The High Affinity Binding Site on Plasminogen Activator Inhibitor-1

The High Affinity Binding Site on Plasminogen ActivatorInhibitor-1 (PAI-1) for the Low Density Lipoprotein Receptor-related Protein (LRP1) Is Composed of Four Basic Residues*

Received for publication, August 28, 2015, and in revised form, October 29, 2015 Published, JBC Papers in Press, November 10, 2015, DOI 10.1074/jbc.M115.688820

Peter G. W. Gettins1 and Klavs DolmerFrom the Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607

Plasminogen activator inhibitor 1 (PAI-1) is a serpin inhibitorof the plasminogen activators urokinase-type plasminogen acti-vator (uPA) and tissue plasminogen activator, which bindstightly to the clearance and signaling receptor low density lipo-protein receptor-related protein 1 (LRP1) in both proteinase-complexed and uncomplexed forms. Binding sites for PAI-1within LRP1 have been localized to CR clusters II and IV. Withincluster II, there is a strong preference for the triple CR domainfragment CR456. Previous mutagenesis studies to identify thebinding site on PAI-1 for LRP1 have given conflicting results orimplied small binding contributions incompatible with the highaffinity PAI-1/LRP1 interaction. Using a highly sensitive solu-tion fluorescence assay, we have examined binding of CR456 toarginine and lysine variants of PAI-1 and definitively identifiedthe binding site as composed of four basic residues, Lys-69, Arg-76, Lys-80, and Lys-88. These are highly conserved among mam-malian PAI-1s. Individual mutations result in a 13– 800-foldincrease in Kd values. We present evidence that binding involvesengagement of CR4 by Lys-88, CR5 by Arg-76 and Lys-80, andCR6 by Lys-69, with the strongest interactions to CR5 and CR6.Collectively, the individual binding contributions accountquantitatively for the overall PAI-1/LRP1 affinity. We proposethat the greater efficiency of PAI-1�uPA complex binding andclearance by LRP1, compared with PAI-1 alone, is due solely tosimultaneous binding of the uPA moiety in the complex to itsreceptor, thereby making binding of the PAI-1 moiety to LRP1 atwo-dimensional surface-localized association.

Urokinase-type plasminogen activator (uPA)2 is one of twoserine proteinases involved in cell surface generation of plasminfrom plasminogen. Although plasmin’s primary role is to

degrade fibrin-containing thrombi, it is also involved in a widerange of other normal and pathological processes that resultfrom its ability to cleave matrix proteins such as laminin andfibronectin. In this way regulation of uPA activity plays animportant role in control of processes such as wound healingand angiogenesis, as well as the growth and dissemination oftumors. For example, inhibition of uPA activity has been shownto block tumor invasion in the nude mouse (1). uPA is localizedto the cell surface by tight binding to its uPA receptor (uPAR)(2) through the N-terminal region of uPA (3, 4).

The principal inhibitor of uPA activity is plasminogen acti-vator inhibitor 1 (PAI-1), a serpin. Inhibition by serpinsinvolves a unique mechanism of kinetic trapping of the acyl-enzyme intermediate formed during cleavage of the serpin’sreactive center loop by the proteinase (5). The mechanisminvolves a massive conformational change in the serpin, withinsertion of the reactive center loop into sheet A of the serpinand translocation and consequent distortion of the proteinaseactive site as the means of kinetic trapping (6 –9). Because uPAis predominantly bound to its receptor, the result of inhibitionby PAI-1 is the formation of a ternary membrane-associateduPAR�uPA�PAI-1 complex. This complex is rapidly internal-ized, as a result of binding to the low density lipoprotein recep-tor-related protein (LRP1), and the uPA�PAI-1 componentdegraded (10, 11). In contrast, uPA alone bound to uPAR ispoorly internalized (12). Cross-competition for binding toLRP1 between uPA�PAI-1 complexes and PAI-1 complexeswith other proteinases suggests that much or all of the bindinginteraction between the uPAR�uPA�PAI-1 complex and LRP1occurs through PAI-1 (13). Additional support for this comesfrom soluble uPAR blocking the binding of uPA to LRP1,although it has minimal effect on the strength of the uPA�PAI-1interaction with LRP1 (12).

Knowledge of the binding site on PAI-1 for LRP1 is thus veryimportant for an understanding of the structural basis for inter-nalization and degradation of uPA in its PAI-1-complexed formand hence for regulation of surface-bound uPA activity. How-ever, no structure of PAI-1 in complex with a fragment of LRP1has yet been determined. Indeed, the only structures of com-plex between a protein ligand and a receptor fragment from anLDL receptor family member are of human rhinovirus HRV2bound to a portion of the VLDL receptor (14), of the thirddomain of RAP bound to a fragment from LDL receptor (15,16), and of reelin R5 and R6 domains bound to a fragment ofapoER2 (17). These structures suggest a common theme ofengagement of protein ligands by LDL receptor family mem-

* This work was supported by National Institutes of Health Grant R01GM54414. The authors declare no conflict of interest with the contents ofthis article. The content is solely the responsibility of the authors and doesnot necessarily represent the official views of the National Institutes ofHealth.

1 To whom correspondence should be addressed: Dept. of Biochemistry andMolecular Genetics, University of Illinois at Chicago, 900 S. Ashland, M/C669, Chicago, IL 60607. Tel.: 312-996-5534; E-mail: [email protected].

2 The abbreviations used are: uPA, urokinase-type plasminogen activator;CRx, complement-type repeat x, with numbering of x relating to the posi-tion of the domain within LRP1 from the N terminus; CR5F6W, CR5W6, andCR5F6F, CR56 species in which the tryptophan(s) in CR5, CR6, or both,respectively, have been changed to phenylalanine; �-ME, �-mercaptoeth-anol; PAI-1, plasminogen activator inhibitor-1; RAP, receptor-associatedprotein; TEV, tobacco etch virus; uPAR, uPA receptor; dansyl, 5-dimethyl-aminonaphthalene-1-sulfonyl; IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid.

crossmarkTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 2, pp. 800 –812, January 8, 2016

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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bers that involves binding of pairs of basic residues (lysine ineach case) to individual CR domains of the respective receptors.These CR domains are small independent modules that occurin clusters within the receptors. This mode of binding is quali-tatively consistent with mutagenesis studies carried out onPAI-1, in which mutation of certain basic residues reducedbinding affinity for LRP1, either alone or in complex with pro-teinase (13, 18 –20). However, there are two major problemswith these mutagenesis studies. The most disconcerting is thatbasic residues identified as being involved in binding result inno more that an 11-fold increase in Kd when mutated andmostly much less, whereas the affinity of PAI-1 for LRP1 is inthe low nanomolar region. Such very small effects are notexpected if basic residues are the dominant contributors tooverall affinity, which has been quantitatively shown to be thecase for the RAP D3/CR56 interaction (21) and for model com-pounds binding to individual CR domains (22). The secondproblem is that there is no consensus on which basic residuesconstitute the binding site, with some residues identified asbeing important in one study (13, 19, 20) and having little or noeffect when mutated in another study (18).

To more definitively establish the binding site on PAI-1 forLRP1, we have developed a sensitive fluorescence assay andexamined the solution binding of wild type and variant PAI-1s,all in their native form, to the fragment CR456 from LRP1,which we and others have shown to be a high affinity bindingsite for PAI-1 and its proteinase complexes within cluster II ofLRP1 (23–25). Comparisons are also made for binding of CR56,CR345, and CR567.

Experimental Procedures

Mutagenesis of PAI-1—All PAI-1 species used were on thestable 14-1B (the very stable variant of PAI-1 containing fourstabilizing mutations (N150H, K154T, Q319L, and M354I)(26)) background that has been shown not to alter the func-tional properties of PAI-1, but it does greatly slow the conver-sion to the latent state (26). Subsequent use here of the termWT PAI-1 implies the 14-1B background. An estimate of the t1⁄2for conversion to the latent state at 4 °C, based on t1⁄2 at 25 °C of22 h (27) and an activation energy for the latent transition of 96kJ mol�1 (28), is well over 200 days for WT PAI-1 and would bevery much longer for the 14-1B variant. For the 14-1B variantused here, the protein thus remains completely in the nativestate, even when kept at 4 °C for several days. The cDNA encod-ing the 14-1B variant was cloned into pQE30 (29) giving a finalproduct containing an N-terminal His6 tag. �W-PAI-1 (PAI-1with all four tryptophans mutated to phenylalanine) was gen-erated by sequentially mutating the four tryptophans (residues86, 139, 175, and 262) to phenylalanine. For FRET measure-ments, single cysteine variants were generated on the�W-PAI-1 background (H2C, G70C, G84C, S127C, G194C,G230C, G264C, and Q312C). For monitoring binding of LRP1fragments from fluorescence perturbation, the H2C mutationwas introduced into PAI-1 14-1B and used as the background togenerate the following variants: K65A, K69A, K65A/K69A,R76A, K80A, R76A/K80A, K88A, K176A, K88A/K176A, andR115A/R118A. All mutagenesis was performed using the

QuikChange protocol (Agilent Technologies), and all variantswere verified by DNA sequencing before protein expression.

Expression and Purification of PAI-1 Variants—PAI-1 vari-ants were expressed in SG13009 cells (Qiagen) in 2YT medium.Cultures were grown at 37 °C to an A600 � 0.6 –1.0 and inducedwith 1 mM isopropyl �-D-thiogalactoside, and the temperaturewas then lowered to 27 °C. Cells were harvested by centrifuga-tion after 4 –5 h and stored at �80 °C until needed. PAI-1 vari-ants were purified as described (30). Cells were sonicated in 50mM sodium phosphate, 300 mM NaCl, and 10 mM imidazole, pH7.4, containing 5 mM �-ME. After centrifugation, NaCl andglycerol were added to 2 M and 5% v/v, respectively. The clearedlysate was loaded on a Ni2�-chelate column. PAI-1 was elutedwith 250 mM imidazole and dialyzed overnight at 4 °C against20 mM Tris-HCl, 1 M NaCl, 0.5 mM EDTA, 5% glycerol, pH 8.0,containing 14 mM �-ME. DTT was added to 5 mM before finalpurification on a Superdex 75 column (860 � 16 mm), withelution in dialysis buffer.

The effect of the lysine or arginine to alanine mutations onPAI-1 function was determined by assessing the ability of thePAI-1 variants to inhibit uPA. Both stoichiometries of inhibi-tion and apparent second order rate constants were determinedby kinetic assay. For all of the variants, the stoichiometries ofinhibition and corrected second order rate constants were verysimilar to those of WT PAI-1, consistent with the alanine sub-stitutions not affecting the structure of the molecule.

IAEDANS Labeling of PAI-1—The peak containing mono-meric PAI-1 was made 10 mM in DTT, concentrated to 1.5–2.5ml using Amicon ultracentrifugal filters, and buffer-exchangedinto degassed 20 mM Tris-HCl, 1 M NaCl, 0.5 mM EDTA, 5%glycerol, pH 8.0, using a Sephadex G25F column (160 � 16mm). The protein concentration was determined using UVabsorption at 280 nm and calculated extinction coefficients of35,410 M�1 cm�1 for WT PAI-1, and 13,410 M�1 cm�1 for�W-PAI-1. Two molar eq of DTT were added to the PAI-1sample, followed by the addition of 12 eq of IAEDANS (Molec-ular Probes). The reaction was allowed to proceed on ice over-night. The next day, the reaction was quenched by adding 14mM �-ME, and the sample was concentrated to 1.5–2.5 mlbefore excess IAEDANS was removed by G25F gel filtration.After concentrating the samples, the concentrations of PAI-1and dansyl were determined spectrophotometrically, using anextinction coefficient of 4700 M�1 cm�1 at 340 nm for dansyland a correction factor of 0.38 for the absorption at 280 nm(A280, corr � A280 � 0.38 � A340). Samples typically contained50 –100 �M PAI-1 and 1.3–1.5 dansyl per PAI-1. A controlPAI-1 sample, without cysteine, was reacted similarly and gave0.3 dansyl per PAI-1. This nonspecific dansyl label was insensi-tive to binding of LRP1 fragments.

Mutagenesis, Expression, Purification, and Refolding of CRConstructs—Expression, purification, and refolding of the var-ious CR fragments was according to procedures we have suc-cessfully used for many years (25, 31–33). All CR constructswere expressed in 2YT medium. CR56 and CR567 were clonedin pGEX-2T, modified to contain a TEV proteinase cleavagesite, and expressed as GST fusion proteins in BL21 cells. Cellswere grown to A600 � 0.6 –1.0 before induction with 1 mM

isopropyl �-D-thiogalactoside and harvested after 5– 6 h at

High Affinity Binding Site on PAI-1 for LRP1

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37 °C. The GST fusion proteins were purified from cleared celllysate by GSH-Sepharose chromatography, and the GST tagwas removed by TEV proteinase cleavage during overnightdialysis against 4 liters of 20 mM Tris-HCl, pH 8.0, 50 mM NaCl,4 mM EDTA, containing 14 mM �-ME. GST and uncleavedGST-CR fusion proteins were removed by passage through theGSH column.

CR345 and CR456 were cloned into pQE-30, modified tocontain a GB1 fusion partner (for increased solubility), and aTEV proteinase cleavage site. These His6-tagged fusion pro-teins were expressed in SG13009 cells containing the plasmidpRARE. The CR fusion proteins were purified from cell lysateby Ni2�-chelate chromatography, and the fusion partners wereremoved by TEV proteinase cleavage during overnight dialysisagainst PBS containing 14 mM �-ME.

Before refolding, all CRs were further purified by Q-Sephar-ose HP chromatography, using a gradient of 0 –1000 mM NaClin 20 mM Tris-HCl, pH 8.0, and 6 M urea. The denatured CRswere diluted with 50 mM Tris-HCl, 50 mM NaCl, 10 mM CaCl2,pH 8.5, containing 14 mM �-ME and 8 mM 2-hydroxyethyldisulfide (refolding buffer). For increased folding efficiency, allCR constructs were mixed with the chaperone RAP as a GSTfusion protein (GST-RAP) at a 1:1 ratio. Final CR concentrationin the refolding mixture was 0.1 mg/ml. The CR species wererefolded by dialysis against refolding buffer for 24 h at roomtemperature with N2 bubbling, followed by 24 h at 4 °C withoutN2. Finally, the mixture was dialyzed twice against 4 liters of 20mM Tris-HCl, pH 7.8, 50 mM NaCl, 1 mM CaCl2 at 4 °C. Therefolding mixture was loaded on GSH-Sepharose, and foldedCR constructs, i.e. those capable of binding to RAP and there-fore retained on the column, were eluted with 40 mM Tris-HCl,pH 8.0, 100 mM NaCl, 8 mM EDTA. Folded CR constructs werefurther purified by Q-Sepharose HP chromatography by saltgradient elution with 50 –1000 mM NaCl in 20 mM Tris-HCl,pH 8.0, and 0.1 mM CaCl2. If traces of GST-RAP were still pres-ent, calcium was removed from the mixture by adding EDTA,and the mixture was passed through a Superdex 75 size exclu-sion column equilibrated in 20 mM Tris-HCl, pH 7.8, 150 mM

NaCl, and 4 mM EDTA, after which calcium was reintroducedto the purified CR. As a quality control between each step ofpurification, samples were analyzed by SDS-PAGE (with andwithout reducing agent) and native-PAGE. The presence of asingle band on SDS-PAGE, which shifts upon reduction, indi-cates homogeneous samples with formed disulfide bridges. Asingle band on native-PAGE suggests the presence of a singlefolding product. The final product was judged by SDS-PAGE tobe more than 95% pure. The effective concentration of func-tional CR fragments was determined by stoichiometric titrationagainst RAP fragment D3. Binding was monitored from theshift in tryptophan fluorescence maximum of the CR domainsupon binding and the high affinity of D3 for all of the CR frag-ments used here (31).

Expression and Purification of RAP—RAP cDNA, cloned inpGEX-2T, was a kind gift from Dr. Dudley Strickland. GST-RAP was expressed in BL21 cells, induced with 1 mM isopropyl�-D-thiogalactoside at A600 � 0.6 –1.0, and harvested after 5 hat 25 °C. GST-RAP was purified by GSH-Sepharose chroma-tography. GST-RAP was then dialyzed overnight against 4 liters

of 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 4 mM EDTA, contain-ing 14 mM �-ME. Free cysteines in the GST moiety wereblocked by treatment with 20 mM iodoacetamide for 1 h atroom temperature, and excess reagent was removed by dialyz-ing twice against 4 liters of 20 mM Tris-HCl, pH 8.0, 50 mM

NaCl, 4 mM EDTA, followed by dialysis against 4 liters of 50 mM

Tris-HCl buffer, pH 8.5, containing 50 mM NaCl and 10 mM

CaCl2.Fluorescence Spectroscopy—All fluorescence measurements

were made on a PTI QuantaMaster spectrofluorimeter,equipped with double monochromators on both excitation andemission sides. For FRET measurements, excitation was at 280nm and emission was monitored from 310 to 650 nm. For directcontact perturbation spectra excitation was at 342 nm andspectra were recorded from 400 to 650 nm. In each case slits of1 nm for excitation and 4 nm for emission were used, with 2-nmsteps, 2 s dwell time, and spectra recorded as the average of fourscans. For single wavelength measurements, used to followbinding of CR fragments to H2C-dansyl-labeled PAI-1 variants,excitation was at 342 nm, and emission was monitored at themaximum for dansyl emission (470 nm). Slits of 1 nm for exci-tation and 8 –10 nm for emission were used, with signal moni-tored for 120 s and averaged.

Samples were 1.5 ml in 1-cm cuvettes and typically contained0.06 –1 �M PAI-1, depending on the anticipated Kd value. ThepH 7.4 buffer contained 20 mM Tris-HCl, 1 mM CaCl2, and 0.1%PEG 8000. NaCl was added from a stock 5 M solution to achievethe desired final ionic strength. The effect of added NaCl on pHwas checked for each sample and found to result in no morethan 0.05 pH unit change. Samples were mixed with a magneticstir bar in the cuvette. Data were corrected for contributionsfrom a buffer blank and for dilution. In all cases dilution was lessthan 2%. Temperature was maintained by a circulating waterbath. All titrations were carried out at 298 K. Kd values weredetermined by non-linear least squares fitting of the bindingdata to a single site binding isotherm using the quadratic equa-tion for equilibrium binding (34). Fitting was done in Kaleida-Graph (Synergy Software).

FRET Measurements—To determine percentage quench oftryptophan fluorescence resulting from FRET to dansyl accep-tor, dansyl-labeled �W-PAI-1 was titrated into a sample ofCR56 until a saturable end point was reached. The intensity ofthe CR56 tryptophan fluorescence in complex was then com-pared with that of unbound CR56 using the intensity at thewavelength maximum. For measurement of the enhancementof dansyl fluorescence from FRET, CR56 was titrated into asample of dansyl-labeled �W-PAI-1 to a saturable end point.Enhancements were determined as the relative increase inintensity at the wavelength maximum, compared with un-bound dansyl-labeled �W-PAI-1, with excitation at 280 nm. Toextract the effect due to FRET, correction was made for directbinding effects when excited at the same wavelength.

Although no quantitative use was made of the magnitude oftryptophan quench in this study, for reasons given under“Results,” there is a direct relationship between the quench andthe efficiency of FRET given by Equation 1,

E � 1 � QDA/QD (Eq. 1)




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where QDA is the donor (tryptophan) quantum yield in thepresence of acceptor, and QD is the donor quantum yield in theabsence of acceptor. E is related to the separation betweendonor and acceptor by Equation 2,

E � R06/�R0

6 � R6� (Eq. 2)

where R0 is the separation for that donor-acceptor pair thatresults in 50% efficiency of FRET, and R is the separation of thepair in the system under study. R0 is a function of the spectralproperties of the donor and acceptor, given by Equation 3,

R0 � 9.7 � 103� J�2n�4D�1/6 (Eq. 3)

where J is the overlap integral, �2 is the orientation factor, n isthe refractive index, and D is the donor quantum yield. Thus,if R0 can be confidently calculated for the system under study, Ecan be used to determine the donor-acceptor separation R.

Results

Fluorescent Reporters of CR56 Binding—Our initial plan fordelineating the binding site for LRP1 on PAI-1 was to use tryp-tophan-to-dansyl FRET between the single tryptophans withineach CR domain and a dansyl fluorophore attached covalentlyto single cysteines introduced within PAI-1. To this end, wecreated a number of single cysteine-containing variants of atryptophan-less PAI-1 (�W-PAI-1) to be able to monitor bothenhancement of acceptor (dansyl) fluorescence and quench ofdonor (CR domain tryptophan) fluorescence. The sites forintroduction of the single cysteines are shown in Fig. 1. CR56was chosen as being a high affinity two-domain fragment fromcluster II (25, 35), with Kd values only 20-fold higher than forthe tightest binding CR456 fragment (25) and with only twotryptophans, one in each domain, to simplify analysis.

We first examined whether any of the dansyl fluorophoresshowed perturbation upon CR56 binding that was due directlyto binding rather than from FRET. Dansyl was excited directlyat 342 nm (well removed from the tryptophan absorption) infree PAI-1 and upon complex formation with CR56. Whereasdansyl at most positions showed small changes in either wave-length maximum or intensity, dansyl attached to His-2–Cysshowed a blue shift of 14 nm and 65% increase in intensity(Table 1 and Fig. 2). Smaller but significant blue shifts were seen

with Gly-84 –Cys and Gly-70 –Cys, although other positionsshowed minimal spectral shifts (Table 1).

The dramatic effect of CR56 binding on dansyl at position 2could be due either to close proximity of the fluorophore to thebound CR56 or CR56-induced conformational change within

FIGURE 1. Structure of native PAI-1 (Protein Data Bank code 3Q02). A and B represent orthogonal views of the structure to show the location of all of thebasic residues mutated in this study, as well as the location of residues mutated to cysteine for incorporation of dansyl reporters. Mutated basic residues areshown in space-filling (cyan and blue) and labeled. Sites of incorporation of cysteine are shown as red spheres on the Cs. C is the same view as A, but it showsonly those residues identified as being involved in binding of CR456, together with the proposed CR domain, in parentheses, that engages each site.

FIGURE 2. Fluorescence emission spectra of Cys-2-dansyl PAI-1. Normal-ized emission spectra are shown with direct excitation of dansyl at 342 nm. 1�M native Cys-2-dansyl PAI-1 alone (solid line), with 1.5 eq of CR56 added (blueshort dashes), or with 1.5 eq of CR456 added (red long dashes).

TABLE 1Effect of binding CR56 on dansyl emission spectra

PAI-1 variant �max ��max �Fmax

nm nm %H2C 481 �14 �65G70C 473 �3 �8G84C 476 �5 �17S127C 475 �1 �18G194C 484 �2 �8G230C 475 0 0G264C 481 �2 �4Q312C 484 0 �4




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the PAI-1. However, we have previously found that binding ofCR fragments to PAI-1 results in only a small perturbation oftryptophan fluorescence (25). From subsequent results pre-sented here (see Fig. 4A), we know that this arises only fromtryptophans in the CR domains. This suggests that binding ofCR56 does not cause significant conformational change withinthe PAI-1 and thus that the large perturbation of the dansylfluorophore upon CR56 binding is due to proximity to the fluo-rophore. To examine whether the fluorophore had itself alteredthe binding of CR56 to PAI-1, we used the direct perturbationof dansyl fluorophore to follow binding by titrating CR56 intolabeled PAI-1. The data were well fitted to a single binding site(Fig. 3A) and gave a Kd of 52 � 2 nM (Table 2) that was indis-tinguishable from the value we found previously for binding ofCR56 to unlabeled cleaved PAI-1, monitored by the less sensi-tive perturbation of tryptophan fluorescence (25). This findingsuggested that the Cys-2– dansyl is an extremely sensitive yetnon-perturbing reporter of CR fragment binding that might beinvaluable for examining binding of both other CR fragmentsand of CR56 or CR456 to variants of PAI-1 that also containedthe Cys-2 mutation.

Use of FRET to Restrict the LRP1-binding Site on PAI-1—Pursuing our initial goal of using FRET to delineate the CR56-binding site on PAI-1, we determined both enhancement ofdansyl fluorescence, from titration of CR56 into dansyl-labeled�W-PAI-1 species and of quench of tryptophan fluorescencefrom titration of dansyl-labeled �W-PAI-1 into CR56. For dan-syl enhancements, correction was made for any direct pertur-bation effects caused by binding itself (see above). In each casea saturable effect was seen, consistent with the dansyl enhance-ment or quench of CR56 tryptophan arising from FRETbetween CR domain tryptophan excited at 280 nm and PAI-1-linked dansyl emitting around 470 nm. By far, the largest effectwas seen for label at Cys-2, with close to 80% quench of CR56tryptophan fluorescence and 440% enhancement (227%enhancement after correction for proximity effects) of dansylfluorescence (Fig. 4). Lesser, but still large, quenches of trypto-phan fluorescence were also seen with label at positions 84

(55%) and 230 (37%). Other positions showed smaller quenchesof tryptophan fluorescence. Significant enhancements of dan-syl fluorescence were seen at all positions except 127 (Table 3).

Although we had hoped to use these FRET measurements todefine the location of the CR56-binding site, we were not able todo so, because of the uncertainties in the analysis, which greatlyreduced the precision possible. The biggest problem was thelarge value for R0 (the Forster distance for 50% efficiency ofFRET) for the tryptophan-dansyl pair of 23 Å (7, 36). Thismade it difficult to find a position on PAI-1 that would give nomeasurable FRET and conversely resulted in many positionsthat were close enough to give quite significant FRET (Table 3).Unfortunately, no other donor-acceptor pair could be foundthat was better (37). A second problem was the length of thelinker between the cysteine sulfur and the dansyl fluorophore(intervening CH2CO-NH-CH2-NH). This gave great uncer-tainty in the location of the fluorophore relative to the cysteinesulfur and consequently might result in a fluorophore at a prox-imal label site pointing away from the CR56-binding site andanother fluorophore at a more distant site pointing toward theCR56 giving similar FRET. Although we examined other label-ing species with shorter linkers, including BADAN, NBD-chlo-ride, and dansyl-chloride, the fluorescence of the derivativessuffered from very low quantum yield (data not shown). Finally,uncertainty in the value of the orientation factor, �2, for eachlabel position introduced another uncertainty in determiningR0 for each donor-acceptor pair. As a result, we were unable touse FRET efficiencies to accurately map the location of CRdomains relative to the mutated cysteine residues. The datawere, however, of more qualitative use in suggesting whetherthe CR domains were near or far from the site of label attach-ment. In this way, we confirmed the observations above fromdirect perturbation of dansyl fluorophore that the CR56-bind-ing site must be close to His-2 and also be in the vicinity ofGly-70, Gly-84, Gly-230, and Gly-264 to account for the largetryptophan quenches due to FRET. This is consistent withother mutagenesis studies that have implicated basic residuesin this general region in the binding of PAI-1 to LRP1 (13,

FIGURE 3. Binding of CR56 and CR456 to Cys-2-dansyl PAI-1. Titrations were carried out at I 0.17 for CR56 (A), using 80 nM PAI-1, and CR456 (B), using 50 nM

PAI-1. The curves shown are the least squares best fits obtained to a single site binding isotherm. Note that the Kd value obtained from this fit for CR456 (1.3 �0.4 nM), even though not considered highly accurate, because the protein concentration is significantly greater than Kd, nevertheless agrees well with the valuein Table 3 obtained by extrapolation from measurements at higher ionic strength.




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18 –20). Although the Gln-312–Cys variant did give a largedansyl enhancement, this was from a very low intensity emis-sion. It is therefore thought that the small tryptophan quench ofCR56 fluorescence is a more reliable indicator of the distance ofthis residue from the binding site.

Affinities of PAI-1 Basic Mutants for CR56 —Guided by thefindings above that the binding site for CR56 must be close toresidues 2, 70, 84, and 230 (see Fig. 1), and the expectation that

individual CR domains engage proximal pairs of basic residues,we identified four possible pairs of basic residues as potentialCR domain binding sites. These were Lys-65 and Lys-69,Arg-76 and Lys-80, Arg-115 and Arg-118, and Lys-88 and Lys-176 (Fig. 1). With the exception of Lys-65 and Lys-176, all ofthese residues have been implicated in LRP1 binding by one orother of the four previous mutagenesis studies (13, 18 –20).PAI-1 variants containing each of these pairs mutated to ala-nine were created on a background of His-2–Cys and 14-1B, topermit introduction of the sensitive non-perturbing dansylreporter at this location.

Binding titrations were carried out for each double basicmutant using direct perturbation of the dansyl fluorophore, asabove. Only three of the pairs of mutations affected the affinityfor CR56 (Table 2). No effect on affinity was seen for the Lys-88/Lys-176 pair. The affinity for the Lys-65/Lys-69 variant wasgreatly reduced, with about a 50-fold increase in Kd to 2.5 �M,whereas the affinity of the Arg-76/Lys-80 variant was so weak-ened that no reliable Kd value could be obtained, because only asmall fraction of the PAI-1 could be bound at the highest con-

TABLE 2Kd values for binding of CR species to WT and variant PAI-1sValues are in given in nanomolar.

CR456 CR56 CR567I 0.02 I 0.17 I 0.52 I 0.17 I 0.17

PAI-1 speciesWT 0.1a

WT 1.5a 52 � 2 28 � 1.8WT 39 � 1K65A/K69A 71 � 9 2500 � 500 84 � 14K65A 18 � 1.5 58 � 4K69A 103 � 9 670a 3900 � 100R76A/K80A 4000 70,000b 8900 � 400b

R76A 130 � 12 845a 2500 � 100K80A 185 � 22 1200a 30,000b

K88A/K176A 16.5 � 1.5 53 � 1.6 24 � 1.7K88A 20 � 0.5K176A 4 � 0.6R115A/K118A 4.5 � 1 15 � 6

a Kd values were obtained by extrapolation from Debye-Hückel plot.b Data were estimated by assuming maximum fluorescence perturbation was the same as for complete titration with tighter binding variants.

FIGURE 4. FRET between CR domain tryptophan and PAI-1-attached dansyl. Titrations show the quench of CR domain tryptophan fluorescence uponbinding Cys-2-dansyl �W-PAI-1 in A and enhancement of dansyl acceptor upon binding CR56 in B. Titrations are shown for addition of 1–3 eq of Cys-2-dansyl�W-PAI to 0.4 �M CR56 (A) and addition of 0.5, 1, 1.5, and 3 eq of CR56 to 0.4 �M Cys-2-dansyl �W-PAI (B). Excitation of CR domain tryptophan was at 280 nm.The spike at 560 nm in B is a secondary scatter peak. Spectra have been corrected for buffer but not for contact enhancements of dansyl emission in B. FRETchanges given in Table 2 make correction for such contact enhancement. Note that in A the tryptophan fluorescence of the CR56 moiety undergoes a blue shift.This shift completely accounts for the changes in tryptophan fluorescence seen when PAI-1 and CR56 form a complex.

TABLE 3Fluorescence changes due to FRET between tryptophan and dansyl

PAI-1 species Quench of Trp Fdansyl increase Fdansyl FRETa

% % %H2C 79 440 227G70C 20 72 87G84C 55 187 145S127C 10 25 52G194C 4 0 9G230C 37 86 86G264C 24 86 93Q312C 13 118 127

a Percent fluorescence increase in dansyl is corrected for change in quantum yielddue solely to complex formation (see Table 1).




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centrations of CR56 used (8 �M). However, assuming similarmaximum fluorescence perturbation obtained for tighter bind-ers, we crudely estimated the Kd value for this variant as 70�M. Surprisingly, the Arg-115/Arg-118 variant was found tobind CR56 more tightly than WT (Kd of 15 nM versus 52 nM),despite a previous report that each of these residues, when indi-vidually mutated, reduced binding of uPA�PAI-1 complexes toVLDL receptor, LRP1, and sorlA, with the Arg-118 mutationhaving about as large an effect on solid-state LRP1 binding asmutating either Arg-76 or Lys-80 (20).

To further define the role of individual basic residues withinthe two pairs whose mutation reduced affinity for CR56, we alsoexamined binding of single Lys-65–Ala, Lys-69 –Ala, Arg-76 –Ala, and Lys-80 –Ala variants. The K65A mutation had noeffect on binding, whereas the Lys-69 –Ala variant had affinityreduced similarly to that of the double K65A/K69A variant(Table 2), suggesting that only Lys-69 of this pair engages one ofthe CR domains of CR56. Mutation of either Arg-76 or Lys-80resulted in large reduction in affinity, with Kd values greatlyincreased to 2.5 �M and an estimated 30 �M, respectively(Table 2), indicating that both of these residues are involved inbinding of PAI-1 to CR56.

Orientation of the CR56 Fragment—Given the proximity ofArg-76 and Lys-80 to one another and their distance fromLys-69 (Fig. 1), it seems likely that the former pair engages oneCR domain of CR56 and Lys-69 engages the other. To deter-mine which of the two domains, CR5 or CR6, engages whichsite, we employed tryptophan-dansyl FRET in a qualitative way,using both WT CR56, in which each CR domain contains asingle tryptophan, and two variants in which one or the other ofthe two tryptophans had been mutated to phenylalanine (vari-ants CR5F6W and CR5W6F).3 When FRET experiments werecarried out using dansyl reporter at Cys-2 (on a �W-PAI-1background) and each of these single tryptophan-containingCR56s, approximately the same reduction (50%) in trypto-phan fluorescence was seen resulting from FRET to dansyl, sug-

gesting equidistant positioning of each CR domain with respectto the Cys-2 dansyl group. However, when the dansyl-labeledCys-230 variant was used, much greater FRET was seen toCR5W6F (58%) than to CR5F6W (17%), compared with 37%when both CR56 domains contained tryptophan. This suggeststhat CR5 is much closer to dansyl attached to residue 230 thanis CR6 (see Fig. 1), and thus that the CR5 domain engages thepair Arg-76/Lys-80 while CR6 engages Lys-69.

Binding of CR456 to WT PAI-1—Although the above experi-ments identified a binding site for CR56, involving residues Lys-69, Arg-76, and Lys-80, we wanted to extend our studies toCR456, because this likely represents the full binding site forPAI-1, having been found to bind with Kd 20-fold smallerthan CR56 and to be the tightest binding three-domain frag-ment from within cluster II (25).

Initially we examined the effect of CR456 binding on directperturbation of dansyl attached to Cys-2 and found similarlarge enhancement of the dansyl fluorescence to that seen forbinding of CR56 (Fig. 2). This is consistent with the CR56 moi-ety in both species binding in the same location. A titration ofCR456 into dansyl-labeled Cys-2 PAI-1, with binding moni-tored using this direct perturbation of dansyl fluorescence, con-firmed the significantly tighter binding of CR456 at I 0.17 (Fig.3B and Table 2).

Ionic Strength Dependence of Binding of CR456 and CR56 —Given the much higher affinity for PAI-1 of CR456 comparedwith CR56, we used the ionic strength dependence of CR456binding at higher NaCl concentrations to obtain an accurate Kd

at I 0.17 by extrapolation. A Debye-Huckel plot of log10Kd

against I0.5 for Kd values obtained between 0.3 and 0.7 M NaClgave a straight line with a slope of 4.3 (Fig. 5A). The Kd at I 0.17,obtained by extrapolation, was 1.5 nM and is actually in goodagreement with the Kd value obtained directly of 1.3 nM (Fig. 2).This compares with the Kd of 54 nM for CR56 obtained directly.

Similar measurements of the ionic strength dependence ofKd were also carried out for CR56 but over the [NaCl] range of0.15 to 0.4 M. The Debye-Huckel plot for these data also gave astraight line but with slope of 2.9 (Fig. 5B). The slope of 2.9 is

3 In the CR5F6W, CR5W6F, and CR5F6F species, the tryptophans in CR5, CR6,or both, were changed to phenylalanine.

FIGURE 5. Debye-Huckel plots for binding of CR456 and CR56 to native Cys-2-dansyl PAI-1. Kd values were obtained from titrations carried out at differentionic strengths and monitored by changes in dansyl emission intensity. A, Debye-Huckel plot for binding of CR456. B, Debye-Huckel plot for binding of CR56.PAI-1 concentrations were between 55 and 105 �M for CR456 titrations and between 30 and 600 �M for CR56 titrations, depending on the expected Kd. Thestraight lines are the least squares best fits to the data.




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close to what would be expected from involvement of only thethree basic residues Lys-69, Arg-76, and Lys-80 in bindingCR56 identified above. Given this, the finding of a higher slopefor binding of CR456 (4.3) suggests that the greater affinity ofCR456 arises from engagement of one or more basic residues inaddition to the three already identified, with the additionalbinding probably involving CR4.

Binding of CR456 to PAI-1 Basic Variants—To better deter-mine whether the CR56-binding site represents a part of the fullCR456-binding site and to determine which, if any, additionalbasic residues are involved in binding CR456, we examined thebinding of CR456 to the same set of four double-basic PAI-1variants, each containing a dansyl reporter at the Cys-2 residue,as for binding of CR56. Where CR56 showed reduced bindingonly to the variants containing mutations at the Lys-65/Lys-69and Arg-76/Lys-80 pairs, CR456 showed reductions for both ofthese pairs, as well as for the variant with mutations at Lys-88/Lys-176 (Table 2). However, the reduction in affinity for thelatter was less than for the other two (about 11-fold versus50 –2700-fold increase in Kd at I 0.17). Interestingly, the variantwith mutations of Arg-115 and Arg-118 showed increasedaffinity compared with WT (Kd values of 4.5 nM versus 39 nM atI 0.52), as was found for CR56 binding. Also similar to bindingof CR56, mutation of the Arg-76/Lys-80 pair had the greatesteffect (2700-fold increase in Kd).

We then examined binding of CR456 to variant PAI-1s withsingle basic residue mutations. Anticipating greatly weakenedbinding, we carried out titrations at I 0.02. Mutation of each ofLys-69, Arg-76, or Lys-80 had very large effects on affinity (Kdvalues of 103, 130, and 185 nM, respectively, at I 0.02) (Table 2).Assuming a slope of three for ionic strength dependence inDebye-Huckel plots of each of these variants (only three of thefour basic residues remained), values of 670, 845, and 1200 nM,respectively, were calculated at I 0.17, implying 447– 800-foldincreases in Kd resulting from mutating these residues. Muta-tion of Lys-88 increased Kd at I 0.17 to 20 nM (13-fold increasecompared with WT). Neither the single K176A nor the K65Amutations perturbed binding compared with WT. Thus CR456appears to use the same basic residues to bind to PAI-1 as does

CR56, but with the addition that CR456 also binds to Lys-88,probably through CR4.

Is There a Preference for CR Domains—Our previous studieshave shown that CR456 binds with about a 10-fold decrease inKd to WT-cleaved PAI-1 than does CR345 or CR567 (25). Thisis consistent with earlier qualitative studies that showed thatPAI-1 does not bind tightly to CR567 but does to a longer frag-ment that includes domains CR3 and CR4 (further refinementof site-specificity was not carried out) (23, 24). Because we haveidentified three well separated binding sites for CR domains(see Fig. 1), the lower affinity of CR345 and CR567 comparedwith CR456 implies that there is a preference at a given PAI-1site for particular CR domain(s).

To examine this, we first confirmed that the lower affinitiesfor CR345 and CR567 hold true here for our slightly differentsystem (native PAI-1 Cys-2-dansylated variant versus cleavedWT). Titrations carried out at I 0.17 gave Kd values of 16.5 � 2and 28 � 1.8 nM for binding of CR345 and CR567, respectively,confirming the much weaker binding of each fragment com-pared with CR456 (Fig. 6).

Next, the effect of binding each fragment on the emissionspectrum of the Cys-2-dansyl fluorophore was examined.CR567 gave a large enhancement and a 13-nm blue shift (Fig. 7)similar to what was seen for both CR456 and CR56 (Fig. 2). Incontrast, CR345 gave a much smaller enhancement and only a7-nm blue shift (Fig. 7B). This suggests that the positional rela-tionship between the dansyl fluorophore and the domain(s) inCR56 that gives rise to the large enhancement and blue shiftremains the same when CR456 and CR567 bind, whereas it isaltered when CR345 binds.

To better determine whether, when CR567 binds, the CR5domain binds to the Arg-76/Lys-80 pair and the CR6 domainbinds to Lys-69 (as in binding of CR56 and CR456), we exam-ined binding of CR567 to the variants containing mutations atthe Lys-88/Lys-176 pair, the Arg-76/Lys-80 pair, and the Lys-65/Lys-69 pair. The affinity of the first was unaffected (Kd of24 � 1.7 nM) compared with WT, whereas the Arg-76/Lys-80-mutated variant had affinity greatly reduced, with Kd of 8.9 �0.4 �M (Table 2). This implies that CR567 does not engage the

FIGURE 6. Binding of CR345 and CR567. Binding of other three-domain CR fragments to native Cys-2-dansyl PAI-1, from perturbation of dansyl fluorescence.A, binding of CR345. B, binding of CR567. Titrations were carried out at I 0.17 using 150 nM PAI-1 for binding of CR345 and 66 �M PAI-1 for binding of CR567.




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Lys-88 site utilized by CR4 in CR456, although it does engagethe site used by CR5, with similar loss of affinity when mutated.Surprisingly, the variant with Lys-65/Lys-69 mutated showedonly a 3-fold reduction in affinity, rather than the expected50-fold. Unfortunately, there was insufficient material to carryout equivalent titrations on CR345. Together, these results aremost simply interpreted as CR5 and CR6 having strong enoughpreferences for the two sites Arg-76/Lys-80 and Lys-69, respec-tively, that they direct binding there in fragments containingthese domains (CR56, CR456, and CR567). When only CR5 ispresent (as in CR345), a higher affinity is presumably obtainedby a shift in register of one compared with binding of CR456,such that CR3, CR4, and CR5 bind where CR4, CR5, and CR6are bound. Although the resulting Kd is 10-fold higher thanfor CR456, binding is presumably tighter than would beobtained if CR4 and CR5 bound in the same way as they do inCR456, and CR3 remained unengaged. This is consistent withthe greater apparent binding contribution from Lys-69 in the“CR6” site (450-fold increase in Kd when mutated) than fromLys-88 in the “CR4” site (14-fold increase in Kd when mutated).The only seemingly contradictory result is the smaller thanexpected reduction in affinity of CR567 when the Lys-65/69pair was mutated. This may, however, result from partial com-pensation for the loss of the CR6 interaction with Lys-69 byinteraction of CR7 with another lysine or arginine that is oth-erwise inaccessible in WT when the CR6 domain is anchored byLys-69. Indeed, such an alternative mode of binding, with con-sequent partial offset of binding losses, might be one explana-tion for the anomalous results of others using the whole ofcluster II in which mutation of residues identified here as con-tributing a large fraction of the binding energy seemingly hadminimal effects (18).

Contribution of Tryptophan in CR Domains to Binding—Thepresence of a highly conserved single tryptophan in CRdomains from ligand binding regions of LDL family receptorshas suggested that the tryptophan makes an important bindingcontribution, probably to the hydrophobic side chain of ligandlysines. Consistent with this, mutation of tryptophan to serinein either the CR5 or CR6 domain of CR56 has been shown togreatly reduce binding to a RAP D3 affinity column (35),although there is the possibility that the drastic mutation mayhave adversely affected folding. Because both single trypto-phan-containingCR56specieshadbeenpreparedforFRETmea-surements, with the more conservative mutation to phenylala-nine, as well as a double variant in which both tryptophans hadbeen replaced by phenylalanine as a control, we determined Kdvalues for binding of each of these species to dansyl-labeledCys-2 PAI-1 to determine the importance of tryptophan in con-tributing to affinity.

Each species produced the expected saturable large enhance-ment in dansyl fluorescence upon binding to PAI-1 (data notshown). The titrations were each well fitted to a single bindingisotherm and gave Kd values at I 0.17 of 130 nM for CR5F6W,120 nM for CR5W6F, and 560 nM for CR5F6F, compared with52 nM for CR5W6W. The mutation of each tryptophan to phe-nylalanine thus reduces overall affinity in an approximatelyadditive manner.

Discussion

Identification of the CR456- and CR56-binding Sites—Using asensitive fluorescent dansyl probe attached to an engineeredcysteine at position 2 in PAI-1, we have determined the affini-ties of basic residue PAI-1 variants for the high affinity LRP1fragment CR456 and shown that the binding site on nativePAI-1 involves the residues Lys-69, Arg-76, Lys-80, and Lys-88(Fig. 1C). The proximity of this site to residue 2 explains theextreme sensitivity of the dansyl fluorophore at this position tobinding of CR456. Independent support for the involvement offour charged residues comes from the slope of the ionicstrength dependence of the affinity (4.3) (Fig. 5A). It should alsobe noted that this is the first time arginine has been identified asbeing involved in binding to an LDL receptor family member,although we have previously shown with model compoundsbinding to CR domains that the strength of the interaction inthat case is nearly the same as for lysine (22).

Based on the effects of mutation on affinity and the nearlyidentical direct perturbation of Cys-2-dansyl fluorescenceupon binding, we have also shown here that CR56 binds toArg-76, Lys-80, and Lys-69 and seems to occupy the same sub-sites as do the equivalent CR domains in CR456. The bindingsite location is also qualitatively consistent with the very largeFRET-derived quench of tryptophan fluorescence when dansylacceptor is at positions 2, 84, or 230 (see Fig. 1). FRET measure-ments on WT CR56 and CR56 variants with each tryptophanmutated separately to phenylalanine indicate that CR5 is muchcloser to residue 230 than is CR6 and thus that the orientationof the CR56 is such that CR5 engages the basic pair Arg-76/Lys-80 while CR6 engages Lys-69. If the CR5 and CR6 domainsin CR456 bind in the same sub-sites as when CR56 binds, thisimplies that CR4 engages Lys-88 as the third binding site.

FIGURE 7. Proximity perturbation from binding CR345 and CR567. Normal-ized dansyl emission spectra of Cys-2-dansyl PAI-1 in the absence (solid line) andpresence of bound CR345 (red long dashes) or CR567 (blue short dashes). Excita-tion was at 342 nm, with emission measured from 400 to 650 nm.




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Examination of the location of these residues in PAI-1 (Fig. 1)shows that this is also consistent with the relative positions ofeach of these basic residues. Thus, binding of the highest affin-ity cluster II fragment CR456 to PAI-1 involves engagement ofCR4 by Lys-88, CR5 by the pair Arg-76/Lys-80, and CR6 byLys-69. Single mutation of each of these residues to alanineresults in increases in Kd of 13, 563, 800, and 447 times,respectively.

Binding Contributions of the Basic Residues—Given theapparent independence of adjacent CR domains (38), resultingfrom their flexible linkers, one might expect binding contribu-tions to a ligand to be additive. Assuming such additivity, thesummed binding contributions of the four basic residues, Lys-69, Arg-76, Lys-80, and Lys-88, amount to 13.65 kcal mol�1 at298 K (calculated from the losses in affinity for each mutation),compared with the measured affinity of CR456 of 12.10 kcalmol�1. This close agreement suggests that we have not onlyidentified basic residues in PAI-1 that are important for bindingto LRP1 but have shown that their binding contributions quan-titatively account for all of the binding energy. In a similar way,we previously showed that engagement of four lysines in RAPD3 accounted fully for the high affinity interaction with CR56(21). It is striking however, that, although the mutationsreported here resulted in extremely large increases in Kd values,the same mutations reported elsewhere had mostly negligibleeffects (18). We have no good explanation for this discrepancy,other than to point out that, whereas the present studies werecarried out in solution using a sensitive fluorescent reporter,other studies relied on solid-phase binding assays, which mightbe subject to complicating nonspecific effects. Another possi-bility is the existence of alternative binding modes when criticalsites are deleted by mutagenesis, as was discussed above forCR567 binding to the Lys-65/Lys-69 variant. Also, in contrastto a previous study (20), we find no evidence for involvement ofeither Arg-115 or Arg-118 in promoting binding of CR456.Indeed, removal of these residues, which lie between the Arg-76/Lys-80 pair and Lys-69, actually increases affinity for bothCR56 and CR456. Finally, using CR56 species containing only asingle tryptophan, we showed that tryptophan within each CRdomain results in 3-fold lower Kd than when phenylalanine ispresent, perhaps explaining why tryptophan is the highly pre-ferred residue at this location in CR domains within ligandbinding regions of LRP1 and other LDL receptor familymembers.

CR Domain Preference—From the respective contributionsof each of the four basic residues to the three CR domain bind-ing sites, it is clear that the strongest interaction by far is

between CR5 and Arg-76 and Lys-80, followed by CR6 bindingLys-69, and the weakest interaction is between CR4 and Lys-88.This preference holds when CR56 binds resulting in CR5 andCR6 occupying the same sites. Even for CR567, the benefit ofCR5 occupying the “middle” site (see Fig. 1C) appears to out-weigh the loss from not occupying the much weaker Lys-88 site.Only with CR345, when occupation of the middle site by CR5would result in the tight “bottom” site (Lys-69) being empty (seeFig. 1C), does there appear to be a shift in register, such thatCR5 probably binds to Lys-69, while CR4 binds to the Arg-76/Lys-80 pair and CR3 binds to Lys-88. We have previouslyshown that the single CR domains CR3, CR5, and CR8 showlittle variation in affinity for the model compounds argininemethyl ester or lysine methyl ester (22). It is thus initially puz-zling that the middle site, containing Arg-76 and Lys-80, seemsto strongly prefer CR5. One possibility is that CR5 has a morenegatively charged surface at the engagement site, with themotif Glu-Xaa-Glu-Glu-Glu present in CR5 compared withGlu-Xaa-Glu-Xaa-Glu for each of the domains CR3, CR4, andCR6 (CR7, CR8, and CR9 also have the latter motif). This mayresult in stronger interaction with the two basic residues in themiddle site.

Sterically Appropriate Location of the Four Basic Residues—Whereas the residues involved in CR456 binding were identi-fied solely by mutagenesis, it turns out that they are also wellpositioned relative to one another to be able to engage the threeseparate CR domains. Guidance is provided by the x-ray struc-ture of the two CR domain fragment from LDL receptor com-plexed to the RAP D3 domain (15) and of the structure of LDLreceptor extracellular domain at low pH in which the YWTDdomain interacts with two CR domains of the ligand bindingregion (39). In the former structure the separation betweenequivalent positions in the two CR domains (C-C betweenPhe-105 and Trp-144) is 22 Å, while in the latter it is 19 Åbetween equivalent tryptophans (Trp-144 and Trp-193). Forthe ligand D3 in the complex, the separation between the -amino groups of the principal lysines involved in binding (Lys-256 and Lys-270) is 21 Å. In a study in which Lys-256 wasremoved from D3, leaving Lys-253 and Lys-270 as the principallysines, with separation between -amino groups of 25 Å, CR56was still able to bind, although with greatly reduced affinity (21).In the highest resolution PAI-1 structure available, that oflatent PAI-1 at 1.8 Å (1LJ5), the separation between the -amino groups of Lys-88 and Lys-80 is 25 Å and between Lys-80and Lys-69 is 19.3 Å. Although only one of these is similar towhat is seen in actual protein complexes, it should be noted thatthe C-C separations for the same residue pairs are 13.9 and

TABLE 4Separation between C� residues of the four basic residues involved in binding to CR456

C�-C�

Native PAI-1 (Protein DataBank code 1DVM)

Cleaved PAI-1 (ProteinData Bank code 3CVM)

Latent PAI-1 (ProteinData Bank code 1LJ5)

Native PN1a (ProteinData Bank code 4DY0)

Å69–76 13.34 13.35 13.23 16.6969–80 18.82 18.91 19.11 12.6369–88 32.16 31.31 31.96 30.1676–80 6.19 6.32 6.42 3.6776–88 13.90 13.43 13.90 16.8580–88 19.41 19.48 20.01 19.55

a For PN1 the four basic residues and in parentheses the positionally equivalent residues in PAI-1 are Arg-63 (Lys-69), Lys-71 (Arg-76), Lys-74 (Lys-80), and Lys-83 (Lys-88).




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19.1 Å (Table 4), so that side chain positioning in solution,which might be different from in the crystal, could easily give19 Å separation even for the pair Lys-88 and Lys-80. Indepen-dent support for the proposed binding site comes from a studyon a peptide inhibitor of PAI-1 binding to LRP1 (40). Modelingof the binding site on PAI-1 suggested that the peptide bindsclose to the residues Arg-115, Lys-69, Arg-76, and Lys-80, thelast three of which we have identified here as part of the CR456-binding site. Significantly, each of the four basic residues con-stituting the CR456-binding site is highly conserved amongmammalian species, with variation only within the other basicresidues (Table 5).

Effect of PAI-1 Conformation on Binding—An importantquestion about our identification of the CR456-binding site iswhether it is the same in all conformational forms of PAI-1,i.e. native, latent, cleaved, and proteinase-complexed. Compar-ison of the relative positions of the four basic residues identifiedin the three states of PAI-1 for which there are x-ray structures(native, latent, and cleaved) (Table 4) shows that there is only asmall variation for any of the C-C separations, which impliesthat CR456 should be able to engage the same four residues inthe same way in each of these conformational states, especiallygiven the length and flexibility of the basic residue side chains.This is in keeping with reports from this laboratory and othersthat native, latent, and cleaved PAI-1 bind with effectively iden-tical affinity to LRP1 or LRP1 fragments (12, 18, 25). It thereforeseems likely that the high affinity binding site we have identifiedon native PAI-1 is present with the same affinity in both latentand cleaved PAI-1, even though latent and cleaved have under-gone major conformational changes relative to native. Impor-tantly, these structural changes do not affect the region of PAI-1where the LRP1-binding site is located (41).

Binding to PAI-1�Proteinase Complexes—An even moreimportant question than whether different conformations ofPAI-1 bind similarly to LRP or its fragments is whether thesame binding site is present in the PAI-1 moiety in covalentcomplex with uPA or other proteinases. Although there is nox-ray structure of PAI-1�proteinase complex to directly answerthe question, there are x-ray structures of other serpin�proteinase complexes, viz. 1-proteinase inhibitor in complexwith either trypsin (9) or porcine pancreatic elastase (8), as wellas NMR structural data on the trypsin complex (42). All of these

show that the secondary structure of the serpin moiety isunchanged from that of the cleaved serpin. Given this, the pres-ence of a “cryptic” LRP1-binding site within PAI-1, which isonly uncovered in complex (13, 18), seems unlikely. The sug-gestion of a cryptic site was based on the greater efficiency ofcellular clearance of uPA�PAI-1 complex compared with any ofthe uncomplexed forms of PAI-1, even the structurally equiva-lent cleaved form. However, it seems more likely that the expla-nation for this lies with the involvement of uPAR as a co-recep-tor to LRP1 in clearance of uPA�PAI-1 complexes but not forcleaved, latent, or native PAI-1, which rely only on LRP1 forclearance. By localizing the PAI-1 to the cell surface, through itscovalent attachment to uPA, which is in turn non-covalentlyassociated with the membrane-anchored receptor uPAR, theinteraction of the PAI-1 moiety with LRP1 would be greatlyfacilitated, even without any alteration in its binding site forPAI-1 or any need to invoke a binding contribution to LRP1from the proteinase moiety. Although it is reported thatuPA�PAI-1 binds to LRP1 with Kd about 10-fold smaller thanPAI-1, the experimental setup does not mimic the situation invivo (12, 18). Thus, the uPA�PAI-1 complex has the N-terminalregion of the proteinase exposed, whereas in vivo, this would beobscured through binding to uPAR (3, 4, 43). Because thisN-terminal region has an exposed single lysine that becomesdeeply buried upon receptor binding (Lys-23) and a pair oflysines that rest at the edge of the binding pocket (Lys-34 andLys-35) and would likely be inaccessible to LRP1, it is quitepossible that high affinity binding site(s) on uPA for LRP1 areonly available when uPA is not in complex with its receptor.This is supported by a study that showed that the N-terminalregion of free uPA is involved in binding to LRP1 and that uPARprotects against such binding (12). Furthermore, the separateaffinities of PAI-1 and uPA for LRP are so high that if bothbinding sites were engaged by LRP when uPA�PAI-1 complexbinds, the Kd would be in the attomolar (10�18) region, whereasmeasured values are only 2 � 10�10 M (20). Taken together,these findings suggest a model for PAI-1�proteinase complexbinding to LRP1 shown in Fig. 8. Here, the orientation of thePAI-1 moiety in complex with uPA, derived from the two x-raystructures of serpin�proteinase covalent complexes (8, 9), alignsthe PAI-1-binding site correctly with the membrane-anchoredLRP1. The only contact between the uPA�PAI-1 complex is

TABLE 5Sequence comparison of PAI-1 from different species from residue 65 to 88The four basic residues identified as composing the CR456 binding site in human PAI-1 are shown in bold.




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with the PAI-1-binding site identified here, but the intrinsichigh affinity (1.5 nM) together with the localization of the com-plex to the cell surface, through being bound to uPAR, wouldensure much more efficient clearance than for PAI-1 alone.

Although the intrinsic high affinity of PAI-1 alone reportedhere for an LRP1 fragment may result in a large fraction of totalPAI-1 being complexed to LRP1 at any given time, it neverthe-less must result in the observed free concentration reported forcirculating PAI-1 of 2 nM (44). Complex formation with uPAwould still result in effective clearance via the proposed co-re-ceptor mechanism, without any further need for a decrease inKd for the uPA�PAI-1 species for LRP1.

Proteinase Nexin1 May Bind Similarly to LRP1—Finally, it isworth considering this model of uPA�PAI-1 binding and clear-ance with respect to a closely related serpin proteinase nexin 1(PN1). This is the second of the two human clade E serpins (45)

and is also capable of inhibiting uPA by the serpin mechanism.It binds about as tightly to LRP1 as does PAI-1 (46) and withsimilar affinity to LRP1 fragments in both native and cleavedstates (25). X-ray structures of PN1 in complex with heparin orS195A thrombin show that there are basic residues, Arg-63,Lys-71, Lys-74, and Lys-83, at structurally equivalent positionsto the four identified here in PAI-1 as composing the LRP1-binding site (Table 4). They are also all highly conserved amongmammalian PN1 species. PN1 may thus use the same bindingsite for binding to LRP1 as does PAI-1, and the same mecha-nism of co-receptor clearance, at least for complexes with uPA.

Author Contributions—P. G. W. G. designed the study, collected,and analyzed the data, and wrote the manuscript. K. D. engineered,expressed, and labeled the proteins. Both authors have read andapproved the final version of the manuscript.

Acknowledgments—We thank Andres Campos for preparing some ofthe PAI-1 species and Steven Olson for helpful comments on themanuscript.

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FIGURE 8. Model of PAI-1 and PAI-1�uPA complex binding to LRP1. A,native PAI-1 binds to CR456 of cluster II of LRP1 through residues Lys-88 (CR4),Arg-76/Lys-80 (CR5), and Lys-69 (CR6) with Kd of 1.5 nM. B, similar binding ofconformationally altered PAI-1 (latent or cleaved). C, uPA is predominantlybound to its cell surface-anchored receptor uPA (red), through the N-terminalregion (yellow), and is therefore unable to bind with high affinity to LRP1. D,PAI-1 reacts with uPAR-associated PAI-1 to form a covalent serpin�proteinasecomplex that is localized to the membrane surface by association of the uPAmoiety with its uPA receptor. The insertion of the PAI-1 reactive center loopinto the serpin body upon complex formation with uPA (7) results in the PAI-1moiety having the same orientation relative to LRP1 as in A and B. Binding ofthe ternary uPAR�uPA�PAI-1 complex to LRP1 still occurs only through thethree sites on the PAI-1, with the same intrinsic affinity, but it results in moreeffective clearance as a result of the two-dimensional rather than three-di-mensional association process. Only the first two CR clusters of LRP1 areshown, with CR domains in red, EGF domains in cyan, and YWTD domains inblack. A dotted line replaces the remainder of the receptor between cluster IIand the membrane-spanning C terminus.




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Peter G. W. Gettins and Klavs DolmerFour Basic Residues

the Low Density Lipoprotein Receptor-related Protein (LRP1) Is Composed of The High Affinity Binding Site on Plasminogen Activator Inhibitor-1 (PAI-1) for

doi: 10.1074/jbc.M115.688820 originally published online November 10, 20152016, 291:800-812.J. Biol. Chem.

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The High Affinity Binding Site on Plasminogen Activator Inhibitor-1