©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Hydrophobic Nature of GroEL-Substrate Binding (*)

(Received for publication, October 31, 1994; and in revised form, November 17, 1994)

Zhanglin Lin (1) Frederick P. Schwarz (1) (2) Edward Eisenstein (1) (3)(§)

From the  (1)Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, and the (2)National Institutes of Standards and Technology, Rockville, Maryland 20850, and the (3)Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21228

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The molecular chaperone GroEL from Escherichia coli is a member of the highly conserved Hsp60 family of proteins that facilitates protein folding. A central question regarding the mechanism of GroEL-assisted refolding of proteins concerns its broad substrate specificity. The nature of GroEL-polypeptide chain interaction was investigated by isothermal titration calorimetry using proteins that maintain a non-native conformation in neutral buffer solutions. A single molecule of an unfolded variant of subtilisin BPN` binds non-cooperatively to GroEL with micromolar affinity and a positive enthalpy change. Additional calorimetric titrations of this chain with GroEL show that the positive enthalpy change decreases with increasing temperature between 6 and 25 °C, yielding a DeltaC(p) of -0.85 kcal mol degree. alpha-Casein similarly binds to GroEL with micromolar affinity and a positive enthalpy change in the range of 15-23 °C, yielding a DeltaC(p) of -0.44 kcal mol degree. The negative heat capacity change provides strong evidence for the role of hydrophobic interactions as the driving force for the association of these substrates with the GroEL chaperonin.


INTRODUCTION

Escherichia coli GroEL is a member of the highly conserved Hsp60 family of molecular chaperones which facilitate protein folding in vivo and in vitro(1) . A key issue in the mechanism by which chaperonins facilitate protein folding is the nature of their recognition of non-native structures. Although in vivo studies have identified a potentially wide range of protein substrates that bind to GroEL(2, 3) , and numerous in vitro studies have demonstrated that many chemically unfolded proteins bind to GroEL(4, 5) , the nature of this interaction remains unclear(6, 7) . Since unfolded proteins possess a high degree of exposed hydrophobic surface, it has been suggested that the association of nonnative chains with GroEL is hydrophobically driven(8, 9, 10, 11, 12, 13, 14) , but definitive experimental evidence in support of this view is lacking. In this study, an unfolded mutant of subtilisin BPN` and alpha-casein were employed in calorimetric experiments to measure directly the energetics of substrate binding to GroEL. These results demonstrate unequivocally that the association of substrates with GroEL is determined by hydrophobic interactions.


EXPERIMENTAL PROCEDURES

Materials

Native subtilisin BPN` from Bacillus amyloliquefaciens (EC 3.4.21.4), a serine protease that belongs to a class of proteins whose folding requires a prosequence to overcome kinetic barriers in the folding pathway(15) , and subtilisin BPN` PJ9, a variant that cannot be refolded in the absence of its propeptide, were cloned, expressed, and purified as described previously(15, 16) . Subtilisin BPN` PJ9, M(r) 27,700, contains the following mutations with respect to native BPN`: D32N, M50F, Y217K, N218S, S221A, and the deletion of residues 75-83 that correspond to the calcium binding site(^1)(15) . groEL was cloned in pKK233-2 (Pharmacia Biotech Inc.) and expressed in E. coli strain JV30 by isopropyl-1-thio-beta-D-galactopyranoside induction of the trc promoter; GroEL was purified to homogeneity using a modification of published procedures(^2)(17) . alpha-Casein, reduced carboxymethylated lactalbumin (RCM-lactalbumin), (^3)and melittin were from Sigma and were used without further purification. All other chemicals were of reagent-grade purity.

Circular Dichroism

Circular dichroism spectra were recorded at room temperature with a Jasco J720 spetropolarimeter using a 10-mm path length cell for far-UV CD spectra and a 1-cm path length cell for the near-UV CD spectra. The protein concentration of native BPN` (in buffer A, containing 50 mM Tris, 100 mM KCl, pH 7.8) for far-UV CD and near-UV measurements were 1.6 and 0.21 mg ml, respectively, and 2.2 and 0.27 mg ml for BPN` PJ9 (in buffer B, containing 50 mM Tris, 100 mM KCl, 1 mM dithiothreitol, 0.1 mM EDTA, pH 7.8). The spectra are averages of 5-7 scans and were corrected for the appropriate buffer base-line values.

Fluorescence

The fluorescence spectra were obtained using a FluoroMax 2000 spectrofluorimeter (SPEX), using an excitation wavelength of 394 nm. The experiments were performed in buffer A for native BPN` and in buffer B (in the absence or presence of 8 M urea) for BPN` PJ9 at room temperature. Reaction mixtures were prepared by mixing 100 µl of 5 µM protein and various amounts of a 47.5 µM bisANS solution to a final volume of 400 µl, which were then incubated for 10 min before acquiring spectra.

Titration Calorimetry

Calorimetric measurements were performed with an Omega titration calorimeter (MicroCal)(18) . In these experiments, the reference cell contained buffer B, the sample cell contained GroEL, and the injection syringe contained non-native polypeptide chains at approximately 150 µM. For experiments with subtilisin BPN` PJ9 conducted at 6.6 and 14.3 °C, a 250-µl syringe was used to inject 16 aliquots of 15 µl of ligand (150 µM) at 4-min intervals into a solution of 12.0 µM GroEL (oligomer M(r) = 805,000), whereas a 100-µl syringe was used to inject 12 aliquots of 8 µl of ligand (150 µM) spaced at 5-min intervals into a 6.4 µM solution of GroEL for the experiment at 24.4 °C. The data presented correspond to experimental traces of heat absorbed versus time (injection number). Experiments using alpha-casein (monomer M(r) 23,600), RCM-lactalbumin (M(r) = 14,200) and melittin (monomer M(r) = 2,840) were performed under similar conditions. Buffer composition (potassium phosphate or Tris-Cl) had no significant effect on observed heat absorption (data not shown). Prior to analysis the data were corrected for the heat of dilution of protein ligand solutions by performing separate titrations against buffer. The resulting, corrected data were evaluated to determine the binding stoichiometry, association constant, and enthalpy change by nonlinear least-squares analysis in terms of the following equation

where Q is the amount of heat exchanged, X is the ligand concentration (in this case, unfolded polypeptides), V is the volume of the cell (1.38 ml), P is the GroEL concentration, 1/r = [X]K(a) and X(r) = [P]/[X](18) . The temperature dependence of DeltaH was analyzed to

where DeltaH is the observed enthalpy change for the association of unfolded chains with GroEL at various temperatures, and DeltaH^0 is the enthalpy change at a reference temperature, T(o). The temperature dependence of the association of BPN` PJ9 with GroEL was estimated from

where DeltaH^0 and K at a reference (14.3 °C) temperature, and the determined value of DeltaC(p), -0.85 kcal mol deg, were used(19) .


RESULTS AND DISCUSSION

Since a quantitative study of the interaction of molecular chaperones with nonnative polypeptides is potentially complicated by the tendency of these chains to spontaneously refold or aggregate in the absence of denaturants, an unfolded variant of subtilisin BPN` was used to measure the energetics of substrate interaction with the GroEL chaperonin from E. coli. Subtilisin BPN` PJ9 is unable to adopt a native-like, folded conformation and remains unfolded in non-denaturing aqueous solution. The nonnative character of subtilisin BPN` PJ9 was established by circular dichroism (CD) and bisANS binding. As can be seen in Fig. 1, the far-UV CD spectrum of subtilisin BPN` PJ9 indicates that the protein is much less structured relative to native BPN`, and the near-UV spectrum shows little indication of native-like tertiary structure(20, 21) . On the other hand, subtilisin BPN` PJ9 is not in a completely random conformation. Residual secondary structure is also evident from the CD spectrum presented in Fig. 1A, which differs from that for subtilisin BPN` PJ9 in the presence of 8 M urea. Additionally, as can be seen in Fig. 2, neither fully folded native BPN` nor the BPN` PJ9 variant that was completely unfolded in the presence of 8 M urea showed measurable affinity for bisANS, which is a probe for solvent-accessible hydrophobic areas. In contrast, about 1 mol of bisANS/mol of BPN` PJ9 is bound with a K(d) 5 µM, indicating the existence of exposed hydrophobic clusters(22) . Subtilisin BPN` PJ9 therefore provides a useful, stable model of an ``unfolded protein substrate'' for GroEL under neutral buffer conditions(23, 24) .


Figure 1: Circular dichroism spectra of native BPN` and subtilisin BPN` PJ9. Molar ellipticity versus wavelength was measured as described under ``Experimental Procedures.'' A, in the far-UV region, the CD spectrum of native subtilisin BPN` (thickline) yields a maximum at 192 nm and minima at about 220 and 210 nm, indicative of the high content of alpha-helical structure in the folded conformation. The CD spectrum of subtilisin BPN` PJ9 (thinline) shows a minimum at about 202 nm, indicating a loss of helical content. The dashedline refers to the spectrum of subtilisin BPN` PJ9 in the presence of 8 M urea. B, in the near-UV region, native BPN` shows a minimum at 278 nm, reflecting native tertiary structure, whereas the region is featureless for BPN` PJ9 in the absence or presence of 8 M urea.




Figure 2: Effect of native and non-native subtilisin on the fluorescence of bisANS. The relative fluorescence emission at 484 nm of bisANS in the presence of various forms of subtilisin BPN` was measured upon excitation at 394 nm. Native subtilisin BPN` in buffer A (squares), and subtilisin BPN` PJ9 in buffer B in the presence of 8 M urea (triangles) show no measurable affinity for bisANS by the lack of fluorescence. Subtilisin BPN` PJ9 in buffer B (circles) shows significant affinity for about 1 mol of bisANS, with the theoretical curve corresponding to simple binding with a K of about 5 µM.



The titration calorimetry results in Fig. 3show that the association of BPN` PJ9 to GroEL occurs with an uptake of heat, and an analysis of the data seen in Fig. 3B yielded a positive enthalpy change of DeltaH= +19.9 kcal/mol, an equilibrium association constant of 5.91 times 10^5M at 14.3 °C, and a stoichiometry of 1.10 polypeptides bound/GroEL. Thus, the binding of this unfolded chain to GroEL is entropy-driven, which suggests a possible role for hydrophobic interactions(25, 26, 27) . The role of hydrophobic interactions was confirmed by performing additional binding experiments as a function of temperature. As can be seen in Fig. 4A, the DeltaH for subtilisin binding to GroEL decreased with increasing temperature. These data were analyzed to to yield a negative value for DeltaC(p) of -0.85 kcal mol deg, which is the hallmark of the hydrophobic interaction(28, 29, 30) .


Figure 3: Exchange of heat on binding of subtilisin BPN` PJ9 to GroEL. A, uptake of heat upon the addition of 15-µl aliquots of a 150 µM subtilisin BPN` PJ9 solution at 4-min intervals to a 12 µM solution of GroEL in buffer A at 14.3 °C. The data correspond to heat absorbed versus time (injection number). B, integrated data after correction for heat of ligand dilution determined in separate experiments, presented as heat exchange versus the subtilisin BPN` PJ9 to GroEL ratio (total concentrations). These data were analyzed as described under ``Experimental Procedures,'' with the theoretical curve corresponding to the parameter values of K = 5.91 times 10^5M, DeltaH = +19.9 kcal/mol, and a stoichiometry of 1.1 polypeptides bound/GroEL.




Figure 4: Energetics of subtilisin BPN` PJ9-GroEL interaction. A, temperature dependence of the enthalpy change for BPN` PJ9 binding to GroEL. The linear dependence of enthalpy versus temperature yields a heat capacity change, DeltaC(p), of -0.85 kcal mol deg. B, a stability curve for the association of BPN` PJ9 with GroEL was determined using as described under ``Experimental Procedures.''



The micromolar binding constants determined calorimetrically for subtilisin BPN` JP9 differ significantly from the picomolar constants estimated for the affinity of chemically unfolded ribulose-1,5-bisphosphate carboxylase with GroEL by its rate of exchange with free chains(4) . This difference may be due to the nature of the unfolded form of the species used in these studies since BPN` PJ9, although largely unfolded under non-denaturing buffer conditions, contains residual structure.

Both the solubility of BPN` PJ9 and instrumental sensitivity limited the binding measurements to the temperature range of 5-25 °C. The stability curve presented in Fig. 4B was constructed using the binding constants determined at three temperatures along with the heat capacity change, DeltaC(p)(19) . This analysis indicates that the maximum association of BPN` PJ9 with GroEL occurs at about 37 °C where, since the enthalpy change is approximately zero, the association of BPN` PJ9 with GroEL is entirely entropically driven. The dissociation constant at this temperature was estimated to be approximately 0.4 µM. The temperature-dependent fashion of GroEL binding suggested by the stability curve provides a possible explanation for the reduction in affinity GroEL shows for various unfolded substrates at lower temperatures(11, 31) . Furthermore, a single chain of subtilisin BPN` PJ9 was bound to GroEL throughout the temperature range investigated, with no evidence for cooperative polypeptide binding to the chaperonin(32) .

It is of interest to note that negligible heat exchange was seen for the association of GroEL with GroES, a cofactor in many refolding reactions(33, 34) . However, similar results to subtilisin BPN` PJ9 were seen for the association of GroEL with alpha-casein, a disordered protein that has been shown to compete with unfolded substrates for binding to GroEL(33, 35) . Titration calorimetry experiments were performed using alpha-casein under similar solution conditions at 15.0 and 22.5 °C. Analysis of the binding isotherms yielded enthalpies of +9.37 and +6.08 kcal/mol, binding constants of 6.2 times 10^5M and 1.9 times 10^6M, respectively, providing an estimate for DeltaC(p) of -0.44 kcal mol deg. Thus it appears that the association of alpha-casein with GroEL is also driven by hydrophobic interactions. However, a more detailed analysis of the calorimetric data for casein was complicated by the unusual binding stoichiometry of approximately 0.5 casein molecule/GroEL oligomer. Additional calorimetric experiments were also performed using RCM-lactalbumin and melittin. No detectable enthalpy change was seen for RCM-lactalbumin addition to GroEL. This indicates RCM-lactalbumin has negligible affinity for GroEL, in agreement with recent results, which indicate that only specific conformers of lactalbumin are able to interact with this chaperonin (14) . On the other hand, melittin, a small, hydrophobic protein, showed a small positive enthalpy change on addition to GroEL, but its association was too weak (K(d) mM) for a quantitative analysis.

The substrate promiscuity of GroEL is an intriguing matter(6) . Previous NMR studies have identified two peptides, STKWLAESVRAGK and KLIGVLSSLFRDK, that bind to GroEL in an alpha-helical conformation, possibly through an apolar face(8, 36) . However, GroEL can facilitate the refolding of an F fragment of an antibody, which contains no alpha-helical structure(9) , and it has been shown recently that the secondary structure of cyclophilin is completely eliminated upon binding to GroEL(11) . Thus, rather than a cognate structural element responsible for GroEL binding(8, 9, 10, 11) , it seems more probable that GroEL recognizes unfolded proteins through their common characteristic of exposed hydrophobic surface, which is not generally present in their folded conformations, and binds these segments through hydrophobic interactions. Not only have hydrophobic amino acids been shown to stimulate preferentially the ATPase activity of GroEL(37) , a detailed mutational study of GroEL also maps a number of polypeptide binding deficient mutants to hydrophobic residues within the central cavity of the two-stacked, seven-membered ringed structure(38, 39) . Independent of whether the association of polypeptides with GroEL are mediated through particular structural elements, however, the energetics of subtilisin BPN` PJ9 and casein binding to GroEL strongly indicate that the major determinant of substrate association with GroEL is governed by the ability to form hydrophobic interactions. The heat capacity changes seen for the interaction of GroEL with subtilisin BPN` PJ9 and casein suggest that a relatively large hydrophobic accessible surface area is buried upon association with the chaperonin. If the correlation between DeltaC(p) and the water-accessible nonpolar surface area is adopted(29) , then roughly 3,040 Å^2 would be buried when subtilisin BPN` JP9 binds to GroEL, whereas 1,570 Å^2 should be buried upon casein binding. The difference in accessible surface buried may be explained by a collapse of BPN` PJ9 when bound to GroEL relative to casein, which is less flexible than unfolded proteins(40) .


FOOTNOTES

*
This research was supported by National Institutes of Health Grant GM49316. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence may be addressed: CARB, 9600 Gudelsky Dr., Rockville, MD 20850. Tel.: 301-738-6244; Fax: 301-738-6255.

(^1)
P. N. Bryan, unpublished observations.

(^2)
Z. White, K. E. Fisher, and E. Eisenstein, manuscript in preparation.

(^3)
The abbreviations used are: RCM-lactalbumin, reduced carboxymethylated lactalbumin; bisANS, 4,4`-bis-1-phenylamino-8-naphthalenesulfonate; deg, degrees.


ACKNOWLEDGEMENTS

We are indebted to P. Bryan for the gift of subtilisin BPN` PJ9 and for helpful discussions.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.