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LETTERS

A reply to Englander andWoodward

There are two areas of disagreementbetween Englander and ourselves. Thefirst is a difference of emphasis. We agreewith Englander that hydrogen exchange isa powerful tool for the study of proteinfluctuations1 and can give informationabout the structure of rarely populated

partly unfolded forms. Indeed, we haverecently used a combination of hydrogenexchange and protein-engineering studiesto demonstrate the existence of a partlyunfolded state of barnase underequilibrium conditions2. However, somereaders might be misled into assumingthat there is an automatic relationshipbetween hydrogen exchange atequilibrium and protein-folding pathways.The principal point of our article3 is thatmeasurements of hydrogen exchange at

equilibrium cannot by themselvesdetermine the order of events in foldingor prove that a partly unfolded state is afolding intermediate rather than a sideproduct. The observation that somepartly unfolded forms and foldingintermediates share similar structuralproperties is interesting but does notprove that they are kinetic foldingintermediates. To determine the order ofevents, kinetic measurements must bemade. As Englander points out, a

correlates. However, our model does notsupport detailed interpretations of fvalues10. We do not suggest that the slow-exchange core resembles folding transition-state structure(s) or that it is an earlynucleation site (turns are likely nucleationsites and tend to exchange rapidly in thenative state and are not generally expectedto be part of the slow-exchange core).Experimental probes for the slow-exchangecore are backbone NH groups, whereasprobes for f values are side chains. Theslow-exchange core and f values arecomplementary parameters that point tocore elements that have native-like, localand non-local structure that is formed earlyduring folding.

A note: the statement of an apparentcorrelation between the slow-exchange coreand folding-core parameters is not aconfusion of thermodynamics and kinetics;it is an observation.

References1 Clarke, J., Itzhaki, L. S. and Fersht, A. R. (1997)

Trends Biochem. Sci. 22, 284–2872 Woodward, C. (1993) Trends Biochem. Sci. 18,

359–3603 Kim, K-S., Fuchs, J. and Woodward, C. (1993)

Biochemistry 32, 9600–96084 Woodward, C., Simon, I. and Tuchsen, E. (1982)

Mol. Cell. Biochem. 48, 135–1605 Barbar, E. et al. (1998) Biochemistry 37,

7822–78336 Pan, H., Barbar, E., Barany, G. and Woodward,

C. (1995) Biochemistry 34, 13974–139817 Bai, Y., Sosnick, T. R., Mayne, L. and Englander,

S. W. (1995) Science 269, 192–1978 Itzhaki, L. S., Neira, J. L. and Fersht, A. R.

(1997) J. Mol. Biol. 270, 89–989 Neira, J. L. et al. (1997) J. Mol. Biol. 270, 99–110

10 Itzhaki, L. S., Otzen, D. E. and Fersht, A. R.(1995) J. Mol. Biol. 254, 260–288

11 Shoemaker, B. A., Wang, J. and Wolynes, P. G.(1997) Proc. Natl. Acad. Sci. U. S. A. 94, 777–782

12 Lazaridis, T. and Karplus, M. (1997) Science278, 1928–1931

13 Daggett, V. et al. (1996) J. Mol. Biol. 257,430–440

14 Jackson, S. E., el Masry, N. and Fersht, A. R.(1993) Biochemistry 32, 11270–11278

15 Li, A. and Daggett, V. (1996) J. Mol. Biol. 257,412–429

CLARE WOODWARD AND RENHAO LI

Dept of Biochemistry, University of Minnesota,1479 Gortner Ave, St Paul, MN 55108, USA Email: clare@biosci.cbs.umn.edu

The slow-exchange coreand protein folding

In a recent article1, Clarke et al. do notappropriately represent our ideas andresults regarding hydrogen-isotopeexchange and protein folding. We proposedthat ‘the slow exchange core is the foldingcore’2,3. The slow-exchange core is acollective term for the elements of packedsecondary structure that carry the peptideNH groups whose protons exchangeslowest by the ‘folded state mechanism’4.The sections of peptide chain thatcorrespond to slow-exchange-core elementstend to be native-like early in folding, whenthe rest of the protein is more disordered.That is, in the ensemble of interconvertingconformations sampled early in folding, themost favored (stable) tend to involvenative-like interactions between stretchesof amino acid residues that eventually foldinto the slow-exchange core. The followingdata support this assertion: slow-exchange-core elements contain NHs protected firstduring folding (15 proteins reported), NHswhose protons exchange slowest inpartially folded proteins (7 proteins), andnon-random structure in unfolded BPTI5,6.

Our model, unlike that of Bai et al.7, doesnot imply the order of folding forsecondary-structural elements outside theslow-exchange core. We do not assume thatintrinsic-exchange rate constants for thefolded-state mechanism are given by smallmodel peptides, or conclude that hydrogenexchange identifies local folding units. Ourmodel implies that, among the multipleparallel pathways for folding, some earlytrajectories are considerably more likelythan others. Subsequent folding of non-coresegments appears to be by multiple,parallel routes of similar probability5.

Does the slow-exchange core correlatewith f values? Yes, in our view – althoughthe data base is limited. For barnase,residues fold into loops or into secondarystructure have experimental f valuesapproaching unity; residues that formsecondary structure correlate with slow-exchange-core elements (loop residues arenot expected to correlate with theseelements, given that they usually exchange

rapidly in the native state). Forchymotrypsin inhibitor 2 (CI2), the slowest-exchanging NHs (at pH 5.3, 338C) are thoseof residues 11, 19–21, 30, 32 and 47–518,9.The slow-exchange-core elements are thusthe a-helix (residues 12–24), and the b-2(residues 28–34) and b-3 (residues 45–51)strands, which pack three dimensionally inthe native structure. The experimental10

and calculated f values11–13 tend to behighest in slow-exchange-core elements. Weconclude that the slow-exchange core andhigh f values correlate in CI2.

By contrast, Ferscht and co-workers1

state that ‘the amino terminus of the uniquea-helix of CI2 constitutes the core of thenucleation site for folding, but it has rapidlyexchanging protons (Figs 2b, 3b). There isno correlation, therefore, between slowexchange and the folding nucleus.’However, Figs 2b and 3b of Ref. 1 do notpermit comparison of f values with slow-exchange-core elements of CI2. The ribbonis color coded, not by f value but by theauthors’ interpretation of complex issuesthat include multiple f values for the sameresidue and/or fractional f values. Thereare several examples of a differencebetween f value and ribbon color in Figs 2band 3b: the blue ribbon (residues 12–18,which are ‘essentially folded in thetransition state’) includes two residues thathave f values equal to or less than those ofresidues 20 and 21; elsewhere, the negativef value of residue 19 is interpreted as f . 114,15; purple-ribbon residues ‘partiallyfolded in the transition state’ includeseveral that have f values greater thanthose of residues in the blue helix. Further,in Figs 2b and 3b, the hydrogen-exchangedata are not accurately represented – forexample, the amide protons of residues 19and 20 in Fig. 3b are numbered incorrectly,and other very slowly exchanging protonsin and near the helix (residues 11, 16, 21and 24) should be indicated as intermediate(i.e. shown in purple). Finally, the existenceof rapidly exchanging protons in a slow-exchange-core helix is not a discrepancy; itis expected for a ‘penetration mechanism’for folded-state exchange4.

A tendency of residues that have higherexperimental and calculated f values to bepart of slow-exchange-core elements addsone more folding parameter to the list of

LETTERS TIBS 23 – OCTOBER 1998

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significant body of kinetic data, in additionto equilibrium hydrogen-exchange data,was needed to map out the kinetic foldingpathway of cytochrome c.

The second area of disagreementconcerns our finding that limitations innative-state hydrogen-exchange methodsmade it impossible to characterize partlyunfolded forms in barnase andchymotrypsin inhibitor 2 (CI2), but thattheir folding pathways could becharacterized fully by other methods.Englander suggests that barnase and CI2are not ‘favourable cases’. However, thesetwo proteins are well behaved and highlycharacterized representatives of the twomost common types of folding system. Agood analytical tool should be able toprobe at least one of these proteins.

Both we and Englander are clearly atodds with Woodward. Although Woodwardsays that we misrepresent her model thatrelates the slow-exchange core to protein-folding pathways, she clearly stated in heroriginal article4 that ‘the folding pathwaywould correspond approximately to thereverse order of native-state exchangerates’, so that ‘since native-state exchangeexperiments are far less complex thanrefolding experiments, they would providea simple way to identify the folding core’.Thus, the model as originally definedclearly implies that fast-exchangingstructural elements fold later than those inthe slow-exchange core. In the modelWoodward now describes, she proposesthat slow-exchange-core elements foldearly but that nothing can be inferredabout secondary-structure elementsoutside the slow-exchange core.Nevertheless, the model still implies thatthe folding of the non-slow-exchange-coreelements occurs ‘subsequent’ to folding ofslow-exchange-core elements. Woodwardsays that the model does not ‘suggest’ thata slow-exchange core is a nucleation siteor ‘resemble[s]’ a transition state. Whateverthe name, both ‘folding core’ and ‘nucleationsite’ refer to a region of structure that is

formed early in folding; further, anythingthat is well formed in the transition statemust fold earlier than something that isunstructured in it. Transition-statestructures therefore also give informationabout the order of folding events.

Independent data are needed to testWoodward’s model. She cites anunpublished database of 15 proteins insupport of the slow-exchange-core model.But, the slow-exchange-core model cannot,however stated, account for the barnaseand CI2 data, which show clearly thatthere are slowly exchanging protons inelements of secondary structure that foldlate. f -value analysis is now the standardbench mark used by many theoreticiansfor testing models of protein folding andfor simulations of protein unfolding5–10. All of the f values have been subject tointense scrutiny, and simulations are inexcellent agreement with experimentalresults5–7,9,11–15. The combination of fvalues and simulation make the transitionstate of CI2 (Ref. 12) one of the best-characterized structures. The order ofevents in the multistep folding pathway ofbarnase is particularly well characterizedby NMR, f values and simulation16.Woodward uses a highly selective choiceof data to compare f values and hydrogenexchange. If a single proton in a helix, suchas the deeply buried residue 14 in the firsthelix of barnase, exchanges slowly, shechooses to put the whole helix into theslow-exchange core – despite the fact thatall the other residues exchange rapidly.Yet, where there is a slow-exchangingresidue in an element of secondarystructure that folds late (such as residue25 in a loop in barnase), or where there isfast exchange in secondary structure thatfolds early (such as b-1 in barnase), shechooses to ignore the data. An analysis ofa helix gives multiple probes along thehelix and provides information at atomicresolution about how the helix folds. Asingle, slowly exchanging residue in a helixcannot give us this information. The single

residue might be atypical, and there can beartefactual reasons why a residue isprotected against exchange. PerhapsWoodward is unaware of the amount ofexperimental data for both barnase andCI2 – given that in a recent papercomparing exchange and f values forbarnase and CI2 (Ref. 17) she cites none ofour experimental papers but only usestheoretical simulations and her letterpublished here as evidence for herinterpretation.

Woodward also raises several specificpoints about the presentation of the datain our original article.

(1) Woodward implies that the colouringof the ribbon diagrams in Figs 2 and 3 ofRef. 3 is based on our interpretation of fvalues and not on the f values themselves.Although there is some scatter in the data,there are distinct regions of high and low fvalues in both CI2 and barnase. Forinstance, f values at those sites thatreport on secondary-structure formationclearly decrease along the CI2 a-helix fromthe N-cap to the C-cap. Thus, the helix isformed from the N-cap to the C-cap, andthe ribbon diagram is shaded from blue tored accordingly. Most of these f valueswere confirmed by the more rigorousAla®Gly scanning approach that allowsunambiguous interpretation of the data.Although the a-helix provides the clearestexample in CI2 that hydrogen exchange atequilibrium and kinetic folding pathwaysare not related, this conclusion is evidentfrom analysis of regions throughoutbarnase, as Fig. 2a shows.

(2) Woodward also says that thehydrogen-exchange data for CI2 are notaccurately represented. This is not thecase. Although there is an error in thelabelling of Fig. 3b in Ref. 3 (residues 19and 20 should be labelled 20 and 21,respectively; residue 18 is hidden in thisview), all the data are representedaccurately. Residues 11 (global), 16 (local)and 24 (local) are all shown correctly18.

In Figs 2 and 3 of Ref. 3, our aim was togive a visual description of hydrogenexchange and f values. However, we canalso represent our data using scatterplots (see Fig. 1) so that f values andhydrogen exchange can be comparedmore rigorously. Such a comparisonmakes it clear that there is no obviousrelationship between f values andhydrogen exchange at equilibrium, foreither barnase and CI2. It is quitepossible, even probable, that thecorrelation between the slow-exchangingcore and early-folding elements holds forsome proteins. However, this correlationdoes not hold for all proteins; equilibriumhydrogen exchange cannot therefore‘provide a simple way to identify thefolding core’4. Englander goes further: ‘the“last-out – first in” hypothesis…cannot bevalid…[because] hydrogen exchange…isdominated by local fluctuations’.

– 5.5

– 5.0

– 4.5

– 4.0

– 3.5

– 3.0

– 2.5

– 2.0

0 0.2 0.4 0.6 0.8 1

log

k ex

f

Barnase

0 0.2 0.4 0.6 0.8 1f

CI2

(a) (b)

Figure 1Plots of f values versus logkex for barnase19 (a) and chymotrypsin inhibitor 2 (CI2)18 (b).Arrows indicate residues that have rates of exchange ,1025 s21.

TIBS 23 – OCTOBER 1998

381Copyright © 1998, Elsevier Science Ltd. All rights reserved. 0968 – 0004/98/$19.00 PII: S0968-0004(98)01264-X

PROTEIN SEQUENCE MOTIFSReferences1 Clarke, J. and Itzhaki, L. S. (1998) Curr. Opin.

Struct. Biol. 8, 112–1182 Dalby, P. A., Clarke, J., Johnson, C. M. and

Fersht, A. R. (1998) J. Mol. Biol. 276, 647–6563 Clarke, J., Itzhaki, L. S. and Fersht, A. R. (1997)

Trends Biochem. Sci. 22, 284–2874 Woodward, C. (1993) Trends Biochem. Sci. 18,

359–3605 Caflisch, A. and Karplus, M. (1994) Proc. Natl.

Acad. Sci. U. S. A. 91, 1746–17506 Daggett, V. et al. (1996) J. Mol. Biol. 257,

430–4407 Lazaridis, T. and Karplus, M. (1997) Science

278, 1928–19318 Onuchic, J. N., Socci, N. D., LutheySchulten, Z.

and Wolynes, P. G. (1996) Folding Design 1,441–450

9 Shakhnovich, E., Abkevich, V. and Ptitsyn, O.(1996) Nature 379, 96–97

10 Gruebele, M. and Wolynes, P. G. (1998) Nat.Struct. Biol. 5, 662–665

11 Li, A. J. and Daggett, V. (1998) J. Mol. Biol.275, 677–694

12 Li, A. J. and Daggett, V. (1996) J. Mol. Biol.257, 412–429

13 Caflisch, A. and Karplus, M. (1995) J. Mol. Biol.252, 672–708

14 Shoemaker, B. A., Wang, J. and Wolynes, P. G.(1997) Proc. Natl. Acad. Sci. U. S. A. 94,777–782

15 TiradoRives, J., Orozco, M. and Jorgensen, W. L.

(1997) Biochemistry 36, 7313–732916 Bond, C. J. et al. (1997) Proc. Natl. Acad. Sci.

U. S. A. 94, 13409–1341317 Barbar, E. et al. (1998) Biochemistry 37,

7822–782318 Neira, J. L. et al. (1997) J. Mol. Biol. 270, 99–11019 Perrett, S., Clarke, J., Hounslow, A. M. and

Fersht, A. R. (1995) Biochemistry 34,9288–9298

JANE CLARKE, LAURA S. ITZHAKI ANDALAN R. FERSHT

Centre for Protein Engineering, MRC Centre,Hills Rd, Cambridge, UK CB2 2QH.

A zinc-binding motifconserved in glyoxalase II,b-lactamase andarylsulfatases

The glyoxalase system represents the mainpathway for removal of cytotoxicmethylglyoxal from cells1–3 and might alsoplay a role in controlling cell differentiationand proliferation4. The system usesglutathione (GSH) as a coenzyme anddepends on two enzymes: glyoxalase I andglyoxalase II.

The zinc metalloenzyme glyoxalase Icatalyses GSH-dependent inactivation oftoxic methylglyoxal. Detailed investigationsof the kinetics and mechanism of thisreaction have been carried out usingenzymes from a variety of biologicalsources and, recently, Cameron and co-workers5 determined and refined thecrystal structure of the dimeric humanenzyme. Glyoxalase II catalyses thehydrolysis of S-D-lactoylglutathione to GSHand D-lactate. The human and Arabidopsisthaliana enzymes have been cloned andexpressed in Escherichia coli6,7, and metalanalysis indicates that the plant enzymecontains two atoms of zinc per monomer8.The human enzyme contains 260 aminoacid residues and shares 54% sequenceidentity with the plant enzyme7.

We searched the SWISS-PROT and EMBLdatabases at the ExPASy site (http://expasy.hchugh.ch/) for previously unidentifiedsequences that share similarity withglyoxalase-II sequences. Alignment of allthe known glyoxalase-II sequencesindicated that a region that spans residues50–65 (of the human enzyme), whichcontains several histidines, is highlyconserved. We therefore performed aBLAST search using the conservedstructural motif T/SHXHXD as a query andfound that this sequence is strictlyconserved in two other hydrolase families:metallo-b-lactamase; and a group thatbelong to the Alteromonas-carrageenovora-ATSA–Escherichia-coli-ELAC family

[see Fig. 1 (over page)]. The residues thatcoordinate the second zinc atom inglyoxalase II, b-lactamase II and thesearylsulfatases are also strictly conserved –with the exception of a cysteine residue,which is substituted by a histidine residuein the arylsulfatases (Fig. 1, lower panel).

Metallo-b-lactamases are zinc-dependenthydrolases, and the recent determination ofthe crystal structure of a b-lactamasepurified from Bacteroides fragilis9 has shownthat this enzyme contains a binuclear zinccenter. The residues that coordinate thetwo zinc atoms are invariant in all themetallo-b-lactamases that have beensequenced, except for two conservativesubstitutions (see Fig. 1b). Despite thepattern of binuclear zinc binding, thecrystal structure of the Bacillus cereusenzyme10 contains only a single zinc atom.The aspartate residue present in theconserved motif lies in the active site and isprobably directly involved in catalysis.

Sulfatases are important physiologicalenzymes that hydrolyze sulfate-ester bondsin a wide variety of structurally differentcompounds. The catalytic activity ofeukaryotic sulfatases depends on the post-translational oxidation of the thiol group(to an aldehyde) of a conserved cysteineresidue. Very recently, Lukatela and co-workers11 determined the crystal structureof the human lysosomal arylsulfatase A at2.1-Å resolution. Unexpectedly, the enzymecontains a magnesium ion in its active siteand is structurally related to alkalinephosphatase – an enzyme that is a typicalmember of a family of hydrolytic enzymesthat contain one or more zinc ions in theiractive sites12.

By contrast, very little is known aboutprokaryotic arylsulfatases. Ourobservation that the zinc-binding motif thatis present in zinc-b-lactamases and theglyoxalase-II enzymes is strictly conservedin bacterial arylsulfatase sequences istherefore very interesting. Other residuessurrounding the zinc-binding region arealso conserved in the three hydrolasefamilies; in particular, some bulkyhydrophobic residues, together with athreonine or serine residue and a glycineresidue, are conserved. These residues

probably play a fundamental structuralrole. To date, glyoxalase II and arylsulfataseproteins have not been crystallized, and noinformation about the spatial arrangementof their polypeptide chains or the presenceof a specific metal is available. Ouridentification of a zinc-binding motif inthese proteins is therefore a new andimportant observation and suggests thatthey, like b-lactamase, are metal-bindingproteins and that the catalytic mechanismsof the three classes of enzyme arosethrough convergent evolution.

References1 Vander Jagt, D. L. (1989) in Coenzymes and

Cofactors (Vol. 34) (Dolphin, D., Poulsen, R. andAvramovic, O., eds), pp. 597–641, John Wiley

2 Thornalley, P. (1993) Mol. Aspects Med. 14,287–341

3 Mannervik, B. (1980) in Enzymatic Basis ofDetoxication (Vol. 2) (Jakobi, W. B., ed.), pp.263–273, Academic Press

4 Szent-Gyorgyi, A. (1965) Science 149, 34–375 Cameron, A. D. et al. (1997) EMBO J. 16,

3386–33956 Ridderstrom, M. et al. (1996) J. Biol. Chem.

271, 319–3237 Ridderstrom, M. et al. (1997) Biochem. J. 322,

449–4548 Crowder, M. W. et al. (1997) FEBS Lett. 418,

351–3549 Concha, N. O. et al. (1996) Structure 4,

823–83610 Carfi, A. et al. (1995) EMBO J. 14, 4914–492111 Lukatela, G. et al. (1998) Biochemistry 37,

3654–366412 Vallee, B. and Auld, D. S. (1993) Biochemistry

32, 6493–6501

SONIA MELINO

Dipartimento di Scienze Biomediche,Università ‘G. D’Annunzio’ Chieti, Italy.

CONCETTA CAPO

Dipartimento di Biologia, Università ‘Tor Vergata’ Roma, Italy.

BEATRICE DRAGANI, ANTONIO ACETOAND RAFFAELE PETRUZZELLI

Dipartimento di Scienze Biomediche,Università ‘G. D’Annunzio’ Chieti, Italy.