Serpin qproteinase inhibitor probed by intrinsic tryptophan ...

Protein Science (1996). 5:2226-2235. Cambridge University Press. Printed in the USA. Copyright 0 1996 The Protein Society

Serpin qproteinase inhibitor probed by intrinsic tryptophan fluorescence spectroscopy

HENRYK KOLOCZEK,' AGNIESZKA BANBULA? GUY S. SALVESEN; AND JAN POTEMPA' ' University of Agriculture, Department of Biochemistry, ul. 29 Listopada 54, 31-425 Krakbw, Poland

Institute of Molecular Biology, Jagiellonian University, Krakow, Poland Burnham Institute, 10901 North Torrey Pines Road, San Diego, California 92037

(RECEIVED June 7, 1996; ACCEPTED August 20, 1996)

Abstract

Various conformational forms of the archetypal serpin human qproteinase inhibitor (a,PI), including ordered polymers, active and inactive monomers, and heterogeneous aggregates, have been produced by refolding from mild denaturing conditions. These forms presumably originate by different folding pathways during renaturation, under the influence of the A and C sheets of the molecule. Because aIPI contains only two Trp residues, at positions 194 and 238, it is amenable to fluorescence quenching resolved spectra and red-edge excitation measurements of the Trp environment. Thus, it is possible to define the conformation of the various forms based on the observed fluorescent properties of each of the Trp residues measured under a range of conditions. We show that denaturation in GuHC1, or thermal denaturation in Tris, followed by renaturation, leads to the formation of polymers that contain solvent-exposed Trp 238, which we interpret as ordered head-to-tail polymers (A-sheet polymers). However, thermal denaturation in citrate leads to shorter polymers where some of the Trp 238 residues are not solvent accessible, which we interpret as polymers capped by head-to-head interactions via the C sheet. The latter treatment also generates monomers thought to represent a latent form, but in which the environment of Trp 238 is occluded by ionized groups. These data indicate that the folding pathway of alPI, and presumably other serpins, is sensitive to solvent composition that affects the affinity of the reactive site loop for the A sheet or the C sheet.

Keywords: alproteinase inhibitor; folding; intrinsic fluorescence; polymerization

Serpins constitute a large family of proteins, many of which inhibit proteinases by rapidly forming tight complexes directed to the enzyme active site. Most serpins are composed of 350400 residues that form a single complex folding unit containing three P-sheets and eight a-helices (Carrell & Stein, 1996). The structural units of serpins cooperate to position a segment of about 20 residues, the reactive site loop, in one of a number of distinct locations in the molecule. The location of the reactive site loop depends on the particular conformation of the serpin; it can exist as a fully exposed loop or it can be fully integrated into the center of the major P-sheet, or it can be partially exposed and partially integrated (Engh et al., 1995). The location of the reactive site loop is critical to inhibition, because this is the part of the molecule to which proteinases dock (Katz & Christianson, 1993). The positions adopted by reactive site loops in a serpin dimer are shown in Fig- ure 1 , and those adopted by several other serpins in Kinemage 1.

The location that the reactive site loop adopts in the inhibitory conformation and whether the various conformations observed in distinct serpins are a general feature of members of this family or

Reprint requests to: Henryk Koloczek, University of Agriculture, De- partment of Biochemistry, ul 29 Listopada 54, 31-425 Krakbw, Poland; e-mail: [email protected].

specific for particular serpins have not been settled. Crystallo- graphic studies have yet to demonstrate clear evidence of what the loop would look like in its inhibitory conformation, but other approaches have provided evidence that partial insertion of the N-terminal end of the reactive site loop into the A sheet is a prerequisite for the formation of a stable inhibitory complex (Skriver et al., 1991; Bjork et al., 1992a, 1992b, 1993; Mast et al., 1992; Lomas et al., 1993a; Lawrence et al., 1994). A combination of these studies has revealed that the reactive site loop exists in an exposed, but noninhibitory conformation that requires considerable adaptation to the proteinase's active site cleft before an inhibitory conformation is attained (Carrell & Stein, 1996). Con- formations in which the cleaved reactive site loop is fully inserted into the A sheet have been demonstrated by crystallography of several serpins (Lobermann et al., 1984; Wright et al., 1990; Bau- mann et al., 1991, 1992; Mourey et al., 1993; Schreuder et al., 1994). Intact, fully inserted, reactive site loops (presumed to represent a latent conformation) have been demonstrated crystallo- graphically for plasminogen activator inhibitor I (PAI-I) and antithrombin I11 (AT3) (Mottonen et al., 1992; Carrell et al., 1994), and predicted for a,proteinase inhibitor (alPI) (Lomas et al., 1995a). Exposed reactive site loops have been demonstrated for three serpins (Stein et al., 1990; Wei et al., 1994; Song et al., 1995), al-

2226

qProteinase inhibitor conformations 2227

14 W

Fig. 1. Stereo view of the AT3 dimer. The AT3 dimer is currently the only molecular structure available for a polymerized serpin, and is shown here to model the positions of Trp residues 194 and 238 of antitrypsin. This stereo view of the dimer is shown in more detail in Kinemage 1, which also contains other serpin structures. The dimer is composed of one molecule in a conformation that is thought to be close to the protease inhibitory conformation (the "i" molecule shown in brick red, with its reactive site loop in red) and one molecule of a latent conformation ("L" shown in blue with its reactive site loop in cyan). The molecules interact in a "head-to-head" manner, where the exposed reactive site loop of the I molecule displaces strand 1C of the other molecule allowing, or forcing, the latter to adopt the L-confonnation. See Schreuder et al. (1994) and Carrell et al. (1994) for descriptions of the molecules. Space-filling representations of Trp 225 (Trp 194 in (uIPI), conserved in all serpins, and Gln 268 (replaced by Trp 238 in (rlPI) are shown in green. Note that the side-chain of residue 268 is solvent exposed in the I molecule, but residue 268 from the L molecule is partly buried by the interface of the two molecules.

though the reactive site loop conformation is different in each case. Together these structures define the limits of the possible conformations of the reactive site loop and demonstrate the importance of the A and C sheets in maintaining each conformation.

Because the reactive site loop can interact with the A and C sheets, it can also interact with these sheets in adjacent molecules, leading to polymerization via a loop-sheet mechanism. Polymer- ization normally requires partial unfolding of the molecules, usu- ally accomplished by mild denaturation, and is dependent on small variations in primary sequence that determine the conformation of the reactive site loop and the A and C sheets (Aulak et al., 1993; Lomas et al., 1993a, 1993b, 1995b; Bruce et al., 1994; Eldering et al., 1995; Kim et al., 1995; Sidhar et al., 1995). Because polymerization involves the reactive site loop, to which proteinases dock during inhibition, the polymers cannot inhibit proteinases.

Evidence from solution studies of serpin conformation imply that the inhibitory form is unstable. Thus, there is an inherent problem in interpreting the serpin inhibitory mechanism from the crystallographic data. None of the crystal structures so far reported are in a conformation that can dock with a proteinase (reviewed by Wright & Scarsdale, 1995, and Carrell & Stein, 1996). Mobility of the reactive site loop is required, which is not observable in the

crystal structures, but valuable predictions can be made based on the location of certain residues in the crystal forms.

In this article, we describe the use of intrinsic fluorescence spectroscopy, based on tryptophan fluorescence that is sensitive to a wide variety of environmental conditions and to different conformational states of aIPI. This serpin contains only two Trp residues, located at positions 194 and 238. The crystallographic structure of this cleaved inhibitor reveals that one of these is located in strand 3 of the A sheet. This would make it inaccessible to quencher molecules, whereas the other is situated in strand 2B and is surface exposed (Lobermann et al., 1984; Song et al., 1995). The equivalent residues in AT3 are in the same relative position in the structure of native, cleaved, and latent forms of antithrombin. The same can be assumed for aIPI, allowing the interpretation of the intrinsic tryptophan fluorescence of the various conformers of the inhibitor using fluorescence methods developed to analyze the environments of a protein that contains only two Trp residues.

Results

The methods used to obtain different conformational forms in- volved denaturation followed by renaturation. Because alPI un-

2228

folds rapidly at about 56 "C in physiologic buffers (Hopkins et al., 1993: Lomas et al., 1993a). and at about 3 M urea at room temperature (Mast et al., 1992), each of the conditions used here would be expected to unfold the protein initially. We do not know the status of the forms in the denaturants. but refolding conditions were empirically chosen to yield a mixture of monomers, polymers, and aggregates. Depending on the denaturing and renaturing conditions, different distributions of conformational forms were obtained. We define these as ( I ) refolded from cold GuHCI, (2) refolded from hot citrate, (3) refolded from hot Tris (see Materials and methods for details).

Electrophoretic characterization of various forms of alPl

Fractions of a l P I conformational forms from cold GuHCl were resolved by size-exclusion chromatography (Fig. 2). and those refolded from hot citrate were resolved by ion exchange chromatography as described previously (Lomas et al., 199Sa). SDS- PAGE of the fractions (not shown) revealed a single band with electrophoretic mobility identical to the native inhibitor, verifying that a l P l had not become degraded during the incubation conditions. The conformational status of protein in each fraction was determined by electrophoresis in transverse urea PAGE (TUG- PAGE: Fig. 3). Significantly, refolding from cold GuHCI, under conditions that were initially believed to induce a stable monomeric form of the related serpin AT3 (Carrell et al., 1991 ), con- verted the majority of a l P l to aggregates (Fig. 2, peak 1 ) migrating as a heterogeneous smear in PAGE (Fig. 3, panel A). Only a small fraction (Fig. 2. peak 2) contained material that migrated with a rate consistent with monomers and small polymers (Fig. 3. panel A). In these conditions. a considerable amount of the inhibitor (Fig. 2, peak 3) retained its native structure and was determined to retain 8S% inhibitory activity. The remaining fractions had no inhibitory activity.

Refolding from hot citrate induced the formation of distinct

I

0.0 I I

I I I I I

16 20 24 28 32 36 40 44 48

number of fractions

Fig. 2. Gel filtration profile of @,PI after incubation in I M GuHCI. Peak I , aggregated protein: peak 2, polymers: peak 3. active monomeric inhibitor.

H. Koloczek et al.

I I

I I

I 3 1 4 - "- - . ._ "

Fig. 3. Conformations of alPl visualized by TUG-PAGE. Approximately 1 0 0 wg portions of various treated a l P l samples were run in TUG-PAGE. followed by staining with Coomassie blue g-250 to identify protein components. 1: Polymer fraction from hot citrate. 2: Monomeric fraction from hot citrate. 3: Polymeric fraction from GuHCI. 4: Total sample from hot Tris, which included high-order polymers and heterogeneous aggregates. Rate of migration is proportional to polymer number (Mast et al.. 1992), with the higher-order polymers migrating most slowly. The material in panel 2 migrated with a rate equivalent to monomeric native alPI (not shown) at the low urea end (left side of gel). but did not exhibit an unfolding transition. It is therefore the same material identified by Lomas et al. (199%) as latent alPI.

monomers, polymers, and heterogeneous aggregates. Separation of these forms by ion exchange resulted in the enrichment of a monomeric form that resisted unfolding in TUG-PAGE (Fig. 3, panel I ) . The form, termed the "latent" form by analogy with a similar form seen to occur spontaneously in PAI-I, is predicted to consist of a molecule in which the reactive site loop is integrated into the A sheet, with distortion of strand 1 of the C sheet (Lomas et al., 199Sa, 199%). This prediction is based on the crystal structures of the related serpins PAI-I (Mottonen et al., 1992) and one of the components of the AT3 dimer (Carrell et al., 1994).

The different denaturinghenaturing conditions generated distinct polymeric forms. Specifically, a l P l refolded from hot citrate contained small polymers consisting of less than S units (Fig. 3, panel 1 ), whereas middle size polymers (10-20 monomers) were obtained following refolding from cold GuHCl (Fig. 3, panel 3). and polymers in excess of 20 monomers were recovered by cooling the protein from hot Tris (Fig. 3, panel 4). The denaturing/ renaturing treatments resulted in variable amounts of large aggregates (see Fig. 3, panel 4, for example). The nature of these aggregates is unknown, although they may represent material produced by hydrophobic collapse of denatured molecules. They were elimi- nated from material to be analyzed by either gel filtration or ion exchange chromatography. We focused on characterization of purified monomers (latent) and polymers from hot citrate, and purified polymers from GuHCI.

Fluorescence quenching of a,PI conformational forms

The fluorescence of the a I P I fractions was quenched with KI and acrylamide, and an example of the data as a Stern-Volmer plot (in

a,Proteinase inhibitor conformations

this case the latent form) is shown in Figure 4. The plot shows a downward curvature that is interpreted in terms of heterogeneous emission from two Trp residues in different environments. In order to resolve the fluorescence emission spectra of alp1 conformational forms, the iodide quenching was measured at different emission wavelengths and the resolved spectra exemplified by the latent form are shown in Figure 5. The data indicate that Trp 194 and Trp 238 have emission maxima at 326 and 330 nm, respectively. These differences distinguish their environments.

The combination of fluorescence quenched resolved spectra (FQRS) and red-edge excitation was employed for a detailed ex- amination of dipole orientational relaxation processes occumng around the excited fluorophores. The dependence of the emission maxima on excitation wavelengths for Trp 238 and Trp 194 of various forms are presented in Figures 6, 7, and 8. The emission shifts on excitation wavelengths of other aIPI forms are summarized in Table 1 .

The emission maxima shift is observed for both tryptophan residues. The shift for polymers obtained after refolding from hot citrate amounts to 14 nm for Trp 194 and 6 nm for Trp 238, whereas the shift for the latent form amounts to 1 1 nm and 5 nm, respectively (Fig. 7). These data indicate that the environment of Trp 194 relaxes in all the forms, but the dipole relaxation process is most significant for polymers from hot citrate. In contrast, the Trp 238 environment relaxes only in polymers from hot citrate and the latent form (Table 1) .

Fluorescence lifetimes of conformational forms

The fluorescence lifetime data and Stern-Volmer constants are summarized in Table 2. We compared the fluorescence fractions of alp1 conformational forms obtained with time-resolved and steady- state measurements and assumed that the lifetime values of frac-

2.5

2.0

k LL

1.5

1 .o

T

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 KI concentration, [MI

Fig. 4. Qpical Stern-Volmer plot for iodide quenching of latent form alPI with excitation wavelength 295 nm and emission at 330 nm. The solid line represents a least-squares fit of experimental data to the two-component model with parameters:fi = 0.65, KI = 4.75 M-l,f2 = 0.35, K2 = 0 M", x2 = 0.8.

4000

.$ 3000

3 c c - 8 c 0 2000 - s 2 LL

1000

0

2229

I I I I I I I I

300 310 320 330 340 350 360 370 380 390

emission wavelength, (nrn)

Fig. 5. Fluorescence-quenching-resolved spectra of alp1 latent form obtained with excitation at 295 nm, using KI as the quencher. The quenchable component has maximum at 330 nm (A) and unquenchable components has maximum at 326 nm (0). 0, total fluorescence spectrum. Iodide quenchable and unquenchable components correspond to the Trp 238 and Trp 194 residues, respectively.

tions similar to steady-state values characterize the iodine accessible Trp 238 residue. Crystallographic data (Lobermann et al., 1984) showed that Trp 238 is solvent exposed in cleaved a lPI and, on that basis, we consider its kq value to be a quantity for solvent exposure of this residue.

315

320

s - 5 9 !T

ui 5 325

.- 330 v) u) '5

335

340 I

Trp194

I I I I I I 1 I

290 292 294 296 298 300 302 304 306 308 excitation wavelengths, nrn

Fig. 6. Excitation-wavelength dependence of the maxima of the fluorescence-resolved spectra of alp1 polymers induced from hot citrate. Quench- able Trp 238 residue, 0; unquenchable Trp 194 residue component, 0. Total shift to red of the quenchable and unquenchable components is equal to 6 nm and 14 nm, respectively.

2230

320

325 s P 5 ui

a, - 2 330 z c 0 u) u)

.-

.- E,

335

340 290 292 294 296 298 300 302 304

excitation wavelengths, nm

Fig. 7. Dependence of latent alPI fluorescence emission maxima of resolved components on excitation wavelength. Quenchable component, .; unquenchable component, 0.

A comparison of the kq values of the conformational forms reveals that Trp 238 is solvent inaccessible in the polymers refolded from hot citrate. The dipole relaxation of Trp 238 that occurs in these forms confirms this conclusion. Judging from the calculated kq values, Trp 238 is solvent exposed in polymers refolded from hot Tris, latent, native, and cleaved aIPI forms. However, Trp 238 in the latent form shows a dipole relaxation reaction that can only be explained either by the presence of

Trpl94

i Trp238

I I I I I I I \

290 292 294 296 298 300 302 304 306 308 excitation wavelengths, nm

Fig. 8. Fluorescence emission maxima of polymers from hot Tris. Depen- dence on excitation wavelength. Quenchable component, 0; unquenchable component, 0.

H. Koloczek et al.

Table 1. Fluorescence emission maxima of a,PI formsa

Excitation wavelength: 292 nm 303 nm

(YIP1 form Trp 238 Trp I94 Trp 238 Trp 194

Polymers-citrate 326 3 20 332 334 Polymers-Tris 330 324 330 330 Virgin 33 1 328 332 333 Latent 32 1 32 1 336 332 Cleaved 330 322 33 1 321

aAll wavelengths are in nm. On the basis of crystallographic data, it was assumed that the Trp 238 residue is accessible to the quencher, whereas Trp 194 is inaccessible. The fluorescence maxima were calculated from resolved spectra obtained in the KI quenching experiments, an example of which is presented in Figure 5 . Only the data for the shortest and longest applied excitation wavelengths are presented.

mobile dipole groups in its environment or by the movement of its indole ring.

In the case of polymers refolded from hot Tris, the kq value is approximately 50% lower than the kq value of cleaved q P I . This suggests some screening of Trp 238 by adjacent fluorescence lifetime quenching group in the polymers, but the lack of a dipole relaxation reaction for this residue gives evidence that the mobility of surrounding dipoles is out of the fluorescence time scale. Taking into account the kq value of 1.2 X lo9 M" s" , it can be con- cluded that this residue is still exposed in these polymers. A similar analysis of the polymers from hot citrate implies that Trp 238 is largely buried in the protein matrix of these molecules.

The CD spectra of the alPI polymers refolded from hot citrate and hot Tris are presented in Figure 9. These spectra reveal that the polymers differ in their secondary structure elements. The polymers induced by temperature contain a smaller portion of a helix compared with the polymers induced by citrate.

Discussion

The various conformations adopted by serpins in their monomeric state are primarily a result of a competition between the A sheet and the C sheet to incorporate the P15"8' region of the reactive site loop (see Kinemage 1). Because these sheets will incorporate the reactive site loops, they are also able to incorporate synthetic peptides corresponding to the appropriate reactive site loop sequences. Although no crystal data are available, less direct data indicate that a synthetic peptide corresponding to portions of the reactive site loop can integrate into the A sheet in place of the protein's own reactive site loop (Schulze et al., 1990, 1992; Carrel1 et al., 1991; Bjork et al., 1992a, 1992b; Mast et al., 1992). In addition, the propensity to incorporate appropriate sequences allows serpins to polymerize. The ordered polymerization of serpins, first illustrated for a,PI by Schulze et al. (1990) and later confirmed by Mast et al. (1992) and Lomas et al. (1992, 1993b), is predicted to represent intermolecular reactions where the reactive site loop joins molecules by interacting with the A and C sheets. Polymerization or the insertion of synthetic peptides does not occur spontaneously under physiologic conditions for wild-type a,PI. It requires denaturing conditions in which the A and C sheets of the protein are probably in a partially unfolded state. Refolding from this state results in distinct distribution of species: aggregates defined as large protein ar- rays that do not enter nondenaturing PAGE gels, ordered polymers

a,Proreinase inhibitor conformations 223 1

Table 2. Fluorescence quenching parameters and lifetimes of the a1 PI forms

Pol-Tris Pol-cit Latent Virgina Cleaved" Aggregates

71 (ns)b 4.62 5.20 3.96 4.04 4.28 5.29 Fraction 48% 58% 38% 53% 49% 60% 72 (ns) 2.32 1.89 2.23 1.94 2.29 3.80 Fraction 52% 42% 62% 47% 51% 58% K , ( M - ~ ' ) ' 2.69 3.90 4.15 5.50 5.32 3.80 Fraction 53% 62% 65% 48% 51% 58% IO-* X k9 ( M - ' s") 12 7.5 21 28 23 I

"Data from Koloczek et al. (1991). Excitation wavelength was 295 nm and emission was through a cutoff filter. Parameters were obtained for a two-component model characterized by minimum values of the reduced x*.

bTime-resolved fluorescence intensities were analyzed in terms of the following decay law: I ( ? ) = I , ZQ, exp( - t / T , ) , where Q and s are the normalized pre-exponential factor and decay time of component i, respectively. The fractional fluorescence intensities of the components were calculated according to the equation, J i = Q , T ; / ~ ( Y , T ~ , and expressed as a percentage of the total fluorescence intensity.

'Stern-Volmer constant ( K , ) and fractional intensity were calculated from the equation in Materials and methods when KI was used as quencher. Values of k9 were calculated by taking the values of lifetimes T that characterized the same fractions.

defined by a "step ladder" appearance in nondenaturing PAGE, and monomeric species in nondenaturing PAGE.

Aggregated form

We define the aggregated form as material that does not migrate in TUG-PAGE. It appears as a fine particulate material in aqueous suspension and is possibly a mixture of high-order polymers and amorphous collapsed protein, and we did not examine it in detait. However, spectral measurements (not shown) revealed an upward curvature of the Stern-Volmer plot, not observed in case of intact or cleaved aIPI (Koloczek et al., 1991). On the basis of excitation

1 J 200 210 220 230 240 250

wavelength [nm]

Fig. 9. CD spectra of a l P I polymers induced by refolding from hot citrate ( . . .), or from hot Tris (-. -).

spectra measurements and Scatchard calculations, it was found that one acrylamide molecule binds weakly (KO 1.1 M") to the aggregates close to Trp 194. The mechanism of acrylamide binding close to Trp 194 is unknown and further investigations are required to explain it. It does, however, imply that the soluble aggregates have a substantial regular structure and are not just amorphous conglomerates.

Polymer forms

We believe that both of the polymers' states are distinct because their CD spectra (Fig. 9) reveal that these forms contain different secondary structure elements. Judging from Figure 9, it is apparent that polymers refolded from hot Tris have less a-helical structure compared with polymers refolded from hot citrate. We speculate that polymers refolded from hot Tris have their reactive site loop inserted into the A sheet and therefore they lose more a-helix structure compared with polymers refolded from hot citrate. In the latter case, the reactive site loop would keep at least a part of its suggested cy-helix structure.

The red-edge data of a I P I polymers refolded from hot Tris and polymers refolded from cold GuHCl presented in Figure 8 suggest that Trp 238 does not show any dipole relaxation. In contrast, the polymers refolded from hot citrate exhibit a kq value consistent with burial of Trp 238. Although it is not possible to characterize the location of Trp 238 in each unit of a polymer, because we are observing a population effect, it is possible to infer the average residue location. These results reveal that this residue is more surface exposed in the polymers refolded from hot Tris and from cold GuHCI, but relatively buried in the citrate-induced ones.

Two pathways have been suggested to account for polymer formation. The head-to-tail polymerization reaction proposed by Mast et al. (1992) suggests that the reactive site loop of one molecule integrates into the A sheet of an adjacent one to form the initial polymerizing unit. The head-to-head reaction suggested by Lomas et al. (1995a, 1995b) proposes that monomers are joined by interactions in their C sheets. In the former model, Trp 238 would be

2232 H. Koloczek et al.

surface exposed, and in the latter model, Trp 238 would be shielded from solvent, at least in 50% of the molecules (Kinemage I ) . Consequently, we suggest that only the A-sheet mechanism of polymerization can be responsible for the lack of a dipole relaxation processes around Trp 238 residue in the polymers refolded from hot Tris and from cold GuHCI.

On the other hand, in the case of polymers refolded from hot citrate, the polymerization reaction can be expected to include a substantial portion of C-sheet dimers of the type seen in the AT3 crystal structure (Carrell et al., 1994). In this case, although polymers greater than two monomers cannot propagate via a C-sheet mechanism due to steric constraints, a mixture of dimers, trimers, and tetramers could include a substantial portion of C-sheet interactions that influence the population. This is apparently the case because Trp 238 is buried in the small citrate induced polymers, as we would predict from the interaction of monomers via the C sheet.

Latent form

The monomeric form of a IPI that is not cleaved and does not unfold in TUG-PAGE, purified from material refolded from hot citrate, is termed the latent form by analogy with the latent form of PAI-I (Hekman & Loskutoff, 1995). Based on the naturally oc- curring latent serpin PAI-I, it is believed that the transition of the native inhibitor to the latent form involves opening of the A sheet accompanying sliding movements of strands 1, 2, and 3 with re- spect to the strands 5 and 6 of the entire A sheet (Mottonen et al., 1992; Carrell et al., 1994). If this is the case, the environment of Trp 194 must be effected in such a transition and the fluorescence properties of the residue may differ from the native inhibitor significantly. Indeed, the KI quenching experiments with red-edge measurements showed that the emission maximum shift of Trp 194 of the latent form is observed starting at an excitation wavelength of 292 nm and amounts to 11 nm at excitation wavelength of 303, whereas the corresponding shift for native and cleaved forms starts at 300 and 295 nm, respectively (Table I ) .

In addition, in the latent form a dipole relaxation process of the environment of Trp 238 was detected, contrasting to the native and cleaved forms of a lPI (Table I ) . This is an unexpected observation because it is known that the residue is surface exposed in native and cleaved inhibitors (Koloczek et al., 1991) and the dipole relaxation rate occurs prior to the fluorescence emission. This result gives evidence that the insertion of the reactive site loop predicted for latent a l P I also influences strand 2B, part of a @-barrel where Trp 238 is located. On the basis of KI quenching measurements of latent aIPI, we suggest that the reactive site loop insertion results in conformational changes of the P-barrel region effecting the Trp 238 microenvironment dipole relaxation.

The only crystal structure available for a l P I is that of a cleaved protein (Lobermann et al., 1984), so it is necessary to compare the structure of other serpins with this to establish the likely environment of Trp 238 in the latent form of aIPI (Kinemage I ) . A comparative analysis of the equivalent position in the crystal structure of the latent component of the AT3 dimer, Gln 238 (Carrell et al., 1994), suggests that the mobility of Trp 238 is restricted (Fig. 1; Kinemage I ) . Therefore, we believe that the flexibility groups of Glu 199, Lys 201, and Asp 222, which surround the tryptophan residue, would be responsible for the dipole relaxation process observed in latent alPI.

Polymerization pathways

The fluorescence data support a polymerization mechanism whereby the reactive site loop inserts into the A sheet of an adjacent molecule to generate an initial polymerizing unit. This propagates by sequential insertions of reactive site loops into the A sheet of adjacent molecules, and so on, by the head-to-tail mechanism proposed by Mast et al. (1992). The C-sheet head-to-head mechanism proposed by Lomas et al. ( 1 995a, 1995b) as a process for building polymers i s difficult to model due to severe steric constraints. However, we agree that a single C-sheet interaction could occur at the end of a polymer chain.

A polymer chain will grow by head-to-tail interactions (see Fig. IO), and only be terminated by cyclization as seen by Mast et al. (1992) or by incorporation of a terminal unit by a C-sheet interaction as proposed by Lomas et al. ( 1995a. 1995b). A prediction following from this proposed pathway is that small polymers, which would have a larger portion of terminating C-sheet interactions, should have a higher portion of inaccessible Trp 238 side chains. This prediction is borne out by the characteristics of the short polymers refolded from hot citrate. Presumably, denaturation/ renaturation under these conditions favors C-sheet interactions, whereas refolding from cold GuHCl or cooling from hot Tris favors head-to-tail interactions. Because the formation of polymers must be a subset of the general kinetics and thermodynamics of

A-sheet dimer latent

C-sheet dimer

A-sheet polymer

Fig. 10. Polymerization pathways. Open shapes illustrate a molecule with its reactive site loop exposed; closed shapes, one with its reactive site loop integrated into its A sheet. The refolding pathway of denatured alp1 can result in any of the products shown above, depending on the buffer conditions. Refolding from hot citrate results largely in the latent form, where the reactive site loop is integrated into its own A sheet, preventing polymerization, or in C-sheet dimers containing one molecule in a latent conformation locked via its C sheet to the reactive site loop of another molecule-presumably in a conformation close to that seen in AT3 dimers (see Fig. I). Refolding from GuHCl or hot Tris results in longer polymers that contain several units built from reactive site loop interactions with the A sheet of adjacent molecules. These molecules may be capped at one end by a C-sheet interaction, as shown, but the polymerization pathway does not require C-sheet interactions. The A-sheet dimer has not been observed in this study, but is theoretically possible by model building.

a,Proteinase inhibitor conformations 2233

protein folding, it is evident that different folding pathways can be followed by a single protein under different conditions.

Our data indicate that the reactive site loop can act as a donor peptide and that both the A and C sheets act as peptide acceptors. The formation of large polymers is driven by the reactive site loop insertion into an opening A sheet, and of small polymers by interactions between the reactive site loop and the C sheet. Both pathways inactivate the proteinase-inhibitory activity of serpins. Serpin mutations that accelerate these pathways, therefore, can result in pathology due to loss of function-as seen in a lPI (Lomas et al., 1992) and CI-inhibitor (Aulak et al., 1993), for example. How- ever, the polymerization pathway has also been described to occur naturally in wild-type PAI-2 (Mikus et al., 1993; Mikus & Ny, 1996), although its consequences are unclear. The propensity of serpins to polymerize in an ordered manner suggests that the physiologic role of some family members, particularly noninhibitory serpins, may be to serve as peptide donors and/or acceptors to other proteins. This role may, for example, explain the mechanism by which the serpin colligin, which is a resident of the endoplasmic reticulum and has no known proteinase-inhibitory activity, acts as a chaperone for certain collagen types (Jain et al., 1994).

Materials and methods

Materials

Human aIPI was purified by the method of Bruch and Bieth (1986). Ultra pure GuHCI, Tris, acrylamide, bis-acrylamide, and all other chemicals of at least analytical grade were purchased from Sigma (St. Louis, MO).

Preparation and electrophoretic analysis of various forms of a,PI

The preparation of a I P I used in this study was more than 95% homogenous and 90% active as assessed by SDS-PAGE and titra- tion with human neutrophil elastase, respectively. Three independent batches of the latent, polymerized, and aggregated forms of a l P I were induced by treatment of the purified inhibitor in mild denaturing conditions, followed by renaturation and purification. Soluble a I P I aggregates were obtained by incubating the inhibitor in 1 M GuHCl at 4 "C for 12 h in basic buffer containing 0.1% P-mercaptoethanol, followed by extensive dialysis and purification by size-exclusion chromatography using a TSK G3000 SWG col- umn (LKB-Produkter AB) equilibrated with 50 mM Tris, pH 7.5, at a flow rate of 2 mL/min. Latent and polymer forms of the inhibitor were induced by heating in citrate buffer and purified as described by Lomas et al. (1995a). Briefly, the inhibitor (0.5 mg/mL) was incubated in 0.7 M sodium citrate, pH 6.5, at 67 "C for 12 h followed by extensive dialysis against 20 mM Tris pH 8.6. Protein was concentrated to 1 mg/mL, reheated at 60 "C for 3 h, and chro- matographed on Mono Q. In addition, polymers of the inhibitor were also induced by heating the protein at 67 "C for 16 h in 50 mM Tris, 50 mM KCI, pH 7.5. In each batch, the position of the protein peaks, resolved by size-exclusion or ion-exchange chromatography and apparently corresponding to native, latent, polymers, and aggregates of alPI, was highly reproducible although the relative amounts of the different forms varied between preparations.

Each fraction of the purified form of a lPI obtained as described above was analyzed by native- and SDS-PAGE, as well as by TUG-PAGE (Goldenberg, 1989) as a modified for serpin analysis

by Mast et al. (1992). In addition, rocket immunoelectrophoresis was used to check protein recovery of the latent and polymerized forms of a lPI following thermal denaturation.

Fluorescence measurements

All of the measurements were performed in 50 mM Tris, 50 mM KCl, pH 7.5, at 23 "C, and repeated at least three times on samples from each batch of a lPI form. In this way, the data presented are an average from at least three independent experiments. Steady- state fluorescence measurements were made on a Hitachi F-4500 spectrofluorometer. Acrylamide and iodide solute quenching studies were performed by adding aliquots from a stock solution of the quencher into a cuvette containing protein. Fluorescence intensities were corrected for any dilution effects and for absorbance by screening acrylamide at excitation wavelengths, and the protein concentration was kept below an absorbance of 0.1 at the excitation wavelengths. The data were analyzed according to the method of FQRS applied to two tryptophan containing proteins, which is described in detail by Wasylewski et al. (1988). Briefly, fluorescence quenching data were analyzed according to the following general form of Stern-Volmer equation:

where FO and Fare the fluorescence intensities in the absence and presence of quencher,fi is the fractional intensity corresponding to component i, Ksy is the dynamic quenching constant (equal to kqT,), where kq is the quenching rate constant and T, is the mean fluorescence lifetime in the absence of quencher for component i, Vi is the static quenching constant for component i, and Q is the total quencher concentration. Quenching data were fitted to the above Stern-Volmer equations by an iterative nonlinear least- squares program (Stryjewski & Wasylewski, 1986).

The dynamic properties of a l P I forms were also investigated by the red-edge excitation using the method described by Demchenko (1988) and applied for two tryptophan containing proteins (Wa- sylewski et al., 1988). Briefly, if fluorescence is excited by the light with energy lower than the average electronic transition energy of entire population of fluorophores, then a selective excitation of fluorophores with the lowest electronic transition energy will occur. Thus, excitation at the longer wavelength edge of ab- sorption band gives a fluorescence emission spectrum with a maximum located at longer wavelength than one obtained when excitation is performed at short wavelength. This phenomenon is called the red-edge effect. It will only occur if the dynamics of the surrounding dipole groups, which can be expressed in terms of relaxation time (TJ, are slower than the excited state lifetime, (T$. More rapid dynamics leads to fluorescence emission following the dipoles' relaxation and the fluorescent spectra are not dependent on the excitation wavelength. It is possible to observe the red-edge effect for proteins with maximum of fluorescence spectra ranging from 325 to 340 nm (Demchenko, 1988), and study the polarity and dynamics of the tryptophan microenvironment (Koloczek et al., 1991).

Fluorescence lifetime measurements of a l P I forms were con- ducted by using the multifrequency cross-correlation phase and modulation fluorometer ISS K2. The excitation wavelength was set at 295 nm, with band width equal to 16 nm using a monochro- mator and an emission was observed through a cut-off filter at 320 nm. The light of a 300 W xenon lamp was modulated over the

2234 H. Koloczek et al.

10-200-MHz frequency by a Pockels cell modulator. As a refer- ence, a glycogen aqueous solution was used. For each frequency used, about 320460 records were collected and analyzed using standard deviation of 0.2% for -rp and 0.004% for 7,. The error of the calculated values of fluorescence decay parameters was assumed to be 5%. The samples were measured at room temperature. Data analyses were performed using ISS software by applying the nonlinear least-squares procedure for two or three discrete lifetimes. In each case, the best fit parameters were obtained by min- imalizing the reduced chi square value for a two-lifetime model. The values of lifetimes fluorescence fraction, which are the same or close to the fluorescence fraction obtained with steady-state measurements, were taken in order to calculate the quenching rate constant kq.

Circular dichroism analysis

CD measurements of alPI forms were recorded on a Jasco-710 spectropolarimeter using a 0.1-cm optical path length cell. Each CD spectrum was run at least five times and the molar residue ellipticities were calculated with the equipped Jasco software.

Acknowledgments

We thank Scott Snipas for technical assistance. Supported by grant 6 P203 009 05 from Committee of Scientific Research (KBN, Poland) to H.K., and NIH grant HL51399 from the National Institutes of Health and a grant from Bayer Corporation, both to G.S.S.

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