Oligomeric Assembly and Ligand Binding of the Members of Protein

Bioconjugate Chem. 2003, 14, 1243−1252

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Oligomeric Assembly and Ligand Binding of the Members of Protein Family YER057c/YIL051c/YJGF Edita Mistiniene,*,† Virginijus Luksa,‡ Jolanta Sereikaite,§ and Vytautas Naktinis‡ Institute of Biotechnology, V. Graicˇiu j no 8, Vilnius 2028, Lithuania, SICOR Biotech UAB, V. Graicˇiu j no 8, Vilnius 2028, Lithuania, and Vilnius Gediminas Technical University, Sauletekio al. 11, Vilnius 2040, Lithuania. Received June 23, 2003; Revised Manuscript Received September 24, 2003

Proteins UK114 and p14.5 are both members of the putative family of small proteins YER057c/YIL051c/ YjgF. The biological role of these proteins is not understood very well, and in addition, their oligomeric structure in solution remains controversial. We therefore investigated the oligomeric structure of UK114 and p14.5 using a number of methods. Both proteins have exhibited a homotrimeric structure in solution. Indeed the trimeric structure of the two proteins appeared to be so similar that when protein subunits derived from different species were mixed, stable heterotrimeric complexes (monomer ratio of 1:2 and 2:1 of UK114 and p14.5, respectively) could be formed in vitro. Furthermore, the trimeric structure of both UK114 and p14.5 proved essential for the stoichiometric hydrophobic ligand, such as fatty acid binding activity of the two proteins.

INTRODUCTION

Perchloric acid- or trifluoracetic acid-soluble extracts from liver or kidney of mammals have been shown to contain regularly identifiable proteins of currently unknown function. UK114 is one of the three proteins found in perchloric acid-soluble extract of goat liver (1, 2). UK114 is located in the cytoplasm of normal cells, while in a wide variety of tumors it is also expressed on the cell surface (3), to suggest that tumors, which express UK114 on the cell membrane, could be subjected to antibody-mediated cytolysis. Indeed, administration of the UK-specific antibodies in vivo specifically suppresses growth of human colon cancer HT29 cells xenografted into nu/nu mice (4, 5). Preliminary evidence on an anticancer efficacy of UK114-containing preparations in humans has also been reported (3, 6). The mode of biological action of these proteins however remains undefined. Melloni et al. demonstrated that UK114 activated the protease µ-calpain and showed its similarity to other activators, such as long chain acylcoAs-binding proteins or activators from bovine brain (7, 8). PSP (perchloric acid-soluble protein), a homologue of UK114, from rat liver and kidney, on the other hand, has been shown to inhibit a cell-free protein synthesis in a rabbit reticulocyte lysate system (9, 10). PSP may also be involved in fatty acid binding and in intracellular metabolism of fatty acids (11). A 14.5 kDa trichloracetic acid-soluble p14.5 protein from human mononuclear monocytes has also been isolated and characterized. Human recombinant p14.5 appears similar to PSP in its ability to inhibit protein synthesis in vitro (12). Hrp12, a homologue from mouse, described by Samuel et al., has specifically been assigned the function of a translation inhibitor, though it also exhibited features of a heat shock protein (13). * Corresponding author. Fax +37052 60 21 16, tel +37052 60 21 19, e-mail: [email protected]. † Institute of Biotechnology. ‡ SICOR Biotech UAB. § Vilnius Gediminas Technical University.

All these proteins were identified as constituting a new hypothetical family of small proteins, the YER057c/ YIL051c/YjgF family (12). The amino acid sequences of the members of this family are highly conserved throughout the evolutionary tree, from prokaryotes to eukaryotes. Approximately 30 proteins sharing sequence similarity of 32-93% have currently been assigned to this family. 3-D structures in crystal are currently available for two bacterial member proteins of the family, a purine regulatory protein from Bacillus subtilis (14) and YjgfF from E. coli (15). In both cases, data obtained was consistent with the proteins forming symmetric trimers in crystals. Recently, the crystal structure of the first mammalian protein UK114 from this family has also revealed the trimeric organization (16). Information about oligomeric structure in solution for members of the YER057c/ YIL051c/YjgF family is controversial. Preliminary X-ray data on PSP was indicative of two molecules being present per asymmetric unit (17), a conclusion supported with size exclusion chromatographic data, which was consistent with the dimeric structure of the protein in solution (9). A monomeric structure of a homologue protein from human mononuclear monocytes, p14.5, has been reported (12). Recently, the first NMR study of the member of YjgF/YER057c/UK114 family HI0719 from Haemophilus influenzae in solution showed trimeric structure (18). There is no clear evidence of oligomeric structures for mammalian members of this family in solution. Such controversy over the oligomeric structure of these very similar and evolutionarily well preserved proteins in solution generates a significant hurdle for efforts made to understand the biological role of these proteins. We therefore made our own routine gel filtration chromatographic measurements and then applied a carefully designed gentle chemical cross-linking method to two proteins of the YER057c/YIL051c/YjgF family, namely, UK114 and p14.5, in solution. We supported this approach by characterizing the conditions of ESI-MS1 (electrospray ionization mass spectrometry) analysis required for identification of the oligomeric state of the

10.1021/bc0341066 CCC: $25.00 © 2003 American Chemical Society Published on Web 11/01/2003

1244 Bioconjugate Chem., Vol. 14, No. 6, 2003

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proteins. In the present study the first homotrimeric solution structure was confirmed for two mammalian members of the YER057c/YIL051c/YjgF family. Furthermore, the trimeric structure in solution was demonstrated to be so similar for the two proteins that stable heterotrimeric complexes could be formed in vitro. Indeed, our evidence indicates that the maintenance of the trimeric structure of these proteins in solution may be critical to their ability to bind hydrophobic ligands, such as free fatty acids, one possible mode of action of these proteins. EXPERIMENTAL PROCEDURES

Materials. Recombinant UK114 and p14.5 expressed in Escherichia coli were a kind gift from SICOR Biotech UAB (Lithuania). Protein molecular weight markers for SDS-PAGE were obtained from MBI Fermentas (Lithuania). Lipidex 1000 was obtained from Packard Instrument Company. 3H-labeled fatty acids were obtained from Amersham Pharmacia Biotech, England. Scintillation cocktail Rotiszint Eco Plus was obtained from ROTH. 1,3,5-Triacryloyl-hexahydro-s-triazine (TAT) was prepared as described by T. L. Gresham and T. R. Steadman (19) and recrystallized from water. Ethylene glycol bis(succinimidylsuccinate) (EGS) and 8-anilino-1-naphthalenesulfonic acid (ANS) were obtained from Sigma. Cross-Linking by TAT. The solution containing 0.025 mM of UK114 or p14.5 in 0.05 M borate buffer pH 9.2 was incubated for 5 min at 30 °C, and then TAT was added to a concentration of 2.8 mM. The reaction mixture was incubated at 30 °C; samples were withdrawn after 5, 15, 30, 60, 120, 180, and 300 min, and the reaction was stopped by acidification to pH 5.0. The samples were analyzed by SDS-PAGE to identify cross-linked protein forms. SDS-PAGE. SDS-PAGE was carried out on the Mighty Small electrophoresis unit (Hoefer Scientific Instruments, San Francisco, CA) according to the method of Laemmli (20). The acrylamide concentration used in the gel was 15%. Proteins were stained with Coomassie Brilliant Blue R-250 or silver. Kinetic Measurements. Kinetics of TAT reaction with glycine amino groups were measured spectrophotometrically in a thermostated cell at 275 nm, under pseudo-first-order conditions. Five kinetic runs were carried out for each experiment. Pseudo-first-order rate constants were calculated with eq 1, using linear-regression program GraphPad:

1 A0 - Ainf k ) ln t At - Ainf

(1)

where A0 is the initial absorbance, At is the absorbance at the time t, and Ainf is the final absorbance, after completion the reaction. Light absorbance of glycine at 275 nm is negligible. Size Exclusion Chromatography. (a) Nondenaturing Conditions. Size exclusion chromatography was performed on Superdex200 HR 300×10 mm column 1 Abbreviations: AEC, anion exchange chromatography; ANS, 8-anilino-1-naphthalenesulfonic acid; DMSO, dimethyl sulfoxide; ESI-MS, electrospray ionization mass spectrometry; Gdn‚ HCl, guanidine hydrochloride; I-FABP, intestinal fatty acid binding protein; RP-HPLC, reversed phase high performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEC, size exclusion chromatography; TAT, 1,3,5-triacryloyl-hexahydro-s-triazine; TNFR, tumor necrosis factor.

(Amersham Pharmacia, Sweden), equilibrated with 10mM potassium phosphate buffer, pH 7.0, 0.3 M NaCl using FPLC Akta-explorer 100 (Amersham Pharmacia, Sweden). Protein samples were applied, and isocratic elution was carried out at a flow rate of 0.5 mL/min. (b) Denaturing Conditions. The column, as described above, was equilibrated with 10 mM potassium phosphate buffer, pH 7.0, 0.3 M NaCl, 7 M GdnHCl. Protein samples were incubated in 7 M GdnHCl for 24 h at ambient temperature and applied on the column, and isocratic elution was carried out at a flow rate of 0.5 mL/ min. Separation of Heterotrimers by Anion Exchange Chromatography. The mixture of 100 µL of UK114 solution (1 mg/mL) in 10 mM phosphate buffer pH 7.4 and 100 µL of p14.5 solution (1 mg/mL) in the same buffer and 200 µL of 3 M urea water solution was incubated for 2.5 h at 30 °C. Then the mixture was diluted to 2 mL with 10 mM glycine-NaOH buffer, pH 9.0, and chromatographed on Mono Q HR 5/5 in a gradient of 30 column volumes in the same buffer, containing 0.1 M NaCl at flow rate of 1 mL/min using FPLC Akta-explorer 100 (Amersham Pharmacia, Sweden). Collected peaks were analyzed using isoelectric focusing. Electrospray Ionization Mass Spectrometry. The method was developed according to the published procedure (21). (a) Mild Conditions. All samples prior to mass spectrometry were dialyzed against 0.1% acetic acid in water. The HP 1100 MSD (mass selective detector) was used for the acquisition of the electrospray ionization mass spectra. Mass spectra were in positive ion mode, over mass range 500-2900 m/z. The samples (protein concentration 0.5 mg/mL) were delivered to the analyzer using a 30 mL/min flow of 1.0% acetic acid. The temperature of drying gas was 150 °C. Nitrogen was used for drying and nebulizing. (b) Denaturing Conditions. All samples prior to mass spectrometry were purified by RP-HPLC using reversed phase column Hi-Pore RP-304 (250 × 4.6 mm, Bio-Rad); solvent A: 0.1% trifluoracetic acid in water, solvent B: 0.1% trifluoracetic acid in acetonitrile/water (90/10, v/v). The column was initially equilibrated with solvent A at flow rate of 1 mL/min. After the injection, the column was eluted with a 4 min linear gradient to 50% B followed by a 64 min linear gradient to 70% B and 65 min to 90% B. Chromatographic analysis was performed on a HP 1100 Series HPLC system. Collected peaks were lyophilized and dissolved in acetonitrile/water/acetic acid (49.5/ 49.5/1, v/v/v). Mass spectra were in positive ion mode, over mass range 500-2900 m/z. The samples (protein concentration 0.05 mg/mL) were delivered to the analyzer using a 50 mL/min flow of acetonitrile/water/acetic acid (49.5/49.5/ 1, v/v/v). The temperature of drying gas was 350 °C. Nitrogen was used for drying and nebulizing. Delipidation by Lipidex Chromatography. Delipidation by Lipidex chromatography was carried out according to the method described by J. F. Glatz (22). Lipidex 1000 was washed free of methanol in a column by elution with 10 bed volumes of 10 mM potassium phosphate buffer (pH 7.4), containing 0.01% NaN3. The gel was transferred to a glass vial and stored at 4 °C as Lipidex-buffer suspension (1/1, v/v). The lipid components in the protein samples were recovered by chromatography on a column (10’×60 mm) of Lipidex1000 equilibrated with 10 mM potassium

Oligomeric Structure of the Proteins UK114 and p14.5

Bioconjugate Chem., Vol. 14, No. 6, 2003 1245

phosphate buffer pH 7.4 at room temperature. All proteins were eluted in a void volume. Free Fatty Acid Binding Assay. For the assay of fatty acid binding activity, protein samples (2 mM) were incubated in 1.5 mL polyethylene test tubes in 10 mM potassium phosphate buffer pH 7.4 with various concentrations of 3H-fatty acid. The final volume was 0.45 mL. After incubation for 10 min at 37 °C, the test tubes were cooled in ice water. Unbound fatty acid was removed from the solution by adding of 0.05 mL of ice cold Lipidex1000/ water suspension, incubated for 10 min at 0 °C, and centrifuged for 2 min at 20000g and 4 °C. An aliquot of supernatant was assayed for radioactivity by liquid scintillation counting. Fatty acid binding was calculated from the amount of radioactivity present in the supernatant and was expressed as mol of fatty acid per mol of monomeric protein. ANS Binding Assay. All fluorescence experiments were performed on a Perkin-Elmer Luminescence Spectrometer LS50B. For the ANS binding assay, protein samples (9.1 µM) in 2 mL 10 mM potassium phosphate buffer pH 7.4 were titrated with increasing ANS concentration. ANS emission spectra (excitation λmax 365 nm) were recorded in the range of 350-650 nm, slit widths 5 nm. RESULTS

Size Exclusion Chromatography. Size exclusion chromatography provides means by which the state of aggregation of proteins can be effectively determined, provided that the subunit-subunit interaction forces are sufficiently strong to withstand such a nonequilibrium analysis. As it is shown in Figure 1A, the UK114 protein behavior on Superdex200 was consistent with the molecular mass of 29kDa. These experiments were performed at an initial protein concentration of ∼70 µM. Reductions of protein concentration to 1.4 µM did not affect the gel-filtration behavior of the protein, to indicate that the oligomeric state of the protein was stable through the range of the concentrations tested. The gelfiltration behavior of the p14.5 human analogue of UK114 was exactly the same as that of UK114. The molecular mass of UK114 derived from the gene sequence has been reported to be 14166 Da (23), to suggest that UK114 may exist in a dimeric state under the experimental conditions described above. Indeed, when UK114 was subjected to gel-filtration using 6 M guanidine hydrochloride (Gdn‚HCl) in a mobile phase, the elution volume was consistent with molecular weight of 14.0 kDa (Figure 1B), a number in good agreement with the expected molecular mass of monomeric subunit. Electrospray Ionization Mass Spectrometry. Significant evidence has now been reported to suggest that proper adjustment of experimental conditions used in the performance of ESI-MS experiments is required to provide insight into oligomeric structure. Without such precisely defined conditions, quaternary structure can be easily disrupted by low pH, high organic solvent content, high temperature of drying gases, and high flow rate of mobile phase (24). When these disruptive forces are not controlled, only covalent protein interactions usually survive, and a precise molecular mass of the protein monomeric subunits is obtained. Quaternary structure of the protein, however, can be maintained throughout the course of analysis by careful adjustment of these disruptive factors, such that the molecular mass of the complex can be established. Initially, we subjected UK114 to ESI-MS analysis under denaturing conditions where disruption of oligo-

Figure 1. Size exclusion chromatography of UK114 on Superdex200 HR 300×10 mm using FPLC Akta-explorer100 (Amersham Pharmacia, Sweden). The column was equilibrated with 10 mM potassium phosphate buffer, pH 7.0, 0.3 M NaCl (A), buffer A also containing 6 M Gdn HCl (B) and buffer A with 0.1% SDS (C). UK114 samples of 200 µL (1 mg/mL) in the A buffer (A), in the B buffer (B) and UK114 3 h cross-linked with TAT in 0.1% SDS (C) were applied and isocratic elution was carried out at flow rate of 0.5 mL/min.

meric state of the protein was expected. Denaturing ESIMS conditions include RP-HPLC with high organic solvent concentration for sample preparation and rigorous ionization parameters (high temperature, high concentration of organic solvent in mobile phase, and high flow rate of mobile phase) for ESI-MS. The data (Figure 2A) demonstrated a typical ESI-MS spectrum of the protein, consisting of multiply charged [M + nH]n+ ions, with charges ranging from +8 to +19. The set of ions detected corresponded to the protein average mass of 14166 ( 1 (mean ( SD) Da. This value was in excellent agreement with the theoretically calculated mass of 14.166 kDa for the monomeric form of UK114. Using denaturing conditions, i.e., RP-HPLC for sample preparation and rigorous conditions for ESI-MS ionization, it is not clear enough which of the factors are mainly responsible for destruction of trimeric structure.


Mistiniene et al.

Figure 2. Mass spectrum of UK114 under denaturing (A) and mild conditions (B).

To preserve noncovalent protein complexes, we used mild sample preparation conditions, i.e., dialysis against 0.1% acetic acid in water, and milder ionization parameters for ESI-MS. Organic solvent was not used in the mobile phase for ESI-MS; lower temperature and lower flow rate of mobile phase were applied. After using mild sample preparation conditions together with mild ionization parameters, a significant change in the overall spectrum was observed (Figure 2B). Two protein components were detected on the spectrum, a major component of 42499 ( 3 kDa which corresponded with the trimeric form of UK114, and a minor component of 14166 ( 1 kDa, which was clearly reflective of monomeric form of the protein. At the same time no dimeric form was detected. Chemical Cross-Linking. The formation of protein oligomers in solution is commonly investigated by crosslinking studies in which closely associated subunits are

cross-linked in such a manner that the so-formed quaternary structure can be subsequently isolated and analyzed. Two cross-linking agents, 1,3,5-triacryloylhexahydro-s-triazine (TAT) (25, 26) and ethylene glycol bis(succinimidylsuccinate) (EGS) (27), were therefore used to probe the quaternary structure of UK114. TAT is a trifunctional chemical cross-linking agent, previously demonstrated to be extremely stable, simple and convenient to use in the generation of cross-linking patterns, which are characteristic of the native structure of the proteins (25, 26, 28). We have previously described its use in concentrations of 4.5-30-fold molar excess of TAT active groups over the concentrations of amino groups in the protein, defining the oligomeric state of such well-characterized monomeric, dimeric, or trimeric proteins, such as human growth hormone, interferon-γ, and tumor necrosis factor alpha (TNF-R), respectively (25, 28).



As it is demonstrated in Figure 3A, the cross-linking profile obtained with UK114 was the same as that of TNF-R. Two predominant species with apparent molecular mass in a range of 48-54 kDa were observed as the ultimate products of cross-linking reaction. It has previously been demonstrated that these two molecular species resemble two different steric isoforms of the crosslinked trimer, the linear and ring-shape isoforms (29). Therefore, the similarity of cross-linking profiles of UK114 with those of the trimeric TNF-R protein was consistent with the trimeric structure of UK114 in solution. Since the most favorable TAT cross-linking conditions included a pH of 9.2, we questioned whether the reaction kinetics and the end product profile were similar under physiological conditions. We reproduced the cross-linking reaction results using ethylene glycol bis(succinimidylsuccinate) at pH 7.2. To further support the observation that UK114 formed trimers in solution, we subjected the products of the TAT 3 h (Figure 3A, lane 8) cross-linking reaction to more detailed characterization. Size exclusion chromatography under mild denaturing conditions (in the presence of 0.1% SDS) revealed that the 3 h reaction mixture was composed of three principal components, with their molecular masses being consistent with monomeric, dimeric, and trimeric forms of UK114 (Figure 1C). The behavior of UK114 in the presence of SDS is no more exceptional: experimental molecular mass of the cross-linked trimer is near the calculated one (42 and 43.2 kDa, respectively). Extending this analysis of the TAT reaction products, we used ESI-MS. Main components of a complex mixture of monomeric, dimeric, and trimeric structures of UK114 3 h cross-linked with TAT (Figure 3A, lane 8) were identified. They are listed in Table 1. Up to seven TAT moieties were attached to the monomeric UK114 form, a number in good agreement with the fact that the UK114 monomer contains seven lysine residues in polypeptide chain. Two principal forms of trimeric UK114 were confirmed in the reaction mixture, with 14 and 15 covalently attached TAT moieties, respectively. All the methods used for probing the quaternary structure led us to conclude that two mammalian proteins from the YjgF/YER057c/UK114 family UK114 and p14.5 exist as homotrimers in solution, like their bacterial homologue HI0719 (18). The Interaction between Subunits in the Trimeric Structure. Having established the TAT crosslinking technique as a reliable tool for probing quaternary structure of UK114, we addressed the issue of the nature of interaction between monomeric subunits in the tri-

Figure 3. SDS-PAGE (15%) of UK114 (A) and TNF-R (B) crosslinked by TAT. A. Lane 1, protein markers; lane 2, unmodified UK114; lane 3-9, samples taken at 5, 15, 30, 60, 120, 180, and 300 min. B. Lane 1, protein markers; lane 2, unmodified TNFR; lane 3-6, samples taken at 30, 60, 120, and 300 min.

Every attempt was made to ensure that the experimental conditions, adjusted to specific characteristics of UK114, exclusively promoted the cross-linking of true molecular contacts rather than accidental protein molecular collisions. First, UK114 was incubated with TAT at a molar ratio of 30:1 of TAT acrylic groups:UK114 amino groups, at pH 9.2. Next, the aliquots of the reaction mixture were withdrawn at increasing time intervals and subjected to denaturing SDS-PAGE analysis. The profile obtained by applying this experimental approach to the well-known trimeric protein, tumor necrosis factor alpha, is indicated in Figure 3B. Characteristically, the TAT interaction with the trimeric protein resulted in an early intermediate of dimeric form. The presence of crosslinked dimers on PAGE gels does not mean that they actually exist in the solution. Cross-linked dimers may be not yet completely cross-linked trimers. If trimer is the main form of a protein in solution, cross-linked trimers become dominant on PAGE at the completion of cross-linking process, as is shown on the Figure 3 for TNF and UK114. Then trimeric structures appeared gradually, and no higher oligomers were detected after long time incubation. A detailed explanation of the kinetics of the cross-linking profile of TNF-R has previously been reported (26, 28). Table 1. Detected Species of TAT-Cross-Linked UK114 ESI-MS experimental data mol mass, Daa

suggested protein structure

14914 15161 15410 15661 15909

+3 TAT +4 TAT +5 TAT +6 TAT +7 TAT

30081 30850 31843

+7 TAT +10 TAT +14 TAT

45988 46244

+14 TAT +15 TAT

a

UK114 cross-linked with TAT pH 9.2, 3 h at 30 °C.

calcd mol mass, Da

percent from the total amount of identified (UK114)i+nTAT

14914 15163 15412 15661 15910

9.4 16.2 16.4 16.7 15.9

30077 30850 31820

4.8 3.9 6.1

45987 46236

5.6 5.0

Monomer

Dimer

Trimer


Mistiniene et al.

Table 2. Pseudo-First-Order Rate Constants for the Reaction of TAT with Glycine Amino Group in KCl Solutiona concentration of KCl, M

k × 104, s-1

0 1 2 3

5.02 ( 0.06b 4.62 ( 0.08 4.75 ( 0.07 4.85 ( 0.05

a Initial concentrations: TAT, 6.9 × 10-4 M; glycine, 0.07 M; 30 °C and pH 9.0. b Standard deviations are given.

meric structure. It was expected that if electrostatic interactions were predominant in forming trimers, then the disruption of such interactions by increasing ionic strength would preclude the formation of trimers in the cross-linking reaction. We added KCl to the cross-linking reaction mixture and monitored UK114 trimer formation by SDS-PAGE analysis. Our results indicated that the elevation of KCl concentration to 0.5 M considerably reduced the formation of the trimeric structure. The further elevation of salt concentration to 2.5 M completely abolishes recovery of the trimeric form. To eliminate the possibility that the inhibitory effect of high salt concentration on the formation of the UK114 trimeric structure was due to the inhibition of the TAT reaction with amino groups, we performed a study where kinetic parameters of the TAT reaction with amino group of model compound, glycine, were measured. As shown in Table 2, pseudo-first-order rate constant of the reaction remained unchanged through the range of KCl concentrations from 0 to 3 M, to suggest that we could exclude the possibility that observed inhibiting effect of elevated salt concentrations on the formation of the trimeric structure of UK114 was due to the inability of TAT to react under these conditions. To explore the possible contribution of hydrophobic interactions in UK114 trimer formation, we added dimethyl sulfoxide (DMSO) to the cross-linking reaction mixture and monitored trimer formation by SDS-PAGE analysis. This chaotropic reagent, when present in concentrations of up to 10%, is known to disrupt hydrophobic interactions of proteins (30). DMSO in concentrations up to 15% had no effect on trimerization, to suggest that the role of hydrophobic interactions in the association of monomeric subunits was insignificant. Heterotrimer Formation. Alignment of the sequence of UK114 with that of p14.5 revealed a striking 86% similarity in primary structure. The cross-linking experiments described above, then repeated using p14.5, generated results very similar to those described for UK114. It was therefore interesting to explore the possibility of creating chimeric structures in vitro. To do so, we exploited the fact that the two proteins, despite significant overall similarity, had very different isoelectric points, pI 7.4 and pI 9.2, for UK114 and p14.5, respectively. We also exploited the fact that we could reversibly dissociate UK114 into subunits and reassociate the trimer structure afterward. If one mixed the two proteins, then subjected the mixture to mild denaturation, to cause the break down of trimers into monomers, and then removed denaturant, monomeric subunits would reassociate into the trimers. Under such conditions, four species of trimers theoretically should form (Figure 4). Two species would resemble the original UK114 and p14.5 trimers, while two would be chimeric structures with monomer ratios of 1:2 and 2:1, UK114 and p14.5, respectively. All four trimeric species would be characterized by different pI values, to allow their separation in a properly selected chromatographic system.

Figure 4. Hypothetical scheme for the formation of heterotrimers of UK114 and p14.5 subunits.

Figure 5. The chromatographic separation of the dissociatedassociated mixture of UK114 and p14.5. Solution of UK114 and p14.5 (1 mg/mL of each) in 3 M Gdn‚HCl was incubated for 2.5h at 30 °C and diluted with 3 volumes of 10 mM glycine-NaOH, pH 9.0. Samples of reaction mixture (2 mL) were chromatographed on Mono Q HR5/5 in a gradient of 30 column volumes in the same buffer, containing 0.1 M NaCl at flow rate of 1 mL/ min using FPLC Akta-explorer100 (Amersham Pharmacia, Sweden).

As indicated in Figure 5, heterotrimer formation was achieved using the scheme described above, and the heterotrimers formed were separable. The nature of all the species formed was confirmed by isoelectrofocusing analysis. Free Fatty Acid Binding. PSP, a protein from rat liver, also a homologue of both UK114 and p14.5, is known as a fatty acid binding protein (11). Because of the significant homologies between these proteins, UK114 and p14.5 may also bind free fatty acids, the possibility we investigated. The principle of the method we exploited involved the incubation of the protein with varying amounts of 3H-labeled fatty acid, removal of unbound free fatty acid on Lipidex1000, and measurement of the remaining labeled complex. Two known proteins were used for comparison: I-FABP, which has previously been demonstrated to stoichiometrically bind 1 mol of fatty acid per mol of protein (31, 32), and TNF-R, a cytokine, whose biological function is apparently not related to fatty acid metabolism (33, 34) and which therefore served as a negative control. The data generated for binding of oleic acid are presented in Table 3. The same results were



Table 3. Binding Activity of 3H-Labeled Oleic Acid protein

Bmax, mol/mol monomeric protein

I-FABP UK114 p14.5 TNF-R

1.10 ( 0.20 0.90 ( 0.15 0.70 ( 0.17 0.09 ( 0.04

obtained in palmitic acid binding experiments. Both fatty acids bind to UK114 (or p14.5) in a saturable manner. This indicates the specific nature of the binding. The stoichiometry of both complexes was very close 1:1 (mol fatty acid per mol monomer protein). As it was expected, the protein known to bind fatty acids specifically (IFABP) manifested 1:1 stoichiometric binding activity, while the binding activity of TNF-R, the negative control, barely exceeded the background level. These results suggest that UK114 maintains three fatty acid binding sites per trimeric molecule. Further, the apparent dissociation constant of the complex UK114fatty acid decreased while increasing the saturation of the protein with ligand, to suggest cooperative character of the fatty acid binding. To exclude the possibility that the free fatty acid binding observed was a random event, we addressed the correlation between structural integrity of the protein and its binding ability. Specifically we evaluated binding at various stages of thermal inactivation. Samples of UK114 were incubated at 37, 60, and 80 °C for 20 min, and at 100 °C for 1 min, and their ability to bind fatty acid was measured as described above. The results of this study revealed that the UK114 gradually lost binding activity with thermal inactivation, starting from 60 °C with only minor activity remaining after heating for 1 min at 100 °C. These data confirmed the specific nature of the binding observed. ANS Binding. Stoichiometric binding activity of fatty acid suggests the presence of three binding sites per trimeric protein. We found that UK114 and p14.5 are able to bind the hydrophobic fluorescent dye ANS in their native state. The fluorescent probe ANS is traditionally used to detect molten globules, i.e. partly folded proteins that accumulate under mild conditions (35). ANS fluorescence is sensitive to the polarity of its microenvironment; upon binding to the apolar surfaces, its emission maximum is shifted to shorter wavelengths and the emission intensity is enhanced. ANS is believed to bind to molten globules due to the presence of solvent-exposed hydrophobic patches, which are a particular characteristic of this protein state (36). Certain proteins also bind ANS in the native state, however, provided this conformation displays exposed hydrophobic sites (37). Fluorescence emission spectra of ANS in the presence or absence of UK114 (or p14.5) are shown in Figure 6A. In water, ANS fluoresce is negligible and exhibits a maximum emission wavelength (λmax) of ∼515 nm. In the presence of UK114 (or p14.5) under native conditions (pH 7.4), ANS fluorescence displays a pronounced increase in intensity with a concomitant shift of λmax to 465 nm, both of which indicate transfer of ANS to a less polar environment. These data show that UK114 and p14.5 bind ANS in the native state. The affinity of the interaction between ANS and UK114 (or p14.5) was examined by titration of UK114 (or p14.5) with ANS while following the ANS emission at 465 nm. The resulting binding curves, displayed in Figure 6B, show that ANS binds to UK114 (or p14.5) in saturable manner. Kd values for UK114 and p14.5 are 2.2 and 2.4 µM, respectively. These experiments yield a binding stoichiometry of 1.1 ( 0.2 mol ANS/mol mono-

Figure 6. A. ANS binding to native UK114. Fluorescence of 9.1 µM ANS in the absence and presence of 9.1 µM UK114 at 25 °C in 10 mM potassium phosphate buffer pH 7.4. ANS fluorescence is a very weak exhibiting a λmax of ∼515 nm (I). Upon addition of UK114 the fluorescence of ANS increase with a concomitant shift of λmax to 465 nm (II). ANS was excited at 365 nm. B. Affinity of ANS to UK114 interaction. Titration of 9.1 µM UK114 with ANS in 10 mM potassium phosphate buffer pH 7.4. ANS was excited at 365 nm. C. ANS binding to UK114 with increasing concentration of Gdn‚HCl. 9.1 µM UK114 was preincubated 5h at 25 °C in 10 mM potassium phosphate buffer pH 7.4 with increasing Gdn‚HCl concentration. ANS binding was monitored by 365 nm excitation and 465 nm emission.

meric protein and support the presumption that proteins UK114 and p14.5 exhibit three ligand binding sites per trimeric molecule. To prove the importance of quaternary structure for ligand binding, we investigated the ANS binding under denaturation of UK114 with GdnHCl. The profile of ANS emission intensity with increasing GdnHCl concentration shows two maxima (Figure 6C). The ANS fluorescence intensity decreases and reaches minimum at 0.25 M GdnHCl. It may be explained by disruption of trimeric structure and intersubunit binding sites. Further increase of emission intensity at GdnHCl concentration above 2.75 M is related with unfolding of UK114 to molten globule, when hydrophobic surfaces of protein are exposed to a greater extent compared with a native state. To confirm disruption of quaternary structure we performed size-exclusion chromatography of UK114 in


the presence of GdnHCl. UK114 was incubated in various concentrations of GdnHCl and applied to a TSK G2000SW FPLC size-exclusion column equilibrated with buffer containing an identical concentration of GdnHCl. In the absence of GdnHCl UK114 eluted as a single peak, corresponding to native trimeric protein, increasing of GdnHCl concentration up to 0.25 M UK114 also eluted as a single peak, but with elution volume, corresponding with monomeric form. This transition from trimer to monomer occurred having the same GdnHCl concentration as the losing of specific binding of ANS (Figure 6C). With further increase of GdnHCl concentration, the peak, corresponding to monomeric UK114, gradually shifted to earlier elution times, suggesting that monomer of UK114 was unfolded and a molecule with increased apparent volume produced. The midpoint of the unfolding curve was 2.5 M. DISCUSSION

The highly conserved protein family YER057c/YIL051c/ YjgF is comprised of ∼30 homologues exhibiting varying degrees of sequence homology from 32 to 93%. Some of them have been identified as proteins soluble in strong acids, but most sequences from this protein’s group were hypothetical and identified by genome projects. Despite the sequence conservation through evolution and the high degree of homology between members of the YER057c/ YIL051c/YjgF family, no common biological function has been allocated to it yet. With the mounting evidence as to the potentially high clinical value of the biotherapeutics being developed on the basis of proteins of this family (6), the deciphering of biochemical mechanisms underlying their clinical actions becomes increasingly urgent. In this paper we describe the oligomeric structures and ligand binding properties of two recombinant DNAderived members of the YER057c/YIL051c/YjgF family: perchloric acid-soluble protein from goat liver, UK114, and its analogue from human monocytes, p14.5, proteins that manifest 86% sequence similarity. Indications as to biological function of these proteins could potentially be derived from the details of their structural organization. However, in the case of the YER057c/YIL051c/YjgF family evidence on oligomeric state in solution has supported conclusions of monomeric to trimeric forms. Gel-filtration data used to demonstrate the native state of the protein yielded results interpreted to be consistent with the dimeric form (9, 12). Indeed, we also initially produced gel-filtration data indicative of the dimeric form for both UK114 and p14.5 in solution. However, we extended our study of the oligomeric state of these proteins by applying a carefully designed crosslinking technique. The formation of UK114 (and p14.5) trimers was observed under a variety of conditions in water solution. Direct evidence of trimeric structure was obtained by using nondisrupting ESI-MS analysis. In fact, our results as well as crystallographic data (16) indicate that these proteins are trimeric under physiological conditions. The apparent conflict between cross-linking, ESI-MS and gel-filtration data may be explained by the UK114 (and p14.5) protein assuming an extremely compact structure in solution, such that effective hydrodynamic radii of the proteins were substantially smaller than the radii of reference proteins used for gel-filtration column calibration. Support of this explanation has now been provided by X-ray study data (16) of the crystal form of UK114, which revealed the dimensions of highly sym-

Mistiniene et al.

metric homotrimeric UK114 of ∼48 × 45 × 43 Å. The apparently more compact spatial organization of UK114 could provide a clue into the misleading gel-filtrational behavior of this protein, compared with conventional reference proteins. To delineate the character of the interaction forces, which keep separate subunits in trimeric form, we exploited cross-linking methodology, where reaction conditions were altered to limit either hydrophobic or electrostatic protein interactions. The overall electrostatic interaction was demonstrated to be the major factor in the trimerization of the protein. Information on monomer: monomer surface interactions retrieved from recent crystal data (16) was consistent with our observation. The UK114 molecule possesses an extensive network of hydrogen bonds and electrostatic interactions throughout the subunits contact surface. The number of complementary interactions between oppositely charged ionizable groups on the contact surfaces between monomers was sufficient to confirm that electrostatic interaction played the major role in the stabilization of UK114 trimer. The critical putative interaction points identified from the 3-D structure now provide a basis for future site-specific mutagenesis studies directed at further biochemical confirmation of the role of these putative contacts in maintaining trimeric organization of the protein. Further, the high degree of sequence similarity between UK114 and p14.5, together with the proven trimeric structure of the two proteins, led us to hypothesize that there could be a high similarity in tertiary structure such that the monomeric subunits from one protein could be interchangeable with the monomeric subunits from the other in their respective trimeric states. We successfully demonstrated that such a possibility could be implemented in vitro by exploring the finding that each of the proteins could be dissociated into monomers under mild conditions and then reassembled into functional trimers by removing the denaturing agent. By mixing the two original proteins, dissociating them into monomers, and later reassociating them, we produced chimeric trimers containing two subunits from one protein and one subunit from another. The heterotrimers formed were sufficiently stable to withstand anionic exchange chromatographic separation, and isoelectric focusing in a way analogous to that of the original homotrimers, to indicate an extremely close if not identical spatial organization of monomers in these two proteins. We plan to explore this observation further in future eukaryote genetic knock-out models (38). Stable trimeric organization could potentially play an important role in the biological function of the protein, specifically its ability to bind hydrophobic ligands, such as free fatty acids. We confirmed that these two proteins bind fatty acid with Kd ∼ 2.0 µM, forming one to one stoichiometric complex (mol of fatty acid per mol of monomeric protein), a finding similar to that of PSP from rat (11). The ability of these proteins to bind hydrophobic ligands in their native state was also demonstrated with fluorescent dye ANS. The binding isotherm is rectangular hyperbolic and reaches saturation, suggesting that binding is specific, i.e. a well-defined number of binding sites exist. Indeed, titrating of UK114 (or p14.5) with ANS at concentrations well above Kd indicates a 1:1 (mol of dye per mol of monomeric protein) binding stoichiometry. Stoichiometric ligand binding activities suggest the presence of three binding sites per trimeric protein. Indeed, trimeric UK114 in a crystalline state contains three hydrophobic solvent-exposed cavities, created between


interacting subunits (16), as it was shown previously for some members of the YER057c/YIL051c/YjgF family (14, 15, 18). It was found that oleic acid displaces ANS bound to UK114, i.e. the two substances compete for the same binding sites. We also demonstrated that ligand binding activity exhibits only trimeric protein. UK114 lost ANS binding ability when trimer was disrupted to monomer under unfolding, induced by GdnHCl. In conclusion, these findings may have important biological implications. Despite the mentioned above multifunctionality of the proteins from YER057c/YIL051c/ YjgF family, high similarity in quaternary structures suggest that these proteins use the same biochemical way for the manifestation of their biological function. Trimeric organization of the two proteins of the YER057c/YIL051c/ YjgF family has been shown to be a critical structural feature in ligand binding, one potential mode of action of these proteins. The structural elements, which direct formation of the trimeric state of the protein, are apparently preserved throughout the evolutionary tree to support a correlation between structural organization and function of these proteins. The data presented therefore could target further studies of the structurefunction relationship of these proteins, to elucidate their respective mechanisms of action. ACKNOWLEDGMENT

We thank prof. G. Dienys for helpful comments and discussion, Barbara Richmond-Smith for critical reading of the manuscript and L.Noreika for technical assistance. This work was supported by Lithuanian State Science and Studies Foundation, grant no. G-053. LITERATURE CITED (1) Bartorelli, A., Biancardi, C., Cavalca, V., Ferrara, R., Botta, M., Arzani, C., Colombo, I., Berra, B., Ceciliani, F., Ronchi, S., and Bailo, M. (1996) Purification and Partial Characterization of Proteins Present in a Perchloric Acid Extract of Goat Liver. J. Tumor Marker Oncol. 11, 57-61. (2) Ceciliani, F., Biancardi, C., Cavalca, V., Ferrara, R., Botta, M., Bailo, M., Arzani, C., Berra, B., Ronchi, S., and Bartorelli, A. (1996) Structural Characterization of the Small Molecular Weight Proteins Present in UK101. J. Tumor Marker Oncol. 11, 63-66. (3) Bussolati, G., Guena, M., Bussolati, B., Millesimo, M., Botta, M., and Bartorelli, A. (1997) Cytolytic and tumor inhibitory antibodies against UK114 protein in the sera of cancer patients. Int. J. Oncol. 10, 779-798. (4) Bartorelli, A., Berra, B., Ronchi, S., Biancardi, C., Cavalca, V., Bailo, M., Mor, C., Ferrara, R., Botta, M., Arzani, C., and Clemente, C. (1994) Immunocytochemical Reactivity of Mammalian Liver Antigen (UK101) in Human Tumors and non Neoplastic Tissues. J. Tumor Marker Oncol. 9, 37-47. (5) Bartorelli, A., Bussolati, B., Millesimo, M., Gugliota, P., and Bussolati, G. (1996) Antibody - dependent cytotoxic activity on human cancer cells expressing UK114 tumor membrane antigen. Int. J. Oncol. 8, 543-548. (6) Mor, C., Garibaldi, A., Bissi, O., Concas Benevelli, D., Di Mattia, D., Gajani, M. R., Gilardoni, E., Liverani, A., Mancini, S., Santi, C., and Bartorelli, A. (1997) On the compassionate use of UK101 in metastatic cancer. J. Tumor Marker Oncol. 12, 29-37. (7) Melloni, E., Michetti, M., Salamino, F., and Pontremoli, S. (1998) Molecular and functional properties of a calpain activator protein specific for µ - isoforms. J. Biol. Chem. 21, 12827-12831. (8) Melloni, E., Averna, M., Salamino, F., Sparatore, B., Minafra, R., and Pontremoli, S. (2000) Acyl - CoA - binding protein is a potent m - Calpain activator. J. Biol. Chem. 1, 82-86.

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Oligomeric Assembly and Ligand Binding of the Members of Protein

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