Proc. Nati. Acad. Sci. USAVol. 82, pp. 5700-5704, September 1985Biochemistry

Model of a2-macroglobulin structure and function(proteinase inhibitor/a2-macroglobulin/trap hypothesis)

STEVEN R. FELDMAN, STEVEN L. GONIAS, AND SALVATORE V. PIZZODepartments of Pathology and Biochemistry, Duke University Medical Center, Durham, NC 27710

Communicated by Irwin Fridovich, May 9, 1985

ABSTRACT A model of a2-macroglobulin is presentedthat is compatible with previous structural, functional, andphylogenetic studies of the protein. The model of the moleculeresembles a hollow cylinder and is comprised of two identicalfunctional halves with three C2 axes ofsymmetry and no mirrorplanes. The "trap mechanism" of this proteinase inhibitor iseffected by slight movement of two trap arms per "half-molecule." Evidence for this model is obtained from the studyof the structure and proteinase binding of the molecule. Byusing this model, predictions are made concerning proteinasebinding ratios, receptor recognition, and "slow-to-fast"conformational change of the molecule.

a2-Macroglobulin (a2M) is unique among the numerousplasma proteinase inhibitors in its ability to inhibitendopeptidases of any class and nearly any specificity (1).The protein has a molecular weight of 725,000 and iscomposed of two noncovalently bonded pairs of identicalsubunits joined by disulfide bonds (1, 2). Each subunitcontains a specific sequence of amino acids termed the "baitregion," which is highly susceptible to proteolytic cleavage(1). When this region is cleaved by proteinase, a conforma-tional change occurs in the inhibitor resulting in irreversiblebinding of the proteinase without blocking of the proteinaseactive site (1). Bound proteinase generally retains activitytoward small substrates, whereas activity toward large sub-strates is greatly reduced. The remaining activity of thecomplexed proteinase may be abolished by active site titrants(e.g., tosyllysine chloromethyl ketone), whereas large pro-tein proteinase inhibitors (e.g., soybean trypsin inhibitor)react slowly, or not at all, with a2M-proteinase complexes.

Covalent binding of the proteinase to a2M is not requiredfor irreversible inhibition; however, such a bond may form byreaction of a lysine residue of the proteinase with a reactivethioester of the macroglobulin (3-5). The thioester may becleaved in the absence of proteinase by small amines. Afterreaction with methylamine, human a2M exhibits a confor-mational change that is nearly equivalent to the changeobserved after reaction with proteinase (6).

After reaction with proteinase or amine, a receptor-recognition site is exposed on a2M and the complex is clearedrapidly from the circulation by hepatocytes and reticulo-endothelial cells (7-9). Specific receptors for a2M exist onmany cell types, as determined in tissue culture studies(8-10). a2Ms that have been allowed to react with proteinaseand amine exhibit a similar affinity for the receptor, whereasnative a2M does not bind to the receptor (11, 12).Human a2M binds many proteinases at a 2:1 ratio of

proteinase to inhibitor (13). There is considerable evidencethat this inhibitor contains two independent functional unitstermed "half-molecules." Functional halves of a2M havebeen prepared by limited reduction and alkylation of theparent protein (14, 15). Furthermore, phylogenetically older

aM (such as from fish) exist as half-molecules in their nativestate (16).

Barrett and Starkey (1) have termed the inhibition mech-anism of a2M "trapping." The sprung or closed form of theinhibitor is more compact than the native inhibitor, asassessed by hydrodynamic measurements and migration inpore-limit gel electrophoresis (6, 17). The electrophoreticbehavior is responsible for the term "slow-to-fast"conformational change. It has been thought that theconformational change involved in compacting the proteineffects proteinase trapping, exposure of the receptor-recog-nition site, and spectral change (particularly, circular dichro-ism) of the molecule (6, 17). In several studies with non-human a2M homologues and chemically modified humana2M, these changes occurred without slow-to-fast conforma-tional change or changes in hydrodynamic parameters (11,18-20).Because of the biologic importance of this general scav-

enger of proteinases and the interest in its unique mechanismfor inhibiting proteinases, several laboratories have attempt-ed to elucidate its structure and the functional correlates ofthis structure. Hydrodynamic studies (6), electron micros-copy (21), and x-ray scattering (22) studies have beenperformed with a2M yet no detailed structural model hasemerged to correlate the structure, proteinase binding, re-ceptor recognition, and slow-to-fast conformational changeof the protein.

THE MODELA model that is compatible with previous studies of thestructure, function, and phylogeny ofa2M is presented in Fig.1. The a2M is composed of four subunits and appearsgenerally cylindrical. A plane perpendicular to the axis of thecylinder and crossing halfway between the ends of themolecule separates the inhibitor into two identical functionalhalves. Each half is made up of a ring with four arms (two permonomer) projecting from one side. Each monomer is con-stituted of one long and one short arm. The other side of thering makes contact with the second functional half. Trappingof proteinases can occur by movement of one long arm permonomer, two arms per functional half. These long arms aretermed the trap arms. As presented, the molecule has three2-fold axes of symmetry (C2) (one through the axis of thecylinder and two perpendicular to it and to each other in themedian plane) and no mirror planes consistent with thepresence of four identical polypeptide chains. This model isin agreement with the available data concerning a2M asdescribed in the four subsequent sections.

Structure. In electron micrographs, native a2M appears asa Cyrillic "H" (XC) (Fig. LA Inset) (21). Reaction withproteinase causes a compacting of the molecule with in-creased curvature of the arms of the XC (Fig. 1B Inset).Computer-averaged electron micrographs show similar fea-tures with greater resolution (21). In the micrographs, there

Abbreviation: aM, a-macroglobulin.

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A B

FIG. 1. Model of a2M. (A) The native molecule accompanied by a schematic of the projection in electron micrographs (Inset). (B) Theconformational change of the molecule with a schematic of an electron micrograph of "fast" a2M (Inset). Small arrows denote the positionspostulated for the receptor-recognition sites. (C) Proteinase trapping by this inhibitor.

appears to be much greater density at the periphery of thecentral portion of the XC. In a2M that has been allowedto react with proteinase, a region of increased densityappears on each side of the central disk aligned linearly alongthe central axis of the molecule.The projections of the model in Fig. 1 are consistent with

the electron micrographs. The arms of the M seen in elec-tron micrographs correspond to the trap arms; the centralportion of the MI corresponds to the central rings of themolecule. The proteinase binding sites are on either side ofthe central rings aligned along the central axis of the mole-cule. In this model, the only conformational change requiredto effect a trap mechanism is a slight movement of the lateralarms of the molecule. Movement of the other two arms of thefunctional domain may occur but is not required for trappingin the model.Low-angle x-ray scattering studies have been performed

with a2M in solution (22). The data obtained in these studiesare most consistent with a representation of the molecule asa hollow cylinder; the model in Fig. 1 presents very nearly asa hollow cylinder.

In a2M that has been allowed to react with proteinase,x-ray scattering studies place the proteinase molecules nextto each other at the central disk of the hollow cylinder (22).The core of the cylinder is filled and the longest distance inthe molecule decreases. The model of a2M presented hereplaces the proteinase molecules within the inhibitor asdescribed by the x-ray-derived data.Pochon et al., employing excitation energy-transfer exper-

iments with a2M and chymotrypsin, demonstrated that theproteinase binding sites are identical and adjacent (23). Fig.1C demonstrates the position of the proteinase molecules inthis model. The binding sites are identical and adjacent.Through the hollow core of the protein, the proteinasemolecules may be close to touching.We next consider the position of the thioesters. Reaction

of the thioesters of a2M with methylamine results in aconformational change in the protein similar to that obtainedby reaction of the molecule with proteinase (6). Pochon et al.(24), employing energy-transfer studies, concluded that (i)the thioester sites are in close proximity to the proteinasebinding sites and (ii) the thioester sites are arranged in twopairs. Further energy-transfer studies utilized a 1:1 complexof chymotrypsin dimer and a2M. The efficiency of transferwas consistent only with a model in which one of the twoproteinase molecules is close to the thioester site. When theavailable data are considered in the context of the present

model, there are a number of constraints placed on thepossible location of the thioesters. Pairs of the thioesterscould occur in close proximity at each end of the molecule,but this seems unlikely to readily explain the extent of theconformational change observed when these bonds are bro-ken. We, therefore, postulate that the thioester is locatednear the hinge of the trap arm ofeach subunit. Cleavage ofthethioester allows the trap arm to swing. In the model, then,reaction with amine can result in the same conformationalchange obtained by proteolytic cleavage of the bait region.The thioesters within a pair are close together (at the base ofadjacent trap arms separated by the plane between thefunctional halves), whereas the two pairs are located about 7nm (the diameter of the ring in this model) apart. In thismodel, the chymotrypsin dimer must be oriented linearlyalong the central axis of the a2M because of the constraintsof the dimensions of the proteinase binding sites (Fig. 2,Table 1). In such a configuration, only one of the subunits ofthe chymotrypsin dimer can be close to the thioesters,consistent with the observed data. This model is consistentwith the observation that binding of a chymotrypsin dimer

FIG. 2. Binding of chymotrypsin dimer by a2M. A chymotrypsindimer is enclosed in the binding site above. A monomer ofchymotrypsin is bound in the second binding cavity. The thioesterbonds (not shown) are in closely apposed pairs, which are locatedabout 7 nm apart near the base of the hinge of the trap or long arms.The distance 7 nm is the diameter of the ring in this model.

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Table 1. Approximate dimensions of a2M and its proteinasebinding sites

Inner Outer CentralLength, diameter, diameter, disk,

Method nm nm nm nm Ref.

Electronmicroscopy 16 5.8 7 4 21

X-rayscattering 19.5 6 9 4 22

Approximate proteinase sizes (assuming a sphere and based onmolecular weight and partial specific volume): chymotrypsin, 4 nmin diameter; plasmin, 6.3 nm in diameter.

does not preclude binding of a chymotrypsin monomer in theadjacent binding site (23).Approximate dimensions of a2M obtained from the above

studies are presented in Table 1. Notably, Barrett andStarkey (1) predicted a trap size similar to that predicted here.

Proteinase Bindikig. Studies on proteinase binding havedemonstrated that many kroteinases bind to a2M in a 2:1proteinase-to-inhibitor ratio (13). Large proteinases such asplasmin and the chymotrypsin dimer bind in a nearly 1:1 ratio(13, 23, 25). Both steric and kinetic explanations have beenextended for this phenomenon, and they are not necessarilymutually exclusive. Fig. 3 demonstrates one mechanism bywhich a large proteinase such as plasmin could not bind in a2:1 molar ratio. Protrusion of one of the domains of plasminacross the hollow core into the second binding site couldprevent sterically the binding of a second proteinase mole-cule.

Functionally active half-molecules ofa2M bind trypsin andplasmin at the same molar ratio (14, 15); this is notablydifferent from native a2M, which binds twice as much trypsinas plasmin. Since the binding sites are uncoupled in half-molecules, this result is not surprising. After reaction withtrypsin or methylamine, the half-molecules reassociate (Fig.4A) (14); however, half-molecules that have been allowed toreact with plasmin do not reassociate (15). This observationis consistent with the model presented here as well. The samesteric hindrance preventing 2:1 proteinase-to-inhibitor bind-ing in native a2M prevents two halves that have been allowedto react with plasmin from reassociating (Fig. 4B).

PREDICTIONS

Receptor Recognition. The model of a2M as a cylindricalstructure with two functional halves and two moving traparms per half-molecule fits the known structural and func-tional data and it makes possible predictions about thelocation of the receptor-recognition sites of the molecule.

FIG. 3. Plasmin binding to a2M. Plasmin bound to the inhibitorin a 1:1 ratio is represented. (Inset) Protrusion of plasmih into thesecond binding site.

FiG. 4. Half-molecules of a2M. (A) Half-molecules of a2M,demonstrating that reaction with proteinase leads to reassociation ofthe "whole-molecule" form. (B) Half-molecules that have beenallowed to react with plasmin. These halves do not undergo completereassociation.

Because the trap arms are the only moving parts ofthe model,it appears that a reasonable placement for the receptor-recognition site would be at the base of the trap arm, near itshinge. Exposure of the site would be effected by the changein conformation of the arm, similar to exposure of the Fcreceptor of immunoglobulins (26). Fig. 1B demonstratesexposure ofthe receptor-recognition site in this model. Thereis one recognition site present on each subunit (one per traparm and two per half-molecule) located at the interfacebetween functionally active halves. Trap closure results inexposure of receptor-recognition sites at the base ring neareach swinging arm of the trap. Marynen et al. (27) demon-strated four receptor-recognition sites per molecule usingmonoclonal antibody to the receptOr-recognition site. Fur-thermore, Strickland et, al. (28), using a probe that changesfluorescence on receptor-recognitidn site exposure, demon-strated that the kinetics of the changes in fluorescence weremost consistent with a model in which each site is exposedindependently. Since the subunits are identical, each musthave a single receptor-recognition site that is exposed inde-pendently.

Half-molecules of human a2M clear faster from the mousecirculation than the native molecule, which may indicatesome interaction of half-molecules with the a2M-proteinasereceptor (11, 14). These data suggest that the receptor-rec-ognition site may be located near or at the interface betweenthe functional halves and is partially exposed when the halvesare separated. Furthermore, half-molecules that have beenallowed to react with plasmin clear faster than the nativetetrameric inhibitor that has been allowed to react withplasmin (15). As mentioned above, plasmin nearly fills thepocket hypothesized in this model and may not allow forcomplete trap closure-that is, swinging of the trap arm andexposure ofthe reocognition site at the base. Better exposureof the recognition site would be obtained in the half-moleculeif the recognition site were at the base of each half asproposed here. Furthermore, these data also indicate that thereceptor-recognition sites do not involve shared determi-nants between two functional halves.More evidence for this model comes from the observation

that whole-molecule aMs from every species studied havethe receptor recognition site, but naturally occurring half-molecules from phylogenetically ancient species do not havethe site (29). In the frog, two distinct aMs have been found:

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one, which is a whole-molecule aM and binds to the receptor,and one, which is a half-molecule aM that does not bind tothis receptor (29, 30). This suggests that the recognition sitecould not evolve until the side ofthe molecule that constitutesan interface of interaction between the functional halves wasburied; otherwise, the native half-molecule would clear fromthe circulation as rapidly as it could be produced. We havepostulated that another mechanism, perhaps carbohydrate-mediated, provides for plasma clearance of the inhibitor inthese ancient species (29).

Differential data obtained from the study of aM whole-molecules from several species also suggest that receptor-recognition site exposure and trap arm movement are inti-mately related. These non-human aMs inhibit proteinase andbind to the mammalian aM receptor similarly to human aM,although they vary in the extent to which they are receptorrecognized or lose proteinase binding activity when they areallowed to react with methylamine.Table 2 lists these aMs and the extent to which they are

receptor recognized and lose proteinase binding activitywhen allowed to react with methylamine. In every caseknown to us, receptor-recognition site exposure is accom-

panied by loss of proteinase binding activity (18, 20, 30, 32).However, as seen in Table 2, slow-to-fast conformationalchange is not so closely correlated to proteinase binding.Furthermore, we have shown previously that structuralchange in the molecule, as measured by changes of itscircular dichroic spectrum, correlates with receptor-recog-nition site exposure and loss of proteinase binding withoutslow-to-fast conformational change (33). Apparently, theclosure of the trap does not effect the compacting of themolecule, which is measured by migration in pore-limit gelelectrophoresis. The nature of slow-to-fast conformationalchange will now be considered.

Slow-to-Fast Conformational Change. Accepted theories ofaM function, based primarily on studies on human a2M,consider a single conformational change responsible formany of the observed property changes after reaction withproteinase or amine. Recent investigations with aMs isolatedfrom other species and with chemically modified human a2Mindicate that this may be an oversimplification (11, 18-20).Bovine a2M undergoes a conformational change after reac-tion with proteinase, which results in slow-to-fast mobilitychange, receptor-recognition site exposure, and trap closure(18). Reaction of the same protein with methylamine causes

trap closure, as evidenced by the loss of proteinase bindingcapacity and receptor-recognition site exposure withoutslow-to-fast conformational change (18).These data indicate that trap closure is insufficient to

explain the change in electrophoretic behavior. Sedimenta-tion velocity studies suggest that the slow-to-fast electropho-retic transition represents significant hydrodynamic com-

pacting of structure (6). A likely mechanism, independent of

Table 3. Stability and slow-to-fast conformational change ofhuman a2M and frog a2M

Increased Slow-to-faststability of conformational

aM tetramer change Refs.

Human a2M 14, 15, 17+ Trypsin + ++ Methylamine + +

Frog ajM 30+ Trypsin + ++ Methylamine -

trap closure, by which aM can become compacted involvesa change in the positioning of the peptide chains at thejunction of the two functional half-molecules. Such a changewould effect a decrease in the apparent separation of thefunctional halves similar to the decrease in the distancebetween the subunits of hemoglobin during oxygenation(34). Many investigators support a significant augmentationin the forces promoting association of the functional half-molecules ofa2M after reaction with proteinase or amine (14,15, 17, 30), but only when slow-to-fast conformational changeis obtained (Table 3). In addition, Barrett demonstratedglutaraldehyde crosslinking between the human a2M subunitpairs only after reaction with amine or proteinase (17).These data suggest that realignment ofjuxtaposed chains of

amino acids at the half-molecule junction site represents amajor structural change that occurs in addition to trapclosure. This realignment might readily yield a more ener-getically favorable positioning of charged and polar aminoacids, explaining the greater stability of the fast form. If it ispossible for trap closure to occur after reaction with meth-ylamine without the realignment of the junction in certainspecies of aM, then the seemingly contradictory data areexplained. For slow-to-fast conformation to occur, realign-ment of structure must occur at the junction of the half-molecules.This hypothesis for the mechanism of slow-to-fast

conformational change requires that a half-molecule aM notexhibit a faster migration in pore-limit gels when treated withproteinase because the required element of quarternarystructure is missing. Indeed, Starkey et al. (35) found this tobe the case. Although fish aM exhibits many of the charac-teristics of aMs such as a trap mechanism and aminereactivity, the molecule does not exhibit a slow-to-fastconformational change when treated with amine or protein-ase.

SUMMARY

The model a2M presented here is consistent with the largebody of known structural and functional properties of the

Table 2. Receptor-recognition site exposure, proteinase binding, and slow-to-fast conformational change of a series ofaM that have been allowed to react with methylamine

Loss ofReceptor- proteinase Slow-to-fastrecognition binding conformational Decreased

aM site exposure activity change Stokes radius Refs.

Human a2M + + + + 6, 8, 17Rat ajM + + + 31Rat a2M - - 31Bovine a2M + + - - 18, 19Chicken aM + + - 32Frog ajM - - 30Human a2M+ 2,4-dinitrophenylthiocyanate + + - 20

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molecule. The model provides a prediction concerning thelocation of the receptor-recognition sites of the molecule thatis supported by presently available evidence. Finally, themodel provides for resolution of the conflict between slow-to-fast conformational change and other aspects ofconformational change of the inhibitor.Although there is not conclusive evidence that this model

is the final representation ofa2M, the model does provide onepossible frame on which to base our knowledge of theprotein. The swinging arms of the model permit an easyinterpretation of the trap hypothesis (1). The space left openbetween the arms of the molecule lends a simple mechanismto the continued activity of proteinases toward small sub-strates and the reduced activity toward large substrates (24).The model is also consistent with the observation thatantibodies directed against plasmin, a large proteinase, reactwith the a2M-plasmin complexes (1, 36) since theseproteinases could easily be envisoned as protruding from thetrap.

We thank Carol Pienta and Nancy Henn for their artistic contri-butions. This work was supported by research Grants HL-24066 fromthe National Heart, Lung, and Blood Institute and CA-29589 fromthe National Cancer Institute. S.R.F. is supported by the MedicalScientist Training Program, National Institute of General MedicalSciences (GM-07171).

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