Proc. Nat. Acad. Sci. USAVol. 72, No. 2, pp. 548-552, February 1975

The Structure and Function of Immunoglobulin Domains: Studies withBeta,-Microglobulin on the Role of the Intrachain Disulfide Bond

(circular dichroism/optical rotation/nucleation/complement fixation)

DAVID E. ISENMAN, ROBERT H. PAINTER, AND KEITH J. DORRINGTON

Department of Biochemistry, University of Toronto, Toronto, Canada, M5S 1A8

Communicated by Charles Tanford, November 8, 1974

ABSTRACT fl-Microglobulin, a low-molecular-weightprotein structurally related to the homology regions ofimmunoglobulins, has been used to study the role of theintrachain disulfide bond in the unfolding of immuno-globulin domains. The intact protein could be reversiblyunfolded in guanidine hydrochloride, as judged by circulardichroism and optical rotation. Similarly, reoxidation ofthe reduced protein, during transfer from high concen-trations of guanidine to neutral aqueous buffer, yielded aproduct with spectral characteristics typical of the nativeprotein. However, if the free SH groups were preventedfrom reoxidizing either by chemical modification or byholding them in the reduced state, the molecule appearedto be in the randomly coiled state even under conditionswhere the intact protein is in the native conformation,judged on the basis of chiroptical measurements. Thecomplement-fixing activity exhibited by native #2-micro-globulin was retained by the reduced and alkylated deriva-tive, suggesting that the site may be formed from a lineararray of amino acids. We suggest a model for the foldingof 02-microglobulin (and immunoglobulin domains) inwhich one of the early folding events results in disulfidebond formation, the latter being an obligatory step forcontinued folding to the native state.

Extensive amino-acid sequence data on kappa and lambdalight (L) chains and heavy (H) chains from several immuno-globulin classes in a variety of animal species, indicate thatthese chains are divided into nonoverlapping homology regions(1). Each region comprises some 110 residues and contains asingle intrachain disulfide bond. On the basis of this informa-tion and a variety of observations regarding the limited sus-ceptibility of immunoglobulin subunits to proteolytic cleav-age, Edelman et al. (2) have proposed that each homologyregion is folded into a compact globular domain. The domainconcept has been substantiated and extended by a number ofchemical and physical studies [see Cathou and Dorrington (3)for a recent review. ]The absolute conservation of the two half-cystines contrib-

uting to the intrachain disulfide bond suggests that this bondplays an important role within the domain. In an earlier study,Lapanje and Dorrington (4) found that extensively reducedand alkylated IgG behaved as a randomly coiled protein evenin low concentrations of guanidine hydrochloride (Gdn. HCl)where the intact protein was in the native state. This observa-tion suggested that the formation of intrachain disulfides is anobligatory step during the refolding of IgG, since if these bonds

were left intact the native state of the IgG (as judged byoptical rotation) was recovered when the guanidine was re-moved.The possible significance of the above observation with

respect to the acquisition of the three-dimensional structure ofIgG during biosynthesis prompted us to seek a simpler systemin which to study domain folding. 032-microglobulin consists ofa single polypeptide chain of 100 residues (molecular weight,11,700), and sequence analysis (5, 6) has shown that the pro-tein is homologous to the constant (C) homology regions ofIgG (i.e., CL, C,,1, CA2, and CI3). A single disulfide bond ispresent, enclosing a loop of residues of similar size to thatfound in IgG domains. The degree of homology between fl2-microglobulin and any one of the homology regions of IgG iscomparable to that between the homology regions themselves.Physical-chemical studies reported by Karlsson (7) indicatethat the protein is compact and globular as judged by hydro-dynamic measurements and that, by these and spectral cri-teria, it shows marked similarity to the proteolytic fragmentsof L chain corresponding to intact domains (8). These datasuggested that j32-microglobulin would be an appropriatemodel for an isolated immunoglobulin domain.

In addition to these structural similarities between 02-microglobulin and IgG domains, Painter et al. (9) have re-

cently shown that 02-microglobulin will interact with the firstcomponent of complement and thereby activate the classicalcomplement cascade. In IgG, this important effector functionis mediated by the C72 domain (10, 11). Although the biolog-ical significance of this observation is far from clear, it hasallowed us to evaluate the dependence of biological activityupon the native conformation of ,32-microglobulin. Thesestudies have enabled us to predict that the complement-fixingsite on the C72 domain is likely to be formed from a contiguoussequence of amino-acid residues rather than from one-

dimensionally distant residues brought into close appositionby tertiary folding.

MATERIALS AND METHODS

The isolation of ,32-microglobulin from the urine of patientswith severe tubular malfunction was performed as describedby Berggard and Bearn (12). Purity was assessed by starch-geland immuno-electrophoresis and ultracentrifugal analysis.Samples of the protein were reduced at 2 mg ml-' in 6.0 MGdn HCl-10 mM Tris- HCl buffer, pH 7.8, for 1 hr with 10mM dithioerythritol at room temperature. The free SH groupswere subsequently blocked with either 25 mM iodoacetamideor 25 mM iodoacetate or 200 mM methyl iodide. The pH was

548

Abbreviations: IgG, immunoglobulin G; L, light chain; H,heavy chain; C, constant region of immunoglobulins; C1, C2 ....components of complement; Gdn HCl, guanidine hydrochloride;CD, circular dichroism.

Folding of Immunoglobulin Domains 549

maintained between 8.0 and 8.4 by the addition of solid Tris.The samples were then dialyzed against either 1.0 M GdnHCI-10 mM Tris* HC1 buffer, pH 7.8, or the Tris buffer alonewithout Gdn HCl. In one experiment, the SH groups were notblocked but held in the reduced state by dialysis against the1.0 M Gdn-HCl buffer, which had been degassed and sub-sequently saturated with oxygen-free nitrogen. Reduced pro-tein (100 4g ml-') in 6.0 M Gdn HCl was reoxidized by ex-haustive dialysis firstly against 1.0 M Gdn HCl-10 mMTris HCJ, pH 7.8, saturated with oxygen and then against theTris buffer alone.

Optical rotation and circular dichroism (CD) were measuredon an ORD/CD-15 spectropolarimeter (Durrum/JapanSpectroscopic Co) equipped with a SS-20 CD modification(Sproul Scientific Instruments) at 240 as described previously(4, 13). In the CD experiments, protein concentrations be-tween 0.8 and 1.0 mg ml-' in cells of 1.00-cm pathlength wereused from 320 to 250 nm and between 0.1 and 0.2 mg ml-I in0.10-cm pathlength cells below 250 nm. Optical rotation wasmeasured at 320 nm at protein concentrations of 0.6 mg ml-'in a 1.00-cm pathlength cell. Solutions were prepared 18 hrprior to optical rotation analysis by weighing into 10 mMTris- HCl buffer, pH 7.8, appropriate amounts of proteinstock solution and solid, dry Gdn * HC1 (Schwarz/Mann ultrapure, lot 3262). Reference solutions contained no protein andfinal Gdn * HCl concentrations were determined by refractiveindex measurements. Studies on the refolding of reduced andalkylated (32-microglobulin were performed by diluting a pro-tein stock solution initially in 6.0 M Gdne HCl-Tris buffer tothe desired final concentration of Gdn HCl with 10 mMTris - HC1, pH 7.8, and allowing the solutions to stand for 18 hrprior to spectral analysis.

Protein concentrations were determined spectrophoto-metrically at 280 nm and using A "7m = 16.8 (12).The direct interaction of native and reduced/alkylated

02-microglobulin with the first component of complement wasmeasured using the method developed by Augener et al. (14)with previously described modifications (9).

RESULTS

The CD spectrum of native j2-microglobulin, shown in Fig. 1,is similar to that published previously by Karlsson (7). Thecomplex series of bands above 230 nm arises from aromaticchromophores located in dissymmetric environments. Therelatively intense positive band at 232 nm has been tenta-tively identified as a tyrosine transition from its behavior atalkaline pH (7). A similar band is associated with the constantregion of some kappa L chains (15, 16). The principal featurein the peptide absorption region is a negative band centerednear 218 nm. A band in this position is also a general feature ofimmunoglobulins and their subunits and has been attributedto peptide bonds in the /8 conformation (17). In 6.0 M Gdn*HCl the characteristic spectral features of the native proteinare lost (Fig. 1). The residual optical activity above 250 nmpresumably reflects the intrinsic activity of the aromatic chro-mophores. Below 250 nm the 218-nm band of the native pro-tein is replaced by intense negative ellipticity. The centerposition of this band could not be seen because of the strongabsorption of the guanidinium ion in this region. In view of thelarge body of evidence that the majority of proteins, includingimmunoglobulins, are randomly-coiled in 6.0 M Gdn-HCl(18), we conclude that this spectrum characterizes the un-

[6-20 U -40

10-3 1009[0

8 ~~~~0.3.0~ ~ ~ 5 -12

40 1~~~~

200 ~~230 250210 230 250 270 290 310

WAVELENGTH (nm)FIG. 1. CD spectra, between 210 and 310 nm, of intact 82-

microglobulin in 10 mM Tris HCl buffer, pH 7.8 (solid line);intact j2-microglobulin in 6.0 M Gdn.HCl, 10 mM Tris-HCl,pH 7.8 (broken line); and reduced and blocked (iodoacetate) (62-microglobulin in 10 mM Tris-HCl, pH 7.8 (dotted line). Thereduced and blocked protein showed a minimum near 200 nm(see inset on left). The mean residue ellipticity, [9]x, is given indeg- cm2 * decimole -. Note the difference in scale for the left andright ordinates.

folded form of 0,2-microglobulin. This was confirmed by opticalrotation studies (see below).The CD spectrum of reduced and blocked (iodoacetate)

#2-microglobulin, recorded in the absence of guanidine, wasstrikingly similar to the spectrum of the intact protein in 6.0M Gdn- HCl (Fig. 1). Certainly the spectral features of thenative conformation were absent. The absence of Gdn*HC1enabled us to determine that the minimum of the band in thefar ultraviolet was located near 200 nm. A band in this positionis typically seen in randomly-coiled polypeptides and proteins.Although the magnitude (about 10,000 deg - cm2 * decimole) isless than that observed for unordered poly-ilysine and poly-L-glutamate, it is similar to several reported values for un-folded proteins (see ref. 19). The spectrum of the reduced andcarboxymethylated protein in 6.0 M Gdn - HC1 was identicalto that of the intact protein in the same solvent. The tentativeconclusion at this stage was that in the absence of the intra-chain disulfide bond 6,2-microglobulin is unfolded, even in theneutral aqueous buffer.One criticism of the above conclusion could be that the

introduction of the bulky, charged blocking groups into thereduced protein was a significant factor in preventing refold-ing. This possibility was tested by replacing iodoacetate witheither iodoacetamide (bulky but uncharged) or methyl iodide(small and uncharged) or omitting blocking altogether andholding the free SH groups in the reduced state by rigorouslyexcluding oxygen. The CD spectra of these various derivativesare shown in Fig. 2. All spectra were recorded in 1.0 M GdnHCl buffer. This change in protocol was introduced becausethe reduced and blocked proteins showed only limited solu-bility in neutral aqueous buffers and the soluble proteintended to aggregate. In 1.0 M Gdn * HCl these problems wereminimized and the intact protein was in the native state asjudged by CD (Fig. 2). None of the modified preparations of02-microglobulin showed spectral characteristics reminiscentof the native intact protein. However, the spectra of the fourderivatives are not identical, suggesting that they may havedifferent (but not native) average conformations. For exam-

Proc. Nat. Acad. Sci. USA 72 (1975)

550 Biochemistry: Isenman et al.

181A~~~ ~ ~ ~~~~8

-3.0

-4.0

210 230 250 270 290 310WAVELENGTH (nm)

FIG. 2. Right: CD spectra of intact P,2-microglobulin (uppersolid line); reduced ,2-microglobulin blocked with either iodo-acetate (lower solid line), or iodoacetamide (-----) or methyliodide (broken line). A sample of the reduced protein was heldin the reducead state under anaetobic conditions (dotted line).Left: The shaded area encloses the CD spectra of the reducedand reduced and blocked derivatives of ,B2-microglobulin. Thesolid line is the spectrum of the intact protein and the broken linerepresents the spectrum of reduced and re-oxidized ,B2-micro-globulin. The final solvent in all cases was 1.0 M Gdn -HCl-10mM Tris * HC1, pH 7.8.

ple, there is a progressive intensification of a positive bandnear 290 nm, probably arising from tryptophan, as the sizeof the blocking group decreases. Below 250 nm all derivativesshow a monotonic change in ellipticity with decreasing wave-length with no evidence for the restoration of the 218-nm bandtypical of the native protein. However, if the SH groups areallowed to reoxidize the 218-nm band is restored (Fig. 2).

Optical rotation at 320 nm was used to follow the unfoldingof intact j32-microglobulin by increasing concentrations ofGdn HCl at pH 7.8 (Fig. 3). The protein underwent whatappeared to be a single-stage conformational transition be-tween 1.0M and 3.5M Gdn* HC1 with mid-point near 2.25 M.Outside this concentration range small monotonic changes in[m'] are seen, presumably reflecting solvent effects on thenative and unfolded states of the protein. Fig. 3 also illustrates

-200-

-300 -

[mI=.

-400.

00 20 4.0 6.0[Gdn HCI], M

FIG. 3. Change in the reduced mean residue rotation of in-tact 62-microglobulin at 320 nm, [Mi']320 nm, as a function of Gdn-HCO concentration (-). A sample of protein in 5.0 M Gdn -HClwas diluted to a final concentration of 2.0 M to test for reversibil-ity as shown by A. The effect of reducing the concentration ofGdn HCl from 6.0 M to 1.0 M on the value of [m' 320nm for re-

duced and alkylated (methyl iodide) fl-microglobulin is alsoshown (U).

the result of progressively lowering the guanidine concentra-tion in a sample of reduced and alkylated (methyl iodide)protein initially in 6.0 M Gdn - HC1. No evidence of refoldingis apparent and the small changes in rotation that do occurcan be accounted for by solvent effects. In contrast, when asample of intact protein in 5.0 M guanidine was diluted to 2.0M reversibility was observed (Fig. 3).The extent to which ,62-microglobulin, reduced and alkyl-

ated with methyl iodide, retained the capacity to interact withC1, the first component of complement, was assessed by themethod of Augener et al. (14). This assay is performed in 4steps leading to the lysis of red cells initiated by C1 that hasnot been bound by the test protein. Serial doubling dilutionsof the test protein were incubated with a preparation of C1containing a limited amount of Clq and an excess of the othersubcomponents of C1. Red cells carrying C4 were then addedfollowed, after washing, by an excess of C2. The red cells werethen lysed by the addition of a reagent (C-EDTA) containingthe terminal complement components (C3-9). In this assayreduced and alkylated (methyl iodide) j32-microglobulin clearlyretained the ability to interact with C1 and showed a dose-response relationship parallel to that of the native protein(Fig. 4) *. However, the log-dose response was displaced toabout a 10-fold lower protein concentration. This enhancedactivity of the ,32-microglobulin derivative is almost certainlyrelated to the aggregation that occurs in neutral aqueousbuffers. The assay cannot be performed in buffers containing1.0M Gdn * HCl, where this aggregation is minimized. It is notpossible, therefore, to compare directly the activities of thenative and reduced and alkylated f32-microglobulin.

DISCUSSION

The results presented above relate to two aspects of the struc-ture and function of immunoglobulin domains: (1) The role ofthe intra-domain disulfide bond in the acquisition and stabili-zation of the native conformation and, (2) the dependence ofbiological function upon this conformation. These two pointswill be discussed separately. The justification for using 32-microglobulin to study these aspects stems from the structuraland functional information, outlined in the introduction, whichstrongly support the view that this protein may be a goodmodel for an isolated domain.Our spectral data indicate that in the absence of an intact

intrachain disulfide the protein does not fold into the confor-mation characteristic of the native state. Furthermore, theproperties of the reduced protein in solvents where the intactprotein is native are similar to those of the latter in the un-folded state (i.e., in 6.0 M Gdn-HCl). The intact protein, incontrast, may be renatured from high concentrations of guani-dine without difficulty. The change in spectral properties ofreduced f32-microglobulin under conditions where the disulfideis allowed to reform indicate that the native conformation can

be substantially recovered.t

* This derivative also inactivated whole complement, as mea-

sured in a classical complement fixation test, as has already beenreported for the native protein (9). The relative potencies of thetwo preparations appear to be equal.t Renaturation and reoxidation of Fab and isolated L chaindomains also results in the recovery of the native conformation(20,21).

Proc. Nat. Acad. Sci. USA 72 (1975)

Folding of Immunoglobulin Domains 551

One of the basic principles of protein chemistry has beenthat the acquisition of three-dimensional structure is con-trolled by thermodynamic factors inherent in the primarysequence. More recently, however, the possibility that kineticfactors may also be important has been receiving much atten-tion. This new awareness was stimulated by the argument thata random search of all possible structures available to even asmall protein would take a biologically unrealistic time (seeref. 22). Consequently it has been suggested that someearly event (frequently called "nucleation") occurs, whichdirects the folding along a kinetically accessible pathway(22, 23). The possible role of short-range structures in theformation of nuclei around which other portions of the poly-peptide might fold has recently been discussed (24). A mecha-nism whereby nucleation might be involved in disulfide bondformation and subsequent folding of 132-microglobulin (and, byextrapolation, immunoglobulin domains) is illustrated sche-matically in Fig. 5. Although the evidence for this hypothesisis based on in vitro data it might also apply equally well to thein vivo situation. Some folding of the polypeptide could occurduring synthesis, although the disulfide cannot form until thechain is more than 80% complete, since the second half-cystine is residue 81. It seems unlikely that disulfide bondformation per se is the nucleation event. Rather, nucleationoccurring elsewhere in the chain brings the two SH groups intoclose apposition, facilitating oxidation. The formation of thisbond stabilizes the nucleus and folding continues. In theabsence of covalent bonding the marginally stable nucleusdissolves and folding aborts. The average conformation ofsuch molecules might approximate the random-coil state, witha large number of possible conformations, as suggested by ourdata. In view of the need for precise orientation of half-cystines for disulfide formation, it is reasonable to supposethat the nucleation site involves regions of the polypeptidechain adjacent to these residues. However this raises a prob-lem, because the half-cystines are separated by 55 residues inthe linear sequence and the nucleation event would thus de-pend upon a random association of one-dimensionally distantregions. Lewis et al. (24) have suggested that one way such aproblem may be overcome is through 3-bends which reversethe direction of the chain and thus may direct nucleation. Inthis regard it is worth recalling that the existence of 1-bends inimmunoglobulin domains has been predicted from sequenceanalysis (25) and observed by high resolution x-ray analysis(26, 27).The "native conformation" depicted in Fig. 5 is a two-

dimensional representation of the "basic immunoglobulinfold" seen by x-ray diffraction (26). Extensive tertiary struc-ture is localized within the loop formed by the disulfide. Acrucial role for this bond in maintaining the overall conforma-tion is not readily apparent. This suggests that once foldinghas reached an advanced state the presence of the disulfidemay no longer be obligatory. This idea might be tested if thebond were available for reduction in the native protein.Unfortunately it is necessary to unfold 132-microglobulin (andimmunoglobulins) before the bond is rendered susceptible toreducing agents.The kinetics of intrachain disulfide bond formation has been

extensively studied in a number of proteins, notably ribo-nuclease (28) and lysozyme (29). In these specific cases morethan one bond is present in the native protein and the forma-tion of intermediates with incorrectly paired disulfides occurs

ic4 10-5 16

Concentration of Protein Incubatedwith C1,M

FIG. 4. The interaction of intact (-) and reduced and alkyl-ated (0) 6@2-microglobulin with the first component of humancomplement. Fab fragment derived from a human IgG1 myelomaprotein was included as a negative control (0). For further de-tails see Results.

during refolding, the reshuffling of which represents a rate-limiting step in the formation of the native conformation (28).Immunoglobulin subunits are also single polypeptide chainswith several intrachain disulfides, but a putative nucleationevent occurring within each homology region would effectivelypreclude mismatching of half-cystines. Independent foldingof discrete regions along the chain represents a mechanism forlimiting the overall time required to fold a large polypeptide.If such a mechanism exists studies on the rate of folding of132-microglobulin in vitro could provide useful insights into thefolding of immunoglobulin chains in vivo.

It is common knowledge that many proteins are devoid ofintrachain disulfides, yet they fold into compact globularstructures. The reason for the important role of such bonds inimmunoglobulins may perhaps be found in their uniquestructural heterogeneity. Unlike most, if not all, other pro-teins, immunoglobulin molecules exhibit a wide range ofprimary sequences, even in a single individual. Despite this

RANDOM CONFORMATION

NUCLEATION

I FOLDING

N C OO-NATIVE CONFORMATION

FIG. 5. The possible role of the intrachain disulfide bond inthe folding of i82-microglobulin and immunoglobulin domains.For details see Discussion. The filled circles represent sulfur atoms.

Proc. Nat. Acad. Sci. USA 72 (1975)

552 Biochemistry: Isenman et al.

heterogeneity the subunits of all molecules, regardless of class,subclass, antibody specificity, etc., are divided into homologyregions which fold into a series of domains. Perhaps the ab-solute conservation of the disulfide ensures a kineticallyaccessible folding pathway in the face of this variability inprimary structure?The retention of a complement-fixing site in the reduced

and alkylated derivative of fl2-microglobulin that our dataindicate to be in an unfolded state raises interesting questionsregarding the structure of this site. Kehoe et al. (30) comparedthe amino acid sequences within the CH2 region of those im-munoglobulin classes and subclasses that are known to interactwith complement and those that apparently do not, but failedto find any linear sequence of residues that might be associatedwith this interaction. On the basis of this observation theyconcluded that complement fixation must depend on a

conformational site composed of residues brought closetogether by tertiary folding. Although we cannot rigorouslyexclude the existence of local regions of tertiary folding inreduced and alkylated f32-microglobulin, our data suggest thatthe complement-fixing site is made up of one-dimensionallyclose residues. This apparent contradiction clearly warrantsfurther study. 0

This study was supported by Grants (MT 1361 and 4259)from the Medical Research Council of Canada.

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