colored blue and gray). The

Fig. 3. A: Stereoscopic drawing of the C a tracing of methylamine dehydrogenase light chain. The coordinates were obtainedfrom the Brookhaven ProteinDa ta Bank (code IMAD ). The N-terminus of the polypeptide chain is located on the pper rightcorner, an d the C-terminus is located in the left center behind the structure. T he color coding of the chain and the disulfide bondsare the same as in Figure 4A. B: Schematic drawing of the molecular topology of m ethylamine dehydrogenase light chain.

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1059opological chiralityof proteinsA ture, whether the chiral D class topology is unique to pro-teins, and whether such topological characteristics couldhave any implications for the folding of polypeptide-"@ chains of globular proteins (Benham & Jafri, 1993) andthus for theprotein folding problem. An intriguing pos-

sibility would behe engineering of topologically interest-ing proteins that could answer some of these questions;one such interesting example wouldbe the replacement ofeach amino acid in scorpion variant 3 neurotoxin by thecorresponding D-amino acid (Milton et al., 1992).B

Fig. 4. A: Covalent structure an d disulfide linkages of m ethylamine de-hydrogenase light chain; cysteine residue numbers are 23, 29, 36, 38,46 ,61 ,77 , 78, 86, 88, 108, and 119 for the protein from Thiobacillusversutus.B: Graph representation of the covalent structure shown in A .Comp arison of this graph with that in Figure 1B shows that the back-bone fragments (a o h in black and i to j in blue) and five of the di-sulfide bonds (in red) represent a m olecular topology identical to thescorpion toxin; tw o additional dges (the disulfide bo nd n yellow andthe backbone ragment in gray) complete the covalent structure for me-thylamine dehydrogenase light chain. This representation can be arrivedat from the structure shown in Figure 3B by rearranging polype ptide seg-ments of the molecule without breaking hem. T he position of dg overcf is identical to that n Figure lB , indicating the chiral D topology forthe structure. C: Alternative arrangement of edges hi an d dg in whichthey are reversed from their relative disposition in the molecular struc-ture schematically represented in B. This graph can e decomposed intotwo linked loops as shown,which is a topologically complex structurediscounted in the classificationscheme for polypeptides n proteins (Mao,1989). Such topologically complex co nstructs are not foun d in B.

structure in Figure 3B can be further rearranged (as ani-mated in Kinemage 2), topologically, into the graphep-resentation in Figure 4B. As shown in Figure 4B, sidefrom theedge hi and theedge d'e', the graph epresenta-tion of this molecule is identical to that of scorpion neu-rotoxin (Fig. lB), and thusbelongs in the same chiralityclass, D. Moreover, the location of the two additionaledges, hi and d'e', maintains the molecule in a topologi-cally simple tructure; had the three-dimensional structureof the molecule been such that the locationsof the edgehi and the edge dg were reversed, then (topologically com-plex) linked oops would have resulted (Fig.C), thus con-tradicting he hypothesis that protein structures aretopologically simple.Given that topological stereochemistry of proteins hasbeen observed only in two instances thus far , it remainsto be seen whethermore such proteins can be found in na-

AcknowledgmentI thank G.M. Maggiora for an editorial comment.

ReferencesAlmassy, R. J., Fontecilla-Camps, J.C., S ud dath , EL ., & Bugg, C.E.(1983). Structure of variant-3 scorpiqn neurotox in from Centruroidessculpturatus Ewing, refined a t 1.8 A resolution. J. Mol. Biol. 170,Benham, C.J. & Jafri, M.S. (1993). Disulfide bonding pattern s and pro-tein topologies. Protein Sci. 2, 41-54.Chen, L., Mathews, F.S., Davidson, V.L., Huizinga, E.G., VeUieux,F.M.D., & Hol, W.G.J. (1992). Three-dimensional structure of thequinoprotein methylamine dehydrogenase from Paracoccus denitri-ficans determined by molecular replacement t 2.8 A resolution. Pro-teins Struct. Funct. Genet. 14 , 288-299.Fontecilla-Camps, J.C., Habersetzer-Rochat, C., & Rochat, H. (1988).Orthorhombic crystals and three-dimensional structure of the po-tent toxin I1 from the corpion Androctonus australisHector. Proc.Kikuchi, T., Nemethy, G., & Scheraga, H.A . (1989). Spatial geomet-ric arrangements of disulfide-crosslinked oops innonplanar proteins.

J. Comput. Chem. 10 , 287-294.Klapper, M.H. & Klapper, I .Z. (1980). Th e 'knotting' problem in pro-teins. Biochim. Biophys. Acta 626, 97-105.Mao, B. (1989). Molecular topology of multiple-disulfide polypeptidechains. J. Am. Chem. SOC. 111, 6132-6136.Milton, R.C.L., Milton, S.C.F., & Kent, S.B.H. (1992). Total chemi-cal synthesis of a D-enzyme: The enantiomers of HIV-1 proteaseshow demonstration of reciprocal chiral substrate specificity. Sci-ence 256, 1445-1448.Richardson, J.S., Richardson, D.C., Tweedy, N.B., Gernert, K.M.,Quinn, T.P., Hecht, M.H., Erickson, B.W., Yan, Y., McClain, R.D.,Donlan, M.E., & Surles, M.C. (1992). Looking at proteins: Repre-sentations, folding, packing, an d design. Biophys. J . 63, 1186-1209.Tho rnton , J.M. (1981). Disulfide bridges in globular proteins. J. Mol.van Tilbeurgh, H., Sarda, L., Verger, R., & Cam billau, C. (1992). Struc-ture of the pancreatic lipase-procolipase complex. Nature 359,Vellieux, F.M.D., H uitema, F., Groendijk, H ., Kalk, K.H., F rank , J.,Jzn., Jongejan, J.A., Duine, J.A., Petratos, K., Drenth, J., & Hol,W.G.J. (1989). Structure of quinoproteinmethylamine dehydroge-nase at 2.25 A resolution. EMBO J . 8, 2171-2178.Walba, D.M. (1985). Topological stereochemistry. Tetrahedron 41 ,White, J.H. & Cozza relli, N.R. (1984). A simp le topological method fordescribing stereoisomers of DNA catenanes and knots. Proc. Natl.Acad. Sci. USA 81 , 3322-3326.

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