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The Journal of Immunology, 1999, 163: 3842-3850.
Copyright © 1999 by The American Association of Immunologists

Mapping the Major Interaction Between Binding Protein and Ig Light Chains to Sites Within the Variable Domain1

David P. Davis2,*,{dagger}, Ritu Khurana2,3,*,{dagger}, Stephen Meredith*, Fred J. Stevens{ddagger} and Yair Argon4,*,{dagger}

* Department of Pathology and {dagger} Committee on Immunology, University of Chicago, Chicago, IL 60637; and {ddagger} Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, Argonne, IL 60439 6S. Aviel et al., submitted for publication.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Newly synthesized Ig chains are known to interact in vivo with the binding protein (BiP), a major peptide-binding chaperone in the endoplasmic reticulum. The predominant interactions between the light chain and BiP are observed early in the folding pathway, when the light chain is either completely reduced, or has only one disulfide bond. In this study, we describe the in vitro reconstitution of BiP binding to the variable domain of light chains (VL). Binding of deliberately unfolded VL was dramatically more avid than that of folded VL, mimicking the interaction in vivo. Furthermore, VL binding was inhibited by addition of ATP, was competed with excess unlabeled VL, and was demonstrated with several different VL proteins. Using this assay, peptides derived from the VL sequence were tested experimentally for their ability to bind BiP. Four peptides from both ß sheets of VL were shown to bind BiP specifically, two with significantly higher affinity. As few as these two peptide sites, one from each ß sheet of VL, are sufficient to explain the association of BiP with the entire light chain. These results suggest how BiP directs the folding of Ig in vivo and how it may be used in shaping the B cell repertoire.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Proper folding of their subunits is an essential step in the biosynthesis of Ag receptors; misfolded subunits are generally incapable of assembly or subsequent expression on the cell surface. While this is true of all secreted and membrane-bound proteins, the control of proper folding is even more crucial for B and TCRs, since the variable domains of these receptors are a product of several genetic mechanisms that diversify the receptor repertoire. Given the mechanisms of somatic recombination and hypermutation, there is heightened likelihood that the resultant Ag receptors will include amino acids that are detrimental to proper folding. Thus, it is not surprising that B and TCRs require interactions with chaperones early in their biosynthesis.

The chaperone BiP5 was first identified based on binding to Ig heavy chain (1, 2) and shown to be the endoplasmic reticulum (ER) member of the hsp70 class of stress proteins. BiP was also shown to interact with the light chain (LC) of Ig. We have previously demonstrated that wild-type LC binds BiP transiently during its folding in the ER, and that the avidity of binding decreases as the two domains of LC fold (3). When coexpressed with a nonreleasing form of BiP, the CL domain of LC is still capable of oxidizing, but the VL domain remains unfolded (4). Many mutant LC, mostly with substitutions in the VL domain, bind BiP more avidly than wild-type LC (5, 6, 7, 8). Several physiological roles for this interaction can be envisioned: promotion of proper folding, coordination of assembly (9), or targeting to degradation (6). Understanding how BiP performs these roles, however, requires that the binding between this chaperone and LC be defined.

Like other members of the hsp70 family, BiP binds incompletely folded proteins by recognizing certain peptides within their sequences (10, 11, 12). Such binding facilitates the process of protein folding, and, in some cases (mitochondrial hsp70 and yeast BiP), the translocation of polypeptides across membranes (13). The hsp70 proteins, including BiP, share a common structure consisting of a C-terminal 27-kDa substrate-binding domain and an N-terminal 44-kDa nucleotide-binding domain (14). The substrate-binding domain consists of two subdomains, one with a shallow groove for transient peptide binding (15). Although the overall conservation of sequence is only about 67%, the residues that contact a bound peptide are well conserved throughout the hsp70 family (15). A second subdomain forms an unusually long {alpha}-helical segment that encloses the substrate-binding region like a latch and may act as a regulatory element (15). The nucleotide-binding domain contains the binding core (16) and accommodates the ATP-Mg complex (14, 17, 18). Binding of ATP transmits a conformational change (14, 19, 20, 21, 22) to the peptide-binding site, decreasing the binding affinity and releasing the peptide (12). Hydrolysis of ATP is thought to convert the protein back to the high affinity-binding state. Individual point mutants of BiP identify the residues that are involved in each of the separate activities: ATP binding, conformational change, and hydrolysis (21). The functional linkage of all three is demonstrated by the common consequence of expressing any of these ATP-binding domain mutants in cells. When each of them is expressed in a cell synthesizing LC, each mutant acts in dominant-negative manner, trapping the LC at an early stage of folding, and therefore reducing its secretion (4). Remarkably, folding is arrested at an intermediate stage, with the CL domain still oxidized, but with the VL domain trapped in a reduced state (4).

A number of in vitro studies to define structural features of hsp70-binding peptides employed either chemically synthesized peptides (12, 24) or affinity panning of a peptide library displayed on bacteriophages (10). The binding peptides consist of either alternate aromatic or long-chain aliphatic amino acids (10), or several successive hydrophobic residues (24). The optimal peptide is seven to eight residues (25) and binds hsp70 in an extended, ß strand conformation (26, 27). The predictive value of these studies was elegantly demonstrated by Knarr et al. (28) in mapping BiP binding sites on Ig heavy chains, and by Rudiger et al. (24) in mapping dnaK binding sites of several proteins.

In contrast to the multitude of peptide-binding studies, the interaction of BiP with natural occurring substrate proteins has scarcely been studied. Since the above-mentioned data with the dominant-negative BiP and with the point mutations in LC suggested that the predominant interaction with BiP is mediated by the VL domain, we set out to reconstitute this interaction in vitro. Work described in this paper shows that VL, a domain important for interaction with BiP in vivo, binds BiP in vitro. This binding is enhanced when the substrate is partly denatured. Furthermore, we map potential BiP-binding peptides within VL and show, surprisingly, that the binding of the entire protein can be accounted for by as few as two BiP-binding peptides. The implications of these results for the progression of folding in the cell and for the selection of the LC repertoire are discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Protein purification

GST-BiP. The pDS78 plasmid, encoding a GST-hamster BiP fusion protein, was obtained from Dr. H. Weissbach (Roche, Nutley, NJ) and grown in Escherichia coli DH5{alpha}. The bacteria were induced for 3 h at mid-log with 1 mM isopropyl ß-D-thiogalactoside and then resuspended in 20 mM sodium phosphate, pH 7.5, and 130 mM NaCl (buffer A) and lysed by sonication in presence of 10% glycerol and 1% Triton X-100, as described by Carlino et al. (29). rGST-BiP was purified by affinity chromatography on glutathione-agarose (Sigma, St. Louis, MO), and eluted with 10 mM reduced glutathione in buffer A. When noted, the GST was cleaved from BiP with 100 U of thrombin per 10 mg of GST-BiP (Calbiochem, La Jolla, CA) in 20 ml buffer A with 5 mM CaCl2. Aliquots of BiP and GST-BiP were stored in 20 mM Tris, pH 7.5, 1 mM EDTA, 100 mM NaCl, and 5% glycerol at -80°C.

His-tagged BiP. His6-tagged mouse BiP was initially obtained from Dr. S. Blond-Elguindi (University of Illinois, Chicago). The protein was expressed in E. coli and purified using affinity chromatography on Ni2+-agarose, as described by the manufacturer (Qiagen, Chatsworth, CA). Following elution from the Ni2+-agarose column, BiP was further purified with an ATP-agarose (Sigma; C-8 linkage) column, as described by Freeman et al. (30). The column was washed with 2 vol of 2 M NaCl and equilibrated with column buffer (20 mM Tris, pH 6.9, 5 mM MgCl2, and 100 mM NaCl), and the bound BiP was eluted with column buffer containing 10% glycerol and 50 mM ATP. His6-tagged yeast BiP (encoded by the Kar2 gene) was obtained from Dr. Jeff Brodsky (University of Pittsburgh) in the form of E. coli strain RR1 transformed with the plasmid pMR2623. It was purified in the same fashion as the murine BiP. The murine version was more active than yeast BiP.

Variable domains of LEN, SMA, and REC {kappa}-chains. The pkIVlen004, pkIVsma007, and pkIVrec006 constructs, which target the {kappa}IV chain (VL) variable domains of LEN, SMA, and REC, respectively, to the periplasm were described before (31). These VL proteins were purified according to the procedure described previously (31) and are referred to in this work simply as LEN, REC, or SMA. Bacteria were grown at 30°C, and induced at mid-log growth with 1 mM isopropyl ß-D-thiogalactoside for 24 h. Periplasmic proteins were harvested by resuspending the bacterial pellet in ice-cold 0.1 M Tris-Cl, pH 8, 0.25 mM EDTA, 0.25 M sucrose, and 0.4 mg/ml lysozyme, and pelleting the spheroplasts. VL was further purified by two successive ion-exchange chromatography steps, first over Econo-Pac High Q cartridge and then a High S cartridge (Bio-Rad, Richmond, CA). Only preparations judged to be >95% pure based on SDS-PAGE were used.

Peptides

All peptides were synthesized by the University of Chicago core peptide facility. Stock solutions were prepared in 100% DMSO and stored at -80°C. Most of the peptides used were water soluble, except for FTLTISS and TDFTLTI, which were only water soluble at concentrations below 600 µM. Concentrations of peptide solutions were determined with the BCA assay (Pierce, Rockford, IL).

Iodination of FYQLALT and VL

The peptide FYQLALT has been demonstrated to bind all the members of hsp70 class of proteins, and its binding to Hsc70 has been studied extensively (28, 32). FYQLALT was iodinated with Iodo-Beads (Pierce) following the manufacturer’s instructions. Usually, 50 µg of peptide in 100 mM NaPO4, pH 6.5, was iodinated with 2 mCi of 125I (Amersham, Arlington Heights, IL) using 3–4 Iodo-Beads. Unincorporated 125I was removed by passing the peptide through a Sep-Pak C18 cartridge (Waters, Milford, MA) and eluting with a 30–50% acetonitrile gradient. The sp. act. of FYQLALT was typically 0.5–1 x 1016 cpm/mol.

VL was iodinated also with the Iodo-Beads, following manufacturer’s instructions. The 125I (1–2 mCi) was activated with the Iodo-Beads in 25 mM Tris-HCl, pH 7.5, and 0.4 M NaCl, and then incubated with 0.5–1 mg of VL. Unincorporated 125I was removed by desalting with a prepoured Bio-Rad Econo-PacI0DG (10 ml bed vol), and the VL was stored at 4°C in 20 mM Tris-HCl, pH 7.5, and 150 mM NaCl. The sp. act. was 2 x 1016 cpm/mol.

Fluorescein conjugation of peptides and proteins

The FYQLALT peptide was suspended in 100 mM borate buffer, pH 9, at 2 mM and coupled to FITC, as suggested by the manufacturer (Molecular Probes, Eugene, OR). The FITC-conjugated peptide was separated from free FITC and unconjugated peptide by chromatography over a C-18 reverse-phase HPLC column. The fractions were collected and lyophilized. Fresh stock solutions of labeled peptide were made in 10 mM borate buffer, pH 9, and the concentration was calculated from the absorbance at 494 and 280 nm. The F/P ratio was 1.

LEN, SMA, and REC were conjugated to 6-fluorescein-5 (and -6)-carboxamido hexanoic acid and succinimidyl ester (SFX) (Molecular Probes) by incubating at a ratio of 5 mg VL to 2 mg SFX, according to the manufacturer’s instructions. Free SFX was separated from SFX-conjugated protein by passage through a Sephadex G-25 column (Pharmacia, Piscataway, NJ) and stored at 4°C in 0.1 M NaHCO3, pH 8.3. Incorporation of SFX yielded an F/P ratio of 3.

Gel filtration assay for binding of peptides and VL to BiP

Binding assays were routinely performed in 25 µl total volume of 50 mM Tris, pH 7.5, 50 mM KCl, 20 mM MgCl2, 0.5% Triton X-100, with 20 µM BiP, 42 µM 125I-FYQLALT, and the appropriate concentration of unlabeled VL or peptide for 1 h at 25°C, unless otherwise noted. When peptides were used to complete the binding, the reactions included 16% final concentration of DMSO. This concentration of DMSO in itself had no effect on the binding of 125I-FYQLALT to BiP (data not shown). BiP-bound 125I-FYQLALT was separated from free peptide by centrifugation for 2 min at 1100 x g through Micro Bio-Spin Chromatography Columns packed with Bio-Gel P-30 (Bio-Rad). To separate BiP-bound VL from free 125I-labeled VL, similar spin columns were packed with Bio-Gel P-60 beads. The radioactivity present in the flow-through was determined by scintillation counting with a Packard Top-Count. Nonspecific binding was determined by the addition of 600-fold excess unlabeled FYQLALT, and was routinely 5–10% of the total.

Kd values were calculated from IC50 values (the concentrations of peptide required to obtain half-maximal inhibition) with the Cheng and Prusoff equation (33):

where Kd* is the dissociation constant of the labeled ligand (derived from the dose-binding analyses, see below), Kd is the dissociation constant of the inhibitor peptides, and L is the concentration of the labeled ligand.

To demonstrate saturable binding, dose-binding assays of 125I-FYQLALT and 125I-labeled VL were performed by adding increasing concentrations of the labeled substrate to a fixed concentration of BiP (20 µM). Data obtained from the dose-binding analyses were fit to a Scatchard plot using the GraphPad Prism computer software to calculate Kd. This program allowed best-fit determinations of the data to either a one- or two-site binding model.

Calculation of the concentration of the BiP-LC complex (C) employed the equation:

where B is the concentration of BiP available for LC binding, L is the concentration of the LC-folding intermediates that can bind to BiP, and Kd is the dissociation constant (34).

ATPase assays

The extent of ATP hydrolysis was measured as previously described (35). In a final volume of 50 µl, 2.5 µg of the appropriate preparation of BiP or hsp70 (a generous gift from R. Morimoto) was incubated at 37°C in the presence of 2 µCi [{gamma}-32P]ATP. Aliquots were removed at various time points and added to activated charcoal slurry. After centrifugation, the amount of 32P in the supernatant was counted in a Packard Top Count.

Gel electrophoresis analysis of the BiP-bound VL

FITC-VL was incubated for 1 h at 25°C with His6-tagged KAR-2 immobilized on Ni2+-agarose beads. After washing, the bound material was eluted with SDS sample buffer and resolved on a 16% acrylamide Tricine-SDS gel (36). Detection of KAR-2-bound FITC-VL was performed by a rapid silver stain method (37).

Prediction of BiP-binding peptides

The distribution of putative BiP binding sites on variable domains was addressed by the use of two computer algorithms. We developed one algorithm based on the BiP-binding scores (weights) determined by Blond-Elguindi et al. (10). These scores were related to the probability of any particular amino acid being located at specific positions within heptameric peptides that had measurable binding to BiP. For all 20 amino acids, negative and positive numerical values were assigned to reflect the preferential exclusion or inclusion of the amino acid at positions 1–7 in BiP-binding peptides. The algorithm analyzes the variable domain sequence seven amino acids at a time by summing the position-dependent scores of each amino acid in the heptamer, assigning the resulting cumulative score to the first residue of the peptide. The algorithm was used to evaluate individual variable domains, as well as to determine an average BiP site distribution by automatically analyzing a database of 122 human V{kappa} domain sequences.

Fluorescence measurements

Intrinsic tryptophan fluorescence was measured with 3 µM of VL in 20 mM Tris, pH 7.5, and 150 mM NaCl in the absence or presence of the appropriate concentrations of Gdn-HCl or DTT (both buffered to pH 7.5). Emission spectra were recorded from 300–400 nm, using a Photon Technology Industries (Princeton, NJ) fluorescence spectrophotometer, at excitation wavelength of 280 nm. Raw data were corrected for the quenching effects of the reducing agents. Measurements of binding of the fluorescent dye ANS to the native or unfolded VL, LEN, were performed by incubating 10 µM ANS with 3 µM of the appropriate LEN in 20 mM sodium phosphate buffer, pH 7. The emission spectra were then recorded from 420–600 nm after excitation at 350 nm. The fluorescence of ANS alone was subtracted from the spectra of ANS in the presence of LEN.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BiP-binding assay

Substrate binding to BiP was determined by reacting the two in solution, followed by separation of bound and free reactants. The peptide FYQLALT, which was previously shown to bind to a variety of hsp70 family members (27, 32), was labeled by iodination or by coupling to FITC, and incubated with recombinant hsp70 proteins produced in bacteria. The reactions were applied to gel filtration spin columns to separate free from bound reactants. The validity of this assay is shown in Fig. 1Go. Of the hsp70 proteins tested, FYQLALT bound best to human hsp70. This binding was inhibited by addition of excess unlabeled peptide and by addition of ATP (Fig. 1Go, A and B), consistent with the known mode of action of hsp70. The iodinated and the fluorescent versions of the peptide behaved similarly. The binding of FYQLALT to hamster BiP, mouse BiP (shown below), and yeast BiP (kar-2) was lower than to human hsp70, but in each case was effectively inhibited by excess cold peptide (Fig. 1GoA). Interestingly, even the GST-BiP fusion protein was active in peptide binding, although less active than the authentic BiP released by thrombin cleavage of the fusion protein.



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  		FIGURE 1. Differential peptide binding and ATPase activity of hsp70 proteins. A, The ability of human hsp70, hamster BiP, GST-BiP fusion protein, and yeast Kar2 to bind 125I-FYQLALT was compared. Open bars = 42 µM 125I-FYQLALT + 20 µM hsp70 or BiP; solid bars = 125I-FYQLALT bound in the presence of 100-fold excess unlabeled peptide. B, ATP-induced release of bound peptide. Complexes of the indicated hsp70 protein with 125I-FYQLALT were purified over a spin column, incubated for 10 min with (filled bars) or without (open bars) 10 mM ATP, and repurified over a spin column. The amount of radioactive peptide remaining in the complex was measured and is presented as relative binding. C, The kinetics of ATP hydrolysis by four recombinant hsp70 proteins. The ability of bacterially produced proteins to hydrolyze [{gamma}-32P]ATP was measured as the radioactivity that fails to bind to activated charcoal slurry, as described in Materials and Methods. A GST-rab fusion protein was used as a negative control for both peptide binding and ATP hydrolysis. D, Binding analysis of 125I-FYQLALT to BiP. 125I-FYQLALT at increasing concentrations was incubated in duplicate for 1 h at 25°C with a constant concentration of BiP. Shown is a representative experiment utilizing 5 µM His-mouse BiP; equivalent results were obtained with 20 µM GST-hamster BiP. Bound peptide was separated from free, as described in Materials and Methods. Data obtained from the dose-binding analysis (inset) were fit to a Scatchard plot using the GraphPad Prism program, to calculate Kd. Peptide binding at higher input concentrations could not be reliably determined due to peptide insolubility.

 
The activity of the nucleotide-binding domain of either BiP, GST-BiP, or hsp70 was determined in two ways. Bound peptides were efficiently released by incubation with ATP in a one-cycle binding assay (Fig. 1GoB), in which complexes were first purified by a desalting column, incubated with ATP, and repurified. This suggested that under conditions of continuous incubation (such as those used in Fig. 1GoA), the observed level of peptide binding is the product of multiple cycles of binding and release. The peptides were released effectively by ATP from the various rBiP, despite their low inherent hydrolytic activity as compared with the recombinant hsp70 (Fig. 1GoC). This is consistent with the notion that nucleotide exchange, not hydrolysis, was important for peptide release of all members of the hsp70 family of chaperones (22). When we measured, instead of rates of ATP hydrolysis, the extent of stimulation of ATPase activity by FYQLALT, both human hsp70 and hamster BiP were stimulated 3–4-fold (data not shown).

The peptide-binding assay was used to measure the affinity of 125I-FYQLALT to BiP (Fig. 1GoD). Binding approached saturation at 500 µM peptide, and Scatchard analysis showed a biphasic binding curve, with a calculated Kd of 2 ± 0.8 µM for the higher affinity site, and a Kd of 163 ± 22 µM for the lower affinity site (n = 4). Since at saturation binding capacity approached a ratio of 1 mol of peptide bound for every mole of BiP, this indicates the biphasic binding is a manifestation of high and low affinity states for the single binding site of each BiP molecule (15, 38, 39). A similar observation was made with the biphasic binding of FYQLALT by the related chaperone hsp70 (32). Competition-binding assays (see Materials and Methods), in which increasing concentrations of unlabeled FYQLALT were used to displace the binding of a constant concentration of 125I-FYQLALT, gave a calculated Kd of 5 ± 2 µM for the high affinity site (Fig. 2GoA). The correspondence between the Kd values of the direct binding and the competition assays validates this spin column assay and shows that iodination of the tracer peptide did not significantly change its binding affinity. Therefore, it was used to measure either direct binding of labeled substrates, or the ability of unlabeled substrates to displace the labeled ones. Because GST-hamster BiP and His6-mouse BiP were found to exhibit equivalent binding affinities and specificities, these rBiP forms were used interchangeably in all subsequent experiments.



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  		FIGURE 2. Binding of folded and unfolded VL to BiP. A, Binding of unfolded VL to BiP. LEN ({blacksquare}) or SMA ({diamondsuit}) was incubated for 2 h at 25°C in the presence of 2 M Gdn-HCl and 40 mM DTT. Increasing concentrations of the appropriate VL were then added to 20 µM GST-BiP and 42 µM 125I-FYQLALT (resulting in final concentrations of 0.8 M Gdn-HCl and 16 mM DTT). As a control for any effect on GST-BiP, unlabeled FYQLALT (•) was used as a competitor in the presence of Gdn-HCl and DTT. B, Dose binding of unfolded VL, showing saturability. Denatured and reduced 125I-labeled SMA was prepared by treatment with 2 M Gdn-HCl and 40 mM DTT. Increasing doses of this preparation were incubated directly with BiP, and the complexes were separated from the free reactants and counted. The data were fit to a nonlinear regression curve with the GraphPad Prism computer software. The percentage of bound VL relative to the amount of input BiP was calculated as 88 ± 24%, while the Kd was calculated as 6 ± 5 µM (n = 4). C, Inhibition of 125I-FYQLALT binding to BiP by LEN ({blacksquare}) or SMA ({diamondsuit}) at 25°C, or Len at 37°C ({square}), as compared with the inhibition by unlabeled FYQLALT (•). D, Gel electrophoresis analysis of the BiP-bound VL. FITC-REC was incubated for 1 h at 25°C with His6-tagged Kar2 immobilized on Ni2+-agarose beads in 96-well plates. After washing, the bound material was eluted with nonreducing SDS sample buffer and resolved on a 16% acrylamide, nonreducing Tricine-SDS gel. Lane 1, Migration of the input VL; lane 2, mock reaction containing VL and beads without Kar2; lane 3, migration of the Kar2-bound VL in native conditions; lane 4, migration of the Kar2-bound VL with Gdn-HCl denaturation. E, Time course of VL refolding, as measured by intrinsic tryptophan fluorescence. Spectra of native LEN (dashed line), LEN denatured with 2 M Gdn-HCl ({square}), or reduced with 40 mM DTT and denatured with 2 M Gdn-HCl ({circ}) were obtained, and then each sample was diluted into the BiP-binding buffer used in A (but without detergent). Emission spectra from 300–400 nm were recorded upon excitation at 280 nm at the indicated times after dilution, and the peak fluorescence was plotted as a percentage of the initial Trp fluorescence of each sample. F, Changes in VL structure upon reduction and denaturation, as measured by ANS binding. A total of 10 µM ANS was added to 3 µM of either folded LEN (dashed line) or unfolded LEN (dotted line) in 20 mM sodium phosphate buffer, pH 7, and emission spectra from 420–600 nm were recorded upon excitation at 350 nm. The spectrum of ANS alone in buffer has been subtracted from each of these spectra.

 
Binding of VL domains of LC to BiP

The LEN {kappa}-chain is a highly soluble Bence Jones protein isolated from a patient who excreted it at a rate of 50 g/day (31). In contrast, the SMA and REC {kappa}-chains were derived from patients diagnosed with light chain amyloidosis. Recombinant LEN and SMA, in the form of VL polypeptides produced as periplasmic proteins in bacteria, were shown to behave like the respective whole {kappa}-chains: LEN is stable and manifests the same monomer-dimer equilibrium typical of purified L chains, while SMA and REC are less stable proteins (31). Recombinant LEN and SMA (as well as REC, not shown) were tested for their ability to bind rBiP in a cold inhibition assay and in a direct binding assay (Fig. 2Go, A and B). Each protein bound best if it was first deliberately denatured (Fig. 2Go, compare A with C). Either treatment with Gdn-HCl or controlled reduction and subsequent alkylation gave rise to populations of unfolded VL that bound BiP. The highest level of binding was observed when LEN, SMA, or REC was both denatured and reduced. That such treatments indeed caused denaturation of VL was shown by increases in the intrinsic Trp fluorescence and binding of the hydrophobic dye ANS (Fig. 2Go, E and F). On the other hand, the structure of BiP itself was not affected at the concentrations of denaturant present in the binding assay (<0.4 M), as judged by the same two criteria (data not shown). Direct binding of 125I-labeled, denatured, and reduced VL was saturable, with 88 ± 24% of BiP bound at saturation (Fig. 2GoB, n = 4). The calculated Kd for LEN, SMA, and REC was similar, 3–11 µM (Fig. 2Go, A and B, and Table IGo). The nature of the label had no significant effect, as similar results were observed with either iodinated or FITC-conjugated VL (data not shown) and the binding of either form of labeled VL was inhibited by unlabeled, denatured VL.


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  		Table I. Binding of VL peptides to BiP1

 
No binding of VL to BiP was detected at 25°C, as demonstrated by the inability to compete for BiP binding with 125I-FYQLALT (Fig. 2GoC). Interestingly, marginal binding of all three VL could be consistently detected in the absence of denaturant when the incubation was performed at 37°C, resulting in an apparent Kd of 300 µM at best (Fig. 2GoC). Analysis of VL eluted from BiP showed that the VL preparations contained a subpopulation that was qualitatively different from the majority of molecules: the BiP-bound VL exhibited a slower mobility than the input material in Tricine-SDS nonreducing gels. A representative experiment is shown in Fig. 2GoD, with either native or Gdn-HCl-denatured REC that was incubated at 37°C with BiP. The mobility of the resulting BiP-bound fractions was the same, i.e., slower than the input REC. Similar results were observed with LEN and SMA (data not shown). We surmise, therefore, that a small fraction of the VL population, as isolated from the bacterial periplasm, is nonnative and leads to the low BiP-binding activity of folded VL. It is likely that this fraction of the protein is larger at 37°C and becomes even larger under unfolding condition. We estimate from the saturation-binding conditions that at least one-half of LEN or SMA can become unfolded enough to interact with BiP under the conditions used.

Kinetic analysis of intrinsic Trp fluorescence of the denatured and reduced LEN showed that it did not diminish substantially upon incubation in the binding buffer (Fig. 2GoE). In contrast, significant reduction in Trp fluorescence was observed upon dilution of Gdn-HCl-denatured LEN (with an intact disulfide bond) into the binding buffer (Fig. 2GoE). This suggests that the form of LEN used as a substrate in these assays did not refold significantly under the binding conditions, whereas in the case of denatured LEN, whose disulfide bond is oxidized, the fraction of the protein capable of BiP binding decreased with time, mimicking a folding reaction. Thus, just as seen in vivo, unfolded forms of VL have higher affinity for BiP than the more mature, folded species.

Identifying potential BiP binding sites in VL

To map BiP binding sites in VL, we searched the sequence for peptides conforming to published motifs that predict likely binder peptides. Based on the data of Blond-Elguindi et al. (10), derived from BiP panning of dodecameric and heptameric peptide libraries displayed by phages, a computer program was written to search any VL sequence. A negative score by this program indicates that the peptide has a low probability to bind BiP, while scores above +5 indicate a significant probability of binding BiP (10). Knarr et al. (28) used a similar approach to test predicted BiP-binding peptides in an Ig heavy chain. Our analysis of the LEN sequence, which is a germline {kappa}IV with one somatic mutation, shows multiple heptameric peptides with the requisite score (Fig. 3GoA).



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  		FIGURE 3. Prediction of BiP binding sites within VL. A, A sequence search of the VL LEN, performed with a computer algorithm based on the data of Blond-Elguindi et al. (10 ). The scores obtained for each peptide using a moving heptamer window are plotted as a function of sequence position. BiP scores of +5 or higher have been predicted to possess significant probability of binding to BiP (28 ). Negative scores indicate that these peptides have little probability of binding to BiP. LEN represents the {kappa}IV family and differs from the germline gene in only one position (Ser for Asn (29 31 )). B, A similar sequence search of the variable domains of 122 human {kappa} light chains. The averages of scores obtained for all of these VL sequences using a moving heptamer window are plotted. The threshold score of +5, which was found to be a reasonable predictor in experimental tests (28 ), is marked with a dashed line. Three regions have significant positive scores (denoted I, II, and III) and span the sequences 31–38, 69–77, and 96–101, respectively, according to the Kabat nomenclature (40 ). C, Binding analysis of VL peptides. VL-derived peptides were assayed for their ability to compete with 125I-FYQLALT for binding to BiP. Increasing concentrations of the appropriate competitor were incubated with constant concentrations of 125I-FYQLALT (42 µM) and GST-BiP (20 µM) for 1 h at 25°C. After separating bound peptides from free, Kd were calculated from the IC50 values, as described in Materials and Methods.

 
More recently, a different motif for peptides that bind the bacterial homologue of BiP, dnaK, was defined via an extensive peptide scan (24). This prediction may be applicable to BiP, because all of the amino acids that contact the bound peptides in dnaK are conserved in BiP (15). The only close match within the LEN sequence to the latter motif was the peptide P(R,K)LLIY (residues 45–50, by the Kabat nomenclature (40)), which is consistently found in all L chain families.

In addition to analyzing individual VL sequences, we analyzed an entire collection of human {kappa} LC, comprising of 122 sequences (F. Stevens, unpublished). The rationale is that scores of individual BiP-binding peptides that are generated by somatic mutation would be dampened by averaging over the entire population, whereas BiP-binding sequences common to most LC would not, and their identification would be more reliable. Some of the results of this analysis are shown in Fig. 3GoB. Two major regions were found to contain peptides with scores above 5 in several registers, indicating significant probability to bind BiP. These regions span amino acids 31–41 (peptide I, Fig. 3GoB) and 69–77 (peptide II) in the framework regions of VL. Both of these regions are conserved in VL sequences. A third region was found in amino acids 102–110, but was not tested in this study because it is highly variable in LC sequences due to joining of various VL-J gene segments.

Comparing affinities of peptides for BiP binding

To test the above predictions, 10 different VL peptides were synthesized (Table IGo). Some peptides were derived from the regions predicted to bind BiP (Fig. 3Go), while others had a fairly low predicted binding probability. The low probability peptide 61–67 was chosen, because previous data from our lab showed that point mutations in this sequence actually enhanced LC binding to BiP in vivo (7, 8). All 10 peptides were tested for their ability to compete with the binding of the labeled peptide FYQLALT to BiP. Examples of the peptide competition assays are shown in Fig. 3GoC, and the experiments are summarized in Table IGo. The apparent dissociation constant (Kd) calculated for the binding of the denatured VL was 6 ± 5 µM, similar to the apparent Kd of the FYQLALT standard. Of the 10 VL peptides, 4 did not bind BiP, including both of the peptides from the predicted negative region 59–67 (DRFSGSG and RFSGSGS). Two heptamers from region I in Fig. 3GoB (NTLAWYQ and TLAWYQQ) bound BiP, while two others (WYQQKPG and YQQKPGQ) did not bind, thus mapping the binding to residues 31–38. Substituting Thr32 with Tyr, the most common residue at this position, did not reduce the binding affinity measurably (data not shown). We also tested BiP binding of an 11-mer peptide 31–41, which contains both binding and nonbinding heptamers, and found its Kd to be 33 ± 13 µM, essentially the same as the Kd of the 31–37 heptamer (Table IGo). In similar fashion, the two predicted binder peptides from the second region, 69–77 (TDFTLTI and FTLTISS), were found to bind BiP experimentally (Table IGo and Fig. 3Go). Apparent Kd were calculated for all the binder peptides from data such as shown in Fig. 3GoC and ranged from 41–220 µM (Table IGo).

Two other heptamers have a lower affinity to BiP. The more N-terminal is peptide 11–17, which in the folded structure forms a tight turn between ß strands A and B (Fig. 4Go). Its apparent Kd (Table IGo) was significantly lower than either peptide regions I or II, in agreement with its barely positive predictive score (Table IGo). The fourth potential binding site is the 45–52 peptide, predicted by Bukau’s algorithm (24) as well as Blond-Elguindi’s algorithm (10). The consensus sequence at this site, PKLLIYAA, could not be tested reliably due to its insolubility. Therefore, we tested PKLLIYWA, which is more soluble and is the exact sequence found in the LEN protein. Its Kd was similar to peptide 11–17, placing these two peptides at the lower end of the affinity spectrum among all of the tested VL peptides.



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  		FIGURE 4. BiP binding sites within VL. A schematic representation of LC folding, adapted from (23 ), with the identified BiP-binding peptides highlighted. The ß sheets in both VL and CL domains are shown in gray, and several key amino acids in VL are indicated by their residue number, for orientation. The disulfide bonds within each domain are shown in black. The two highest affinity BiP-binding peptides are shaded black, and the two intermediate affinity sites are shaded dark gray.

 
The two best BiP binders of all the heptamers tested, peptide 31–37 and peptide 71–77, each form an extended ß strand. Peptide 31–37 is in strand C, spanning the diameter of the VL domain (Fig. 4Go). It is buried in the core of the native protein and includes Trp35, which packs against the internal disulfide bond (Fig. 4Go, top). The other BiP binding site, peptide 71–77, forms part of ß strand E (Fig. 4Go, bottom). Thus, each ß sheet of the variable domain contains one high affinity BiP binding site.

The disparity between the apparent affinity of each peptide and the affinity of the entire protein for BiP could be due to several factors. The heptamers in solution may only contain a fraction of the total population that is in a binding-competent conformation, whereas when they are constrained in the polypeptide they may all be in a similar conformation. The observation that the 11-mer 31–41 bound slightly better than the heptamer 31–37 and considerably better than the 32–38 heptamer (Table IGo) is consistent with this idea. Alternatively, it is possible that each VL is bound at more than one site and that binding of one BiP molecule to one site increases the likelihood of another BiP binding to the second site. Experiments designed to detect such possible cooperativity are in progress.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This work describes the in vitro reconstitution of interactions between Ig L chain and one of its known in vivo chaperones, BiP. This reconstitution mimics the in vivo binding specificity of BiP for the immature forms of L chain and the sensitivity of binding to the nucleotide state of BiP. The reconstituted binding was then used to map binding sites within the LC, leading to the conclusion that as few as two peptides are sufficient for this interaction.

One mechanism that is often envisioned for BiP binding to newly synthesized LC is attachment of multiple BiP molecules to appropriate peptide sites displayed along the length of the unfolded polypeptide. Data for the related hsp70 family protein dnaK show that in various random proteins, sites are found on average every 36 amino acids (24). If these data are extrapolated to BiP, there can be six binding sites per LC. However, this number is almost certainly an underestimate, because unlike the collection of substrate proteins used to generate the data for dnaK, LC consists entirely of ß sheets, with no {alpha} helices, and BiP binds to peptides in the extended ß conformation (26). Attachment to multiple sites along the polypeptide is also inferred from the mode of action of BiP or mitochondrial hsp70 in the translocation of proteins across membranes: the power stroke of hsp70 is proposed to mediate vectorial transport across the membrane, either as a ratchet mechanism or as a mechanochemical motor (41, 42, 43).

An alternative mechanism for the action of BiP in protein folding is attachment to key peptides that must not be left exposed lest folding take an unproductive pathway. According to this view, there are a few dominant binding sites among the many potential sites. Previous work from this lab and others (44, 45, 46)6 pointed to the VL domain as the major determinant in the association of LC with BiP during folding in the cell. In this study, we present direct evidence that the VL domain indeed binds to BiP in vitro, and we map two likely dominant BiP binding sites.

The data presented in this study demonstrate that two VL proteins, LEN and SMA, bind BiP in vitro with similar affinities. Furthermore, their binding is a property of partially unfolded intermediates and not of the native VL, as shown by the vastly increased affinities after denaturation of the recombinant proteins. This feature recapitulates the in vivo behavior of newly synthesized LC that only associate with BiP as long as they have not completed their disulfide bonding (3, 44). LC binding to BiP in vitro is saturable, sensitive to ATP as expected from BiP’s mode of action, and is inhibited by heptameric peptides that themselves bind to BiP.

Our in vitro studies identified four BiP-binding peptides in the sequences of LEN and SMA, out of the collection of predicted peptides. Of these four peptides, two bind BiP with affinities that are reasonably close to the affinity of the entire VL protein. These two peptides are in ß strands that are buried in the hydrophobic core of the folded VL and are well conserved among LC sequences. One site is in strand E of the four-stranded ß sheet, and a second site is in strand C of the five-stranded ß sheet (Fig. 4Go). We propose that binding of BiP to these two peptides is sufficient to account for binding of the entire protein. Binding of BiP to one or both of these dominant sites could maintain the two halves of the ß sandwich in an open conformation, with Cys23 and Cys88 in a reduced state. Three lines of evidence are consistent with this interpretation. First, maximal binding to BiP occurred when VL was not only denatured, but also reduced. Second, coimmunoprecipitation data from metabolically labeled cells enabled an estimation that 2–4 mol of BiP were bound per mole of LC (3). Third, such binding accounts for the in vivo observation that prolonged association with BiP retards the oxidation of the VL domain (4).

The binding to peptide 11–17 is intriguing: in the native state, this peptide forms a tight turn between strands A and B. This turn is a conserved feature of the Ig fold, and mutations disrupting this turn are associated with various aggregation-prone LC (Ref. 46 and footnote 6). If the binding of this peptide to BiP persists, the formation of the four-stranded ß sheet is expected to be delayed, leaving the VL domain in a very unfolded state. More likely, given the low affinity of this site, it may be the first of the peptides to fold, enabling the folding of the two ß sheets, while BiP binding to the other two sites delays the formation of the disulfide bond between Cys23 and Cys88. Like the 11–17 peptide, the 45–50 peptide, whose binding affinity in vitro is intermediate, is largely solvent exposed even in the native structure. In most {kappa} families, this peptide is P(K,R)LLIY, a sequence that is predicted (24) to bind BiP even less avidly than the PKLLIKY sequence found in the LEN and SMA proteins. We therefore propose that the 11–17 and 45–50 peptides are not the important BiP binding sites in vivo.

The presence of two dominant BiP binding sites in the VL domain does not rule out the possibility of other BiP binding sites in the VL domain or in the CL domain. We predict, however, that because CL folds and oxidizes even in the presence of a nonreleasing BiP (4), only low affinity BiP-binding peptides exist in this domain. The data presented in this study suggest that binding of BiP to a newly synthesized polypeptide in the cell should not necessarily be viewed as a process of coating a string of amino acids with multiple BiP molecules. Such binding to multiple low affinity peptides may mediate nascent chain translocation across the ER membrane, one of the physiological functions of BiP (47). This role is analogous to that of mitochondrial hsp70 in translocation of proteins across the organelle (42, 48). Instead, we suggest that once the proteins are in the lumen of the ER, association of BiP during folding can be explained by selective binding to higher affinity sites identified in this work.

How much of the LC in the ER is bound to BiP? The concentration of the incompletely folded intermediates of LC within the ER is 0.36–0.9 µM (49, 50). The total BiP concentration is estimated at 100 µM (49), and it is possible that only a fraction of the total pool is available to bind LC. Together with the Kd values derived in this study, it can be calculated that 32–50% of the LC intermediates should be bound to BiP when ATP is present, and 48–92% when ATP is depleted. These calculations are consistent with our data showing that only ~1/3 of the total LC can be immunoprecipitated with BiP (51), particularly considering that some substrate is likely to dissociate during the immuno-isolation procedure. The fraction of BiP-bound LC is also consistent with the kinetics of LC folding: given that LC bound to BiP cannot complete its folding, between 10% and 14% of the F1 intermediate (the one disulfide intermediate) (4) must be free at any given time to account for the rate constant of its conversion to the fully oxidized form (derived from the t1/2 of the F1 intermediate and assuming first order reaction).

When the human germline V{kappa} segments are examined, the sequences of the two peptides with the highest affinity for BiP are highly conserved. The hydrophobic residues in the 71–77 sequence are invariant, and the variability is limited to a Lys-for-Thr74 substitution in the {kappa}II family, Arg-for-Ser77 in the {kappa}II and two {kappa}III genes, and a few other genes with Asn instead of Ser76 or Ser77. In the 31–37 sequence, Trp35 is invariant, as is the tetrapeptide WYQQ, except for the {kappa}II family again, displaying a few conservative substitutions (Phe or Leu for Tyr36; Leu for Gln37). Position 34 is almost always occupied by a residue with a small side chain (Ala, Gly, or Ser) and occasionally Asn or Asp, while position 31 is always either Ser or Asn. This sequence conservation of the BiP-binding peptides is not due to protection from the somatic mutation mechanism: a number of mutations within the sequences have been identified in LC transcripts from Peyer’s patch B cells, showing that mutations in these residues do occur (52) (C. Milstein, personal communication). Therefore, the conservation is due to selection at the level of the expressed protein, presumably because these peptides are important in the proper folding of the domain.

BiP binding to framework peptides is most likely an important, yet little appreciated, selective force that participates in shaping the expressed repertoire of Ig V genes. Proper folding of variable domains depends heavily on an extensive network of hydrogen bonding (53), so many somatic mutations disrupt entire constellations of amino acids. Even mutations in the hypervariable loops are known to have global destabilizing effects on the whole domain (54). Therefore, mutations in many positions could lead to persistent exposure of the conserved BiP-binding peptides, causing prolonged BiP-LC interactions. In addition, somatic mutations may also create new, high affinity, BiP-binding peptides causing prolonged interactions. If the overall avidity for BiP is not too high, the mutant LC may actually benefit from the shielding from water and other proteins provided by BiP, manage to fold, and be included in the expressed repertoire. If, on the other hand, the mutation is catastrophic in terms of proper folding, the persistent association with BiP could delay folding, inhibit Ig assembly, and ultimately target somatic mutants for degradation. Finally, occupancy of BiP by mutant proteins is most likely used in an ER-to-nucleus signal transduction pathway, termed the unfolded protein response, and characterized in yeast (55). Thus, BiP could be a sensor that monitors V region diversity and reports back to the genetic apparatus whether to undergo further gene rearrangements.

In conclusion, our reconstitution of BiP-substrate interactions strongly supports the hypothesis that during folding in vivo BiP binds to only few higher affinity sites per substrate that are located in important elements within the folding module. By binding to them, BiP retards the folding, thus helping to ensure progress along the productive pathway. This binding mechanism is a sophisticated tool that may be used by B lineage cells to monitor the outcome of gene rearrangements and somatic mutations during the diversification of the Ig repertoire.


   Acknowledgments
 
We thank Helene Auer for synthesizing most of the peptides used in this work, and the BSD Protein-Peptide Core Facility for synthesis of the other peptides. N. Christoforou diligently purified the REC and SMA proteins. Drs. J. Brodsky, R. Morimoto, S. Blond, and H. Weissbach generously provided constructs and purified hsp proteins, and C. Milstein kindly shared unpublished Ig sequences from mouse Peyer’s patch B cells. We thank Drs. T. Pan and T. Sosnick for the use of the fluorescence spectrophotometer, and all members of our group for vigorous discussions and comments on this work.


   Footnotes
 
1 D.P.D. was supported by the Baron Fellowship of the Unicorn Foundation. This work was supported in part by the U.S. Department of Energy, Office of Health and Environmental Research, under Contract W-31-109-ENG-38 (F.J.S.), and by U.S. Public Health Service Grants DK43757 (F.J.S.) and AI30178 (Y.A.). Back

2 D.P.D. and R.K. contributed equally to this paper. Back

3 Current address: Department of Chemistry and Biochemistry, University of California, 1156 High Street, Santa Cruz, CA 95064. Back

4 Address correspondence and reprint requests to Dr. Yair Argon, Department of Pathology, University of Chicago, 5841 South Maryland Avenue, MC 1089, Chicago, IL 60637. E-mail address: Back

5 Abbreviations used in this paper: BiP, binding protein; ANS, 1,1'-bi(4-anilino)nahpthalene-5,5'-disulfonic acid; ER, endoplasmic reticulum; Gdn-HCl, guanidine hydrochloride; 125I-FYQLALT, 125I-labeled FYQLALT; LC, light chain; SFX, succinimidyl ester. Back

Received for publication April 26, 1999. Accepted for publication July 26, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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