Interaction of ATP and lens alpha crystallin characterized by equilibrium binding studies and intrinsic tryptophan fluorescence spectroscopy

et Biophysics &ta

ELSEVIER

Biochimica

et Biophysics

Acta 1246 (1995) 91-97

Interaction of ATP and lens alpha crystallin characterized by equilibrium binding studies and intrinsic tryptophan fluorescence spectroscopy * David V. Palmisano

a, Barbara Groth-Vasselli b, Patricia N. Farnsworth Mayani C. Reddy ’

aybp *,

aDepartment ofPhysiology,UMD-New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103, USA b Department of Ophthulmology, UMD-New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103, USA ’ Department of Surgery; UMD-New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103 USA Received 12 June 1994; accepted 28 August 1994

Abstract a-Crystallin, the most prevalent protein in vertebrate lenses, is a high molecular weight aggregate composed of cuA and (YB subunits. Evidence is presented that ATP, a major phosphorus metabolite of the lens binds to a-crystallin extracted from calf lenses. The following parameters were obtained from equilibrium binding studies conducted at 37°C: binding sites per 400 kDa aggregate = 10 and K, = 8.1 . lo3 M-‘; and an essentially identical K, of 7.84. lo3 M-’ and 22 binding sites were determined for a 850 kDa aggregate. The cooperativity parameter, ~~11,approximates unity which denotes that the binding of ligand is at independent sites. Binding was not significant at 22°C and was absent at 4°C. The specificity of the binding site for ATP was established by intrinsic tryptophan fluorescence spectroscopy. In the presence of increasing concentrations of ATP (0.05-0.3 mM), tryptophan fluorescence decreases in a concentration dependent manner to a minimum at 0.2 mM above which there is a non-linear response. Quenching of fluorescence was not evident with Pi, AMP or ADP. GTP elicited a minimal quenching of fluorescence only at the highest concentration (0.30 mM). Modulation of both supramolecular organization and lens metabolism is predicted as a consequence of ATP/a-crystallin binding. Keywords:

Lens; Protein-ligand

binding;

cu-Crystallin; ATP; Intrinsic tryptophan

1. Introduction The optical properties of the transparent eye lens are dependent upon a diverse group of cellular proteins called crystallins. a-Crystallin, the predominant protein in all vertebrate lenses, and the /Yy superfamily of crystallins account for 200-400 g N 1-l and provide the supramolecular order that maintains transparency. The supramolecular order of the outer, newly formed lens cortical fiber cells differs substantially from the older nuclear cells [l-4]. Therefore, fiber cells that differentiate, mature, and are

* This study was presented in palt at the Association for Research in Vision and Ophthalmology at Sarasota, FL, May, 1993 and at the International Cooperative Cataract Research Group meeting at Bethesda, MD, November, 1993. * Corresponding author. 0167-4838/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0167-4838(94)00176-6

fluorescence

spectroscopy

displaced inward throughout life require a continuing process of dynamic modeling and remodeling of their supramolecular organization. An important goal in our studies of lens structural chemistry is the identification of factors which normally stabilize and/or modulate the orientation and interaction of the crystallins. It is critical to distinguish normal physiological factors from those that induce abnormal aggregation which causes loss of transparency associated with the medical condition of cataract. Early observations on lens homogenates by Hammer and Benedek [51 showed that endogenous small phosphorous metabolites can significantly alter lens transparency by modifying protein-protein and protein-water interactions. This is of particular importance since a decrease in metabolism and consequent decrease in metabolite concentrations occurs between the outer cortical and inner nuclear regions [6-91. These data led to our investigation of metabolite/crystallin binding as a possible control mecha-

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nism for lens supramolecular order. Initially, indirect evidence for metabolite/crystallin binding in the lens was provided by 31P-NMR spectroscopy which revealed an increased rotational diffusion time for ATP and phosphomonoesters in lens homogenates [9]. Subsequently, the interaction of L-cr-glycerophosphate (a-GP), a major lens metabolite, and y-crystallin was characterized by radioactive equilibrium binding [lo], 31P- NMR spectroscopy [ 111, and computer-assisted molecular modeling [12]. CX-GPhydrogen bonds to y-crystallin with moderately high affinity and in a positive cooperative manner. The reversible nature of the interaction permits a rapid response to the decreased concentration of metabolites that occurs during lens fiber cell maturation. More recently, indirect evidence for ATP/cY-crystallin binding was determined by 31P-NMR spectroscopy [13]. In addition, this study revealed that the association of a-crystallin with purified fiber cell membranes is significantly enhanced by addition of ATP. In the present investigation, the binding of ATP to cy-crystallin was further characterized by equilibrium binding and intrinsic tryptophan fluorescence spectroscopy. The binding study revealed a temperature dependence, a K, of 8.1 * lo3 M-’ at 37°C and an estimated 10 binding sites per 400 kDa aggregate. An essentially identical K, of 7.84 * lo3 M-’ and 22 binding sites were determined for an 850 kDa aggregate. In addition, indirect evidence for the binding of ATP to cr-crystallin was provided by a decrease in intrinsic fluorescence of tryptophan residues in the presence of ATP. This denotes a change in the environment of one or more of these amino acids. Quenching of fluorescence was not evident in the presence of Pi, AMP or ADP while GTP produced a minimal effect only at an elevated concentration. The data from both fluorescence and equilibrium binding studies provide evidence for the specificity of the binding sites for ATP.

2. Material and methods

2.1. Protein preparation

and purification

a-Crystallin was prepared from the water soluble protein fraction of 6 to 8 week old calf lenses according to Bloemendal [ 141. Lenses were enucleated, decapsulated and homogenized in 50 mM Tris-HCI buffer containing 5 mM 2-mercaptoethanol, 100 mM KCI, 1 mM EDTA (pH 7.2) (Sigma, St. Louis, MO) and 0.02% sodium azide (Fisher, Fairlawn, NJ). The homogenate was centrifuged (DuPont Sorvall S uperspeed Refrigerated Centrifuge, Model RC 5, Wilmington, DE) at 4°C for 20 min at 17000 X g to remove insoluble albuminoid. The homogenate was chromatographed using a 2.5 cm X 200 cm glass column (Pharmacia, Sweden) packed with Sephadex G-200 superfine gel chromatography medium (Pharmacia, Sweden). The elution buffer was the Tris-HCI homogenization buffer, Following fractionation, the a-crystallin

peak was collected and concentrated with Centriconconcentrators (Amicon incorporated, Model 10, Beverly, MA) to a final volume of 5 ml and rechromatographed on a second 2.5 cm X 200 cm glass column containing Bio-Gel A-5M gel chromatography media (Bio-Rad, Richmond, CA). To ensure purity, the central 90% of the single a-crystallin peak was collected and concentrated with Centricon-10 concentrators to remove buffer, and frozen in liquid nitrogen prior to lyophilization (Labonco, Model 75200, Kansas City, MO). The purity and composition of the protein preparation was assessed by SDS-PAGE [15]. Protein concentrations were determined according to Bradford [16] by using bovine serum albumin (BSA) (Sigma, St. Louis, MO) as a standard. 2.2. Equilibrium binding study Equilibrium binding measurements of ATP to a-crystallin were performed following the procedure of Farnsworth et al. [lo] as adapted from Paulus [17]. The procedure was further modified by using microconcentrators (Amicon, Beverly, MA) with a molecular weight cutoff of 30 kDa to achieve partition between free and bound ligand. The Tris buffer used for lens homogenization and subsequent protein purification was also used in all procedures in the binding studies. Aliquots containing 2.5 nmol a-crystallin in buffer were introduced into the upper chamber of a prerinsed microconcentrator. 14C-ATP (specific activity of 51 mCi/mmol) was purchased from Amersham, Arlington Heights, IL). Approx. 3.0 nM 14C ATP was appropriately diluted with buffer and added to the chamber. Total ligand concentration was varied by adding up to 100 nM unlabelled ligand (Sigma, St. Louis, MO) followed by adjustment of the reaction mixture volume to 0.5 ml with buffer. To determine non-specific binding, an identical reaction mixture was prepared except that excess (2.5 mM) unlabelled ligand was added [181. The experimental sample was incubated for a 2 h period at 4, 22 or 37°C. Following incubation, the samples were centrifuged (Brinkmann Eppendorf Microcentrifnge, model 5414, Westbury, NJ) at 10000 X g for 30 min to achieve partition between free and bound ligand. The experimental temperature was maintained throughout all procedures. To ensure that no free ligand was retained on the disc, 10 ml of buffer was added and the contents were recentrifuged. The tubes were dismantled, the filtering discs containing protein-ligand complex removed, and placed in liquid scintillation vials containing 10 ml of Ecoscint A (National Diagnostics). The vials were counted using a Packard Tri-Carb 300 scintillation counter (Packard, model A-3000, Downers Grove, IL). The amount of bound ligand retained on each filter was calculated from the measured radioactivity after corrections for counter efficiency using standards of known 14C-ATP concentrations and for non-specific and background binding. Free ligand concentration was calculated from the total of the labeled and unlabeled ligand. The

D.V. Palmisano et al. /Biochimica

et Biophysics Acta 1246 (1995) 91-97

calculated values of free ligand concentration and fraction bound were submitted to Scatchard and Hill plot analyses to determine binding parameters. 2.3. Intrinsic tryptophan fluorescence spectroscopy Samples containing 0.1 rag/ml a-crystallin in the absence and presence of 0.05, 0.1, 0.2, and 0.3 mM ATP in buffer described above were incubated for 2 h periods at 37°C (pH 7.2). Samples were transferred to quartz cuvettes with a 3.0 cm path length. Spectra were recorded at 37°C using a SPEX FluoroMax spectrofluorometer (SPEX, Edison, NJ) equipped with a temperature controlled refrigerator-circulator (Model 900, Fisher, Fairlawn, NJ). Intrinsic tryptophan fluorescence was monitored from 310 to 380 nm (maximum emission band for tryptophan is 340 nm) with an excitation wavelength of 295 nm and 1 nm excitation and emission bandwidths. All spectra were corrected for background emission Raman scattering. To test the specificity of ATP binding, similar experiments were performed using Pi, AMP, ADF’ and GTP.

3. Results

3.1. Equilibrium binding study Equilibrium binding stud:ies of ATP/a-crystallin conducted at 37, 22, and 4°C revealed temperature dependence of the interaction of ATP and cr-crystallin. Binding occurred at 37°C but was not lsignificant at 22°C and absent at 4°C. The absence of binding at the lower temperatures may be due to the observed modifications of a-crystallin between 25 to 37°C. On raising the temperature of an a-crystallin solution, Seizen et al. [19] demonstrated that

0.00 0

2

4

6

8

10 l2 14 16 16 20 22 24 u

93

aggregate s~,,~ reached a minimum of 14.5s at 37°C and Walsh et al. [20] observed that fluorescence intensity of MAINS labeled cy-crystallin was 35% higher at 37°C than that labeled at 25°C. Since MAINS is a probe that does not fluoresce in aqueous buffer, but does so when reacted with thiols of a protein, this increase in SH reactivity of (Ycrystallin denotes a change in conformation. The data for the binding of ATP to a-crystallin conducted at 37°C is presented in Fig. 1 in the form of a Scatchard plot, U/[L] vs. Z where U is the fraction bound and [L] is the concentration (PM) of free ligand. Due to differences in reported aggregate molecular weights of a-crystallin at 37°C the data has been analyzed for both 400 [21] (Fig. 1B) and 850 kDa (Fig. 1A) [19] aggregates. Linear regression analysis of the experimental data yields the function (solid line), Y = 8.1 . lo-‘-8.2 * 10m3X (R2 = 0.87) calculated for a 400 kDa aggregate (Fig. 1B) and Y = 16.9. 10p2-7.84. 10e3X, R2 = 0.81 for the 850 kDa aggregate (Fig. 1A). The respective linear downward plots are characteristic of independent single site binding of ligand to protein. From the slope and intercept of both plots, a K, of 8.2 * lo3 M-l and 10 binding sites were determined for a 400 kDa aggregate (Fig. 1B) and a K, of 7.84 . lo3 M-l and 22 binding sites were determined for an 850 kDa aggregate (Fig. 1A). Assuming a 400 kDa aggregate and a 3:l ratio of (YA to aB [22], 10 molecules of ATP bind to an aggregate composed of 15 aA and 5 aB subunits (molecular weight for each subunit is = 20 kDa). Or 22 molecules bind to an aggregate composed of approx. 30 (YA and 10 aB subunits. The K, calculations obtained in the present study exhibit moderately high affinity under conditions similar to those encountered physiologically and are comparable to the K, of several ligand/protein interactions. The K, of a-GP to y-crystallin is 6.2 . lo3 M-l [lo]. Benesch et al. reported that

0.00 0

2

6

4

8

IO

I

Fig. 1. Scatchard plot, S/[L] vs. Z, where ij is fraction bound and [L] is free ligand concentration (PM). From the slope and intercept of these plots, a K, of ATP binding to a-crystallin at 37°C is 7.84. lo3 M-l with an N value of 22 binding sites per aggregate of 400 kDa (A) and a K, of 8.2. lo3 M-r and N of 10 (B) for an 8.50 kDa aggregate. Open circles denote the mean of three separate determinations with S.E.M. ranging from 0.16 to 0.24. The straight lines represent linear regression analysis of the data.

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D. V. Palmisano et al. / Biochimica et Biophysics Acta 1246 (I 995) 91-97

b-8 In

/a-

Lnh/F-

11

11

Fig. 2. Hill plot, In - [L] vs. In - [(n/5) - 11, where n is 22 (A) or 10 (B) binding sites derived from the Scatchard plots in Fig. 1. Open circles denote the mean of three separate determinations with S.E.M. ranging from 0.0023 to 0.0041. The cooperativity index, CYH, derived from linear regression analysis of the data points (straight line) approximates 1.0 in both curves and corroborates ligand binding at independent sites.

2,3-bis-phosphoglycerate binds to hemoglobin with a K, of 4.8 . lo4 M- ’ [23]. Additionally, the K, of Nacetylneuraminic acid to Limax fluvus agglutinin (a lectin) is 3.8 * lo4 M- ’ [24]. The equilibrium binding data was further analyzed using a Hill plot, In N [L] vs. In _ [(n/E) - 11 (Fig. 2) where n is 10 (Fig. 2B) and 22 (Fig. 2A) binding sites obtained from the Scatchard analysis and V and [L] are described in Fig. 1. Linear regression analysis of the experimental values reveals the function, Y = 4.84 - 1.01X, CR2 = 0.954) for a 400 kDa aggregate (Fig. 1B) and Y = 4.84 0.982X (R2 = 0.970) for an 850 kDa aggregate. A Hill plot provides an index of cooperativity; aH > 1 and < 1 indicates positive and negative cooperativity, respectively. An index of 1 suggests that the binding of ligand and protein occurs at independent sites [25]. The CYH values derived from the slope of the respective linear functions approximate unity and corroborate the independent site interaction of ATP and a-crystallin predicted in the Scatchard analysis. 3.2. Intrinsic tryptophan fluorescence

spectroscopy

ATP. Additionally, at 0.20 mM ATP, the wavelength at peak intensity shifts from 341 to 336 nm. The decrease in fluorescence and the shift to the shorter wavelength are attributed to a change in the position of tryptophan residues to a more compact, hydrophobic environment [27]. This may reflect alterations in conformation in the secondary structure and/or aggregative properties of the subunits [28]. To test the specificity of ATP as a modulator of cY-crystallin, the ITF peak emission intensities of 0.1 mg/ml a-crystallin alone and in the presence of 0.05-0.3 mM Pi, AMP, ADP, and GTP were recorded. At all concentrations, no effects on the peak emission intensities by Pi, AMP or ADP were observed. The absence of effects by Pi eliminates the possibility of non-specific binding of ATP phosphates to cu-crystallin. Our previously reported

(ITF)

The intrinsic fluorescence of tryptophan provides a reporter amino acid to monitor conformational changes induced by protein perturbation, such as ligand/protein (ATP/cw-crystallin) binding [26]. The effect of increasing concentrations of ATP (0.05 to 0.30 mM) on the tryptophan fluorescence emission spectra of a-crystallin (0.1 mg/ml) were determined (Fig. 3). With reference to 100% peak fluorescence intensity assigned to a-crystallin alone, fluorescence declines 4% (Student t-test, P = O.OS>, 13% (P = 0.01) and 35% (P = 0.01) at 0.05, 0.10 and 0.20 mM ATP concentrations, respectively. A near maximal decrease is reached at 0.20 mM ATP concentration and is not significantly different from the 36% decline at 0.30 mM

320

340

Wavelength

360

380

400

(nm)

Fig. 3. Representative relative intrinsic tryptophan fluorescence spectra ranging from 310 to 380 nm. The emission spectra of 0.1 mg/ml cx-crystallin alone is presented in curve A and with added 0.05, 0.10, 0.20 and 0.30 mM ATP (curves B-E).

D.V. Palmisano et al. / Biochimica et Biophysics Acta 1246 (I 995) 91-97

31P-NMR data revealed

a strong involvement of the Pr and a minor involvement of Pp of ATP upon binding to a-crystallin. These observations are in accord with the absence of effects by AMP or ADP; therefore, measurable binding of ATP apparently requires Py. To test the specificity of the purine binding site, the effects of GTP were studied. The specificity of ATP and GTP protein binding sites is well established in biological systems. For example, ATP is specific in its effects on actin polymerization while GTP is required for microtubule assembly. GTP produced only a 5% decrease in fluorescence (P = 0.05) at the highest concentration studied (0.30 mM). The data implies that the binding site in a-crystallin is specific for adenine and requires Py.

4. Discussion The highly tions provides

selective nature of protein-ligand a sensitive and rapid mechanism

interacfor the

95

modulation of protein activity in response to a changing cell environment. In an exhaustive study of environmental effects on a-crystallin quatemary structure, Siezen and co-workers [19] concluded that there may be unidentified cofactors present in lens cells necessary for its structural stability. We suggest that ATP is one of these factors. In the present study we have provided evidence for the binding of ATP to cu-crystallin. Since binding is concentration dependent in vivo, ATP would be released from binding during decreased metabolic activity as fiber cells mature. In vitro, ATP would be lost during the extensive dilutions required in protein preparation and/or at the lower temperature. The K, for binding approximates that of other important protein-ligand interactions [10,23,24]. A moderately strong binding constant ensures the reversibility of binding during in vivo fluctuations in ligand concentrations and other physiological conditions, i.e., temperature, pH, ion concentration and composition. This is of particular impor-

aA/aB Complex Fig. 4. Computer-generated working model of the backbone structure of the CIA (black) and crB (gray) subunits and their complex. In the significant portion of the C-terminal domain of aA (arrows) may be inaccessible for ATP binding. Individual ‘working’ models of bovine subunits were constructed based on secondary structure sequence based prediction algorithms [41] and experimental global secondary structure building, docking and minimization to obtain the lowest energy structure were performed on a Silicon Graphics Workstation using Sybyl software.

complex, a cu-crystallin [33]. Model version 6.0

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D.V. Palmisano et al. /Biochitnica

tance in the changing lens fiber cell environment during differentiation and maturation. The decreasing metabolic activity and consequent metabolite gradient as the fiber cells are displaced inward is concomitant with supramolecular reorganization [3]. These processes lead to the formation of a stable metabolically inert nucleus where a major portion of a-crystallin is in the water insoluble fraction of lens proteins. The present equilibrium binding and intrinsic fluorescence data agree well with our evidence for ATP/a-crystallin binding determined by 31P-NMR spectroscopy [13]. The NMR chemical shifts as well as the Tr and T2 values indicate that the Pr of ATP is of prime importance in its binding to a-crystallin. This is in agreement with our intrinsic fluorescence data that protein/ligand binding occurred in the presence of ATP, but was absent with either AMP or ADP. The absence of significant fluorescence quenching by GTP also establishes the specificity of the binding site for adenine versus guanine. The temperature dependence of binding may be related to an altered secondary/quaternary structure resulting from modification of a-crystallin aggregation and/or conformation of the binding sites. The 10 or 22 binding sites predicted from Scatchard analysis of our data for respective 400 kDa and 850 kDa aggregates imply that only half of the subunits per aggregate are available for ATP binding. One possible explanation may be related to serine phosphorylation of 20-30% of the subunits [29-321. This post-translational modification has the potential for inhibiting/potentiating binding by altering protein surface charge distribution and/or conformation. A second possibility is the masking of aA binding sites due to formation of aA/aB complexes predicted by our computer-assisted molecular modeling (Fig. 4) [33]. Individual aA and CYB subunits as well as the complex are amphipathic; i.e., each possesses distinct hydrophobic N-terminal and hydrophilic C-terminal domains. In the complex, a substantial portion of the Cterminus of aA subunit is bound in the cleft of aB and may alter the accessibility of ATP binding sites. Therefore with respect to the 400 kDa aggregate, assuming a 3:l ratio of (uA/~B in bovine lenses [22] and five crA/aB complexes, only 10 aA subunits are available for binding. Considerable experimental data and our molecular modeling of a-crystallin support the micellar quaternary structure originally suggested by Augusteyn and Koretz [34]. Thus, the remaining 10 subunits would have equivalent accessibility for binding at the hydrophilic C-terminal domain. A similar relationship applies to the 850 kDa aggregate. In summary, the binding of ATP to cY-crystallin adds another facet toward understanding the role of a-crystallin in the differentiation and maturation of lens fiber cells. In differentiation, the dynamics of cell architecture are undoubtedly related to the association of cytoskeletal actin filaments with cu-crystallin [35], glycolytic enzymes [36] and the plasma membrane [13,37,38]. This ensures the

et Biophysics Acta 1246 (1995) 91-97

proximity of the ATP producing glycolytic pathway and the ATP binding sites of both a-crystallin and actin. The interaction with ATP stimulates actin polymerization [39] and modulates a-crystallin. Since the subunits of a-crystallin have been identified in other tissues under both normal and pathologic conditions [40], the relevance of our investigation reaches beyond the confines of the lens and cataractogenesis.

Acknowledgements This study was supported by an NE1 Grant, EY05787. Additional support was from Research to Prevent Blindness, Inc. and the Lions Sight Foundation of New Jersey. We appreciate the expert assistance of Drs. M. Balasubramanyam and Jeffrey Gardner in the intrinsic tryptophan fluorescence study. Dr. Goutam Chakraborty provided invaluable assistance in the radioactive equilibrium binding study.

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