- Email: [email protected]
Folding of the human immunoglobulin G3 Kus core hinge into the thirteenth globular domain
Immunology Letters 90 (2003) 43–47
Folding of the human immunoglobulin G3 Kus core hinge into the thirteenth globular domain Vladimir M. Tischenko a , Galina A. Zav’yalova b , Vladimir P. Zav’yalov b,∗ a
Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino 142290, Russia b Russian Research Center for Molecular Diagnostics and Therapy, Moscow 113149, Russia Received 15 June 2003; accepted 9 July 2003
Abstract Earlier, the electron microscopy and hydrodynamic studies revealed the transformation of the globule-like form of the human (h) IgG3 Kus hinge into a rod-like shape under non-denaturing perturbations [Eur. J. Biochem. 190 (1990) 393]. In this work, it is shown with the differential scanning calorimetry (DSC) that the melting of a globule-like form of the hIgG3 Kus hinge is a co-operative process. The ‘two-state’ model accepted for small globular proteins well describes the transition. Thus, in the hIgG3 Kus molecule, the core hinge folds into the thirteenth globular domain. The model of folding of four double poly-l-proline (PLP) helices of the core hinge into the compact tertiary structure similar to ‘a folded camp bed’ is suggested. © 2003 Elsevier B.V. All rights reserved. Keywords: Human IgG3 core hinge; Thirteenth globular domain
1. Introduction An IgG molecule is composed of two identical Fab portions with the antigen (Ag) binding site and Fc region, which determines most of the effector functions of antibodies (Ab’s), in particular, complement and Fc receptor (R) binding. The Fab and Fc subunits are joined through a flexible hinge that permits the molecule to be functionally biAbbreviations: hIgG3, human immunoglobulin G3; Kus, family name of patient; m, mouse; H-chain, heavy chain; L-chain, light chain; Fab, antigen-binding fragment consisting of the light chain and half of the heavy chain (VH , VL , CL and CH 1 domains); Fc, two C-terminal halves of the heavy chain (two CH 2 and CH 3 domains) with the interheavy chain disulfide bond intact; Fch, fragment from hIgG3 containing two CH 2 and CH 3 domains and part of the hinge region; VH , VL , CL , CH 1, CH 2 and CH 3 domains, regions comprising about 110 amino acid residues with an “immunoglobulin fold” structure; Ab, antibody; Ag, antigen; FcR, Fc receptor; S–S bond, disulfide bond between two cysteine residues; PLP, poly-l-proline; HIV, human immunodeficiency virus; MW, molecular weight; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CD, circular dichroism; DSC, differential scanning calorimetry; Tm , melting temperature, corresponding to the maximum for the heat absorption peak; Hcal , calorimetric enthalpy of melting; Heff , effective or van’t Hoff enthalpy of melting; Cp , partial heat capacity; S20,w , sedimentation coefficient ∗ Corresponding author. Tel.: +7-95-113-23-65; fax: +7-95-113-23-65. E-mail address: [email protected] (V.P. Zav’yalov). 0165-2478/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0165-2478(03)00161-5
valent for multivalent Ag’s [1–3]. The analysis of crystal structure of hIgG1 b12 [4] has revealed that its hinge extends for 17 residues. They can be assigned to three separate regions: the upper hinge defined as the region between the Fab L-chain–H-chain S–S bond and the first S–S bond of the core hinge, the core hinge, including two pairs of Cys and two pairs of Pro residues, and the lower hinge. The upper hinge may exhibit a high degree of flexibility [5]. In the structure of hIgG1 Kol, the core hinge forms a double PLP helix stabilized by two S–S bonds [6]. The hIgG3 molecule has the hinge of 62 amino acid residues encoded by four exons [7–9]. The lower hinge of hIgG3 participating in the FcR-binding [10] has the sequence identical to that of hIgG1. The core hinge of hIgG3 [3] consists of one short (CPRCP) and three long (PPPCPRCP) sequences that, like hIgG1 [4,6], may form potentially four double PLP helices. The PLP domains are separated by three identical hydrophilic sequences (EPKSCDT), in which a few residues are statistically preferable for turns [11,12]. An extended rod-like shape of the hIgG3 core hinge of 90–100 Å has been proposed on the basis of the small-angle X-ray scattering data for some hIgG3 paraproteins [13] and the electron microscopy study of chimeric m/hAb’s with the hIgG3 constant region [14]. For a few other hIgG3 paraproteins, the same methods have
44
V.M. Tischenko et al. / Immunology Letters 90 (2003) 43–47
demonstrated a globule-like form of the hinge [15–18]. The electron microscopy and hydrodynamic studies revealed the transformation of a globule-like form of the hIgG3 Kus hinge into a rod-like shape under non-denaturing perturbations [16,17]. The recent DSC and CD studies have shown that the secondary structure of the hIgG3 Kus core hinge consists of four independent co-operative blocks of the extremely stable double PLP helix separated by sites of non-co-operative structure [19]. These sites permit the hinge to fold into a compact globule-like structure. The hIgG3 subclass represents 5–10% of the common IgG level in sera of blood of the healthy persons. The hIgG3 hinge region confers to the Ab’s enhanced HIV-neutralizing ability [20]. Therefore, of doubtless interest are the functional role of the extreme length of the hIgG3 hinge and the puzzling situation with its conformational features. Furthermore, from an applied point of view the rapidly growing number of humanized and total hIgG Ab’s for therapy of different diseases [21] increases the urgency of such studies. The aim of this work is to study the process of transformation of the globule-like form of the hIgG3 Kus hinge into a rod-like shape using the DSC and hydrodynamic methods. It was demonstrated that the DSC method has sensitivity to the domain organization both globular and fibrillar proteins [22–24]. 2. Materials and methods 2.1. Preparation of purified hIgG3 and isolation of Fab, Fc and Fch fragments The hIgG3 Kuc was obtained from myeloma serum as was described earlier [25]. The isolated protein had the MW of 170 kDa and specifically reacted with the anti-sera to hIgG3. The protein did not display reactivity with staphylococcal protein A. The Fc was prepared by digesting hIgG3 Kuc with trypsin according to reference [26]. The Fab was prepared by the same manner but the time of digestion was 1 h. It was purified using DEAE-Sephadex [27]. The F(ab )2 and Fch, which contain the Fc region and a part of the hinge were prepared as was described earlier [8]. The fragments were isolated according to [28]. The preparations were often slightly contaminated with Fc that is the terminal product of the Fch digestion with papain. The fractions were further purified by affinity chromatography on a column with immobilized Ab’s against the hinge. According to the data of immunoelectrophoresis [29] and SDS-PAGE by Laemmli [30], the hIgG3 samples and their fragments were homogenous. 2.2. Production of anti-sera and purification of antibodies and their Fv fragments against the hIgG3 hinge region, Fab and Fc fragments, CH 2 and CH 3 domains Anti-serum to the hIgG3 hinge was raised in rabbit according to [31]. F(ab )2 was used as Ag. Anti-hinge Ab’s purification was performed using the immunosorbent tech-
nique [32]. Anti-sera to the hIgG3 Fab and Fc fragments, CH 2 and CH 3 domains were raised in rabbit, using the Fab, Fc and pFc fragments as Ag’s. Their purification was performed using the immunosorbent technique. The Fv fragments of Ab’s were obtained according to the earlier described approach [33]. 2.3. Biophysical studies The MW was determined by two equilibrium centrifugation methods [34,35] using a MOM (Hungary) and a Beckman Model E (USA) ultracentrifuges with an interference optical system at 0.3 mg/ml concentration of the proteins. The sedimentation velocity measurements were made using Beckman ultracentrifuges in the protein concentration range of 2–8 mg/ml (Schlieren optics) and of 0.15–0.3 mg/ml (absorption optics). The DSC experiments were performed using a computer driven version of a DASM-4A microcalorimeter [24] with a cell volume of 0.47 ml at a heating rate of 0.5–2 K/min. Gel-filtration on an ACA-34 Ultragel column equilibrated with a corresponding buffer was used prior to the measurements of the samples. The protein concentrations in the experiments varied in the range of 0.5–5.0 mg/ml. The Cp , Hcal and Heff were calculated from the calorimetric data as described previously [24,36]. The solution viscosity was measured in an Ostwald viscosimeter with a water flow time of about 200 s at 20 ◦ C. The protein intrinsic viscosity was determined by extrapolation to zero concentration of the reduced viscosity measured at the corresponding temperature. Extrapolation was done from 5 to 6 points obtained at different concentrations in the range from 1 to 8 mg/ml. The model of the hIgG3 core hinge conformation was built using the DeepView-Swiss-PdbViewer software (GlaxoSmithKline Research and Development S.A., Geneva).
3. Results The hIgG3 Kuc purified in gentle conditions at a neutral pH area exists in a state characterized by S20,w of 6.0 S [16]. The transition in a state with a lower S20,w value (5.1 S) is induced by incubation at an acidic pH area (4.0) or heating to 55 ◦ C, which does not unfold the co-operative structure of globular domains [16,19,25,33,37,38]. In accordance with the electron microscopy data [16], these changes are caused by the transformation of the compact globule-like form of the hinge into an extended rod-like shape. A prolonged incubation at acidic pH area makes the transformation into the extended state nonreversible. Therefore, it is possible to compare the melting of the hIgG3 Kus extended and compact forms at neutral pH area. The solid curve in Fig. 1 shows the melting of the hIgG3 Kus extended form at pH 7.5. When the hinge has a rod-like shape, the Fab and Fc subunits are disposed at a distance
V.M. Tischenko et al. / Immunology Letters 90 (2003) 43–47
Fig. 1. Temperature dependence of the molar heat capacity of the hIgG3 Kus extended (solid curve) and compact (dashed curve) forms at pH 7.5. The dotted curve shows the melting of the compact form in the complex with the Fv fragment of Ab’s against the hinge.
more than 100 Å [6,14,16]. Therefore, even in the intact hIgG3 molecule they represent independent subsystems of globular domains. Consequently, the melting curve of the intact hIgG3 Kus molecule with a rod-like hinge can be obtained as a result of summarising the curves of melting of the Fab and Fc fragments [19,25,33]. This allows relating each of the observed peaks at the hIgG3 Kus melting curve to the co-operative melting of definite globular domains in the protein using the results of analysis for the isolated Fab and Fc fragments [19,25,33,38]. The melting curve of the hIgG3 Kus compact form (Fig. 1, dashed curve) is significantly different from that obtained for the extended form. First, the low-temperature heat absorption peak appears. Second, the heat absorption peak relating to the CH 1, CL and CH 2 domains melting is bisected. The common enthalpy of the hIgG3 Kuc compact form melting is significantly higher than that of the extended form. This indicates that the melting of a compact form is accompanied by disruption of a greater number of non-covalent bonds than that of the extended form. Since the Tm and the intensity of all of the observed peaks do not depend on the concentration of the protein studied, one can conclude that they indicate only disruption of intra-molecular bonds. The question arises: What are the hIgG3 structures melting in two additional peaks of heat absorption? To answer this question, we used Fv fragments of Ab’s against different portions of the hIgG Kus. An interaction of the Fv fragment with a definite domain induces an increase of thermal stability of the co-operative block formed by this domain. The data obtained by this approach coincide with the results of identification of the co-operative blocks in the extended form of hIgG3 Kus using its fragments. The application of this approach to the hIgG3 Kus compact form allowed relating the first low-temperature peak of heat absorption to the melting of a compact globule-like form of the hinge. The second peak was related to the melting of CH 2 domains. The further confirmation of the correct identification of the first low-temperature peak of heat absorption was obtained
45
Fig. 2. Temperature dependence of intrinsic viscosity (circles) and a fraction of the hIgG3 Kus hinge denatured state according to the DSC data (solid curve).
with the study of temperature dependence of the hIgG3 Kuc intrinsic viscosity. Fig. 2 shows that the increase of temperature up to 55 ◦ C causes an increase of this parameter in parallel with the increase of a fraction of the denaturing state of the hinge according to the calorimetric data. According to the electron microscopy data, just in this temperature range the transition from the compact globule-like form to the rod-like shape of the hinge was observed [16]. According to the CD measurements, the transition is not accompanied by any changes in the secondary structure that is a PLP helix [17]. The co-operative melting of the PLP helix accompanied by characteristic changes in the CD spectra at 220–230 nm was observed for the hIgG3 Kus in a significantly higher temperature range [19]. Therefore, one can relate the changes of intrinsic viscosity to the co-operative melting of the tertiary structure of the hinge. The Heff value, calculated from the half-width of transition, coincides with Hcal (210 and 202 kJ mol−1 , respectively). Thus, one can describe the transition by a ‘two-state’ model accepted for small globular proteins [22]. The temperature dependence of Hcal (Fig. 3) is also typical for small globular proteins [22]. The stability of the tertiary structure of the
Fig. 3. Temperature dependence of Hcal of the hIgG3 Kus hinge melting.
46
V.M. Tischenko et al. / Immunology Letters 90 (2003) 43–47
hinge is decreasing in the narrow pH range. The melting of the tertiary structure of the hinge was observed earlier at pH 5.5 [25] as an abnormal pre-denaturing change of the heat capacity, but it was already absent at pH 5.3.
4. Discussion The obtained data indicate that a compact form of the hIgG3 Kus hinge exhibits the features typical for a small globular protein [22]. Thus, at physiological conditions the hIgG3 Kuc hinge may be considered as the thirteenth globular domain. But there are some differences between the thermodynamic properties of the globule-like structure of the hinge and the small globular protein or typical globular domain, in particular, any other IgG globular domain [33,38–42]. The tertiary structure of the hinge should be formed of the co-operative blocks of the double PLP helix, which is the main secondary structure of the core hinge [6,17,19,43,44]. The sites of the non-co-operative structure separate the PLP helices [19]. Potentially, these sites can form turns and, consequently, permit the folding of four double PLP helices into a globule-like structure. According to the CD measurements, the unfolding of the hinge tertiary structure is not accompanied by any change in the secondary structure that is a double PLP helix [17]. The preservation of the main secondary structure of the core hinge after the melting of tertiary structure may explain why the free energy of its stabilization is a low as 13 kJ mol−1 . This value is significantly lower than the averaged 50 ± 20 kJ mol−1 for most of typical globular proteins [22]. This indicates that relatively small perturbations can accent the equilibrium between the globular structure and the rod-like shape of the hinge, e.g. acidification of the medium in the inflammation sites, which is usually accompanied by an increase of the body temperature. The Ag binding by hIgG3 Ab’s also may induce the transition [14] because the average S20,w value for the intact m/hIgG3 Ab’s is 6.09 S, which practically coincides with the S20,w value of 6.0 for the hIgG3 Kus compact form [16]. The globular form of the hinge affects the conformation of CH 2 domains (see Fig. 1) that correlates with less effective inhibition of the complement activation by the hIgG3 compact form [18]. Thus, the 13th globular domain modulates the hIgG3 effector functions. Co-operative unfolding of this domain increases the fraction of molecules with higher biological activity. As was mentioned in Section 1, the core hinge of hIgG3 consists of four sequences that, like hIgG1, may form potentially four double PLP helices. The PLP domains are separated by three identical hydrophilic sequences, in which a few residues are statistically preferable for turns. Fig. 4 shows the model of compact packing of four double PLP helices of the hIgG3 core hinge into the tertiary structure. Two identical turns between adjoining double PLP helices are connected in the middle by S–S bond (Fig. 4A). Consequently, two adjoining double PLP helices can fold
Fig. 4. Model of the tertiary globule-like structure of the hIgG3 core hinge. (A and B) Two different projections of the structure, in which the segments of poly-l-proline helix are shown by cylinders and the side chains of cystines are shown by spherical models. Sulphur atoms are displayed as dark shaded spheres. The letters N and C indicate the N- and C-termini of the hinge sequence. (C) The molecular surface model of the structure. The figures were generated using PyMOL (DeLano Scientific LLC).
in the compact tertiary structure in the symmetrical antiparallel position to each other (Fig. 4A). As a result, the tertiary structure similar to ‘a folded camp bed’ is formed. The hydrophobic core of the structure is composed of eight cystines (Fig. 4B), which are the constituents of the double PLP helices. Fig. 4C demonstrates that the tertiary structure formed by the double PLP helices is dense packed. Fig. 5 shows the compact and extended states of the hIgG3 molecule. These models are in agreement with the images of
V.M. Tischenko et al. / Immunology Letters 90 (2003) 43–47
Fig. 5. The compact (A) and extended (B) states of the hIgG3 molecule, which are shown by the molecular surface models. The models of the Fab and Fc subunits and the lower hinge were built using the PDB atomic coordinate file 1HZH for hIgG1 b12 [4]. The figures were generated using PyMOL (DeLano Scientific LLC).
the compact and extended forms of the hIgG3 Kus molecule visualized by the electron microscopy studies [16]. Acknowledgements This work was carried out under Contract No. 403662-AK2 with the Pacific Northwest National Laboratory of the US Department of Energy and was also supported by the Russian Foundation for Basic Research (RFBRr2001moscow region 01-04-97010). References [1] R. Nezlin, Adv. Immunol. 48 (1990) 1–40. [2] D.R. Burton, J.M. Woof, Adv. Immunol. 55 (1992) 1–84.
47
[3] O.H. Brekke, T.E. Michaelsen, I. Sandlie, Immunol. Today 16 (1995) 85–90. [4] E.O. Saphire, R.L. Stanfield, M.D. Crispin, P.W. Parren, P.M. Rudd, R.A. Dwek, D.R. Burton, I.A. Wilson, J. Mol. Biol. 319 (2002) 9–18. [5] J.L. Dangl, T.G. Wensel, S.L. Morrison, L. Stryer, L.A. Herzenberg, V.T. Oi, EMBO J. 7 (1988) 1989–1994. [6] M. Marquart, J. Deisenhofer, R. Huber, W. Palm, J. Mol. Biol. 141 (1980) 369–392. [7] T.E. Michaelsen, J.B. Natvig, FEBS Lett. 28 (1972) 121–124. [8] T.E. Michaelsen, B. Frangione, E.C. Franklin, J. Biol. Chem. 252 (1977) 883–889. [9] S. Huck, P. Fort, D.H. Crawford, M.-P. Lefranc, G. Lefranc, Nucl. Acid Res. 14 (1986) 1779–1789. [10] S. Radaev, P. Sun, Mol. Immunol. 38 (2001) 1073–1083. [11] C.M. Venkatachalam, Biopolymers 6 (1968) 1425–1436. [12] J.L. Crawford, W.N. Lipscomb, C.G. Schellman, Proc. Natl. Acad. Sci. U.S.A. 70 (1973) 538–542. [13] L. Gregory, K.G. Davis, B. Sheth, J. Boyd, R. Jefferis, C. Naves, D.R. Burton, Mol. Immunol. 24 (1987) 821–829. [14] M.L. Phillips, M.-H. Tao, S.L. Morrison, V.N. Schumaker, Mol. Immunol. 31 (1994) 1201–1210. [15] F. Kilar, I. Simon, S. Lakatos, F. Vonderviszt, G.A. Medgyesi, P. Zavodsky, Eur. J. Biochem. 147 (1985) 17–25. [16] S. Ryazantsev, V. Tishchenko, V. Vasiliev, V. Zav’yalov, V. Abramov, Eur. J. Biochem. 190 (1990) 393–399. [17] V.M. Tischenko, Biochemistry (Moscow) 65 (2000) 1227–1230. [18] V.M. Tischenko, Biochemistry (Moscow) 66 (2001) 1352–1355. [19] V.M. Tischenko, V.P. Zav’yalov, Immunol. Lett. 86 (2003) 281–285. [20] O. Scharf, H. Golding, L.R. King, N. Eller, D. Frazier, B. Golding, D.E. Scott, J. Virol. 75 (2001) 6558–6565. [21] M.A. Van Dijk, J.G.J. Van de Winke, Curr. Opin. Chem. Biol. 5 (2001) 368–374. [22] P.L. Privalov, Adv. Protein Chem. 33 (1979) 167–241. [23] P.L. Privalov, Adv. Protein Chem. 35 (1982) 1–104. [24] P.L. Privalov, S.A. Potekhin, Methods Enzymol. 131 (1986) 4–51. [25] A.I. Denesyuk, V.M. Tishchenko, V.M. Abramov, V.P. Zav’yalov, Mol. Biol. (Moscow) 17 (1983) 1262–1271. [26] B. Sjoberg, E. Rosenqvist, T. Michaelsen, S. Pap, R. Osterberg, Biochim. Biophys. Acta 625 (1980) 10–17. [27] B. Frangione, E.C. Franklin, H.H. Fudenberg, M.E. Koshland, J. Exp. Med. 124 (1966) 715–732. [28] T. Michaelsen, J.B. Natvig, Scand. J. Immunol. 2 (1973) 299–312. [29] A. Jounstone, R. Thorpe, Immunochemistry in Practice. second ed., Blackwell Scientific Publication, Oxford, 1987. [30] U.K. Laemmli, Nature 277 (1970) 680–683. [31] T. Michaelsen, J.B. Natvig, Immunochemistry 8 (1971) 235–242. [32] T. Michaelsen, J.B. Natvig, Scand. J. Immunol. 1 (1972) 255–263. [33] V.P. Zav’yalov, V.M. Tishchenko, Scand. J. Immunol. 33 (1991) 755–762. [34] K.E. Van Holde, R.L. Baldwin, J. Phys. Chem. 62 (1958) 734–743. [35] D.A. Yphantis, Biochemistry 3 (1964) 297–317. [36] V.M. Tischenko, A.M. Ichtchenko, C.V. Andreyev, A.V. Kajava, J. Mol. Biol. 234 (1993) 654–660. [37] A. Schmidt-Kessen, A. Lustig, Hoppe Seylers Z. Physiol. Chem. 362 (1981) 18–19. [38] V.M. Tischenko, V.M. Abramov, V.P. Zav’yalov, Biochemistry 37 (1998) 5576–5581. [39] V.P. Zav’yalov, G.V. Troitsky, N.N. Khechinashvili, P.L. Privalov, Biochim. Biophys. Acta 492 (1977) 102–111. [40] V.M. Tischenko, V.P. Zav’yalov, G.A. Medgyesi, S.A. Potekhin, P.L. Privalov, Eur. J. Biochem. 126 (1982) 517–521. [41] O.I. Loseva, V.M. Tischenko, Z. Olsovska, F. Franek, V.P. Zav’yalov, Mol. Immunol. 23 (1986) 743–746. [42] V.M. Tischenko, V.P. Zav’yalov, Immunol. Lett. 84 (2002) 241–245. [43] P.M. Johnson, T.E. Michaelsen, P.M. Scopes, Scand. J. Immunol. 4 (1975) 113–119. [44] V.M. Tischenko, Mol. Biol. 34 (2000) 95–100.
Folding of the human immunoglobulin G3 Kus core hinge into the thirteenth globular domain Vladimir M. Tischenko a , Galina A. Zav’yalova b , Vladimir P. Zav’yalov b,∗ a
Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino 142290, Russia b Russian Research Center for Molecular Diagnostics and Therapy, Moscow 113149, Russia Received 15 June 2003; accepted 9 July 2003
Abstract Earlier, the electron microscopy and hydrodynamic studies revealed the transformation of the globule-like form of the human (h) IgG3 Kus hinge into a rod-like shape under non-denaturing perturbations [Eur. J. Biochem. 190 (1990) 393]. In this work, it is shown with the differential scanning calorimetry (DSC) that the melting of a globule-like form of the hIgG3 Kus hinge is a co-operative process. The ‘two-state’ model accepted for small globular proteins well describes the transition. Thus, in the hIgG3 Kus molecule, the core hinge folds into the thirteenth globular domain. The model of folding of four double poly-l-proline (PLP) helices of the core hinge into the compact tertiary structure similar to ‘a folded camp bed’ is suggested. © 2003 Elsevier B.V. All rights reserved. Keywords: Human IgG3 core hinge; Thirteenth globular domain
1. Introduction An IgG molecule is composed of two identical Fab portions with the antigen (Ag) binding site and Fc region, which determines most of the effector functions of antibodies (Ab’s), in particular, complement and Fc receptor (R) binding. The Fab and Fc subunits are joined through a flexible hinge that permits the molecule to be functionally biAbbreviations: hIgG3, human immunoglobulin G3; Kus, family name of patient; m, mouse; H-chain, heavy chain; L-chain, light chain; Fab, antigen-binding fragment consisting of the light chain and half of the heavy chain (VH , VL , CL and CH 1 domains); Fc, two C-terminal halves of the heavy chain (two CH 2 and CH 3 domains) with the interheavy chain disulfide bond intact; Fch, fragment from hIgG3 containing two CH 2 and CH 3 domains and part of the hinge region; VH , VL , CL , CH 1, CH 2 and CH 3 domains, regions comprising about 110 amino acid residues with an “immunoglobulin fold” structure; Ab, antibody; Ag, antigen; FcR, Fc receptor; S–S bond, disulfide bond between two cysteine residues; PLP, poly-l-proline; HIV, human immunodeficiency virus; MW, molecular weight; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CD, circular dichroism; DSC, differential scanning calorimetry; Tm , melting temperature, corresponding to the maximum for the heat absorption peak; Hcal , calorimetric enthalpy of melting; Heff , effective or van’t Hoff enthalpy of melting; Cp , partial heat capacity; S20,w , sedimentation coefficient ∗ Corresponding author. Tel.: +7-95-113-23-65; fax: +7-95-113-23-65. E-mail address: [email protected] (V.P. Zav’yalov). 0165-2478/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0165-2478(03)00161-5
valent for multivalent Ag’s [1–3]. The analysis of crystal structure of hIgG1 b12 [4] has revealed that its hinge extends for 17 residues. They can be assigned to three separate regions: the upper hinge defined as the region between the Fab L-chain–H-chain S–S bond and the first S–S bond of the core hinge, the core hinge, including two pairs of Cys and two pairs of Pro residues, and the lower hinge. The upper hinge may exhibit a high degree of flexibility [5]. In the structure of hIgG1 Kol, the core hinge forms a double PLP helix stabilized by two S–S bonds [6]. The hIgG3 molecule has the hinge of 62 amino acid residues encoded by four exons [7–9]. The lower hinge of hIgG3 participating in the FcR-binding [10] has the sequence identical to that of hIgG1. The core hinge of hIgG3 [3] consists of one short (CPRCP) and three long (PPPCPRCP) sequences that, like hIgG1 [4,6], may form potentially four double PLP helices. The PLP domains are separated by three identical hydrophilic sequences (EPKSCDT), in which a few residues are statistically preferable for turns [11,12]. An extended rod-like shape of the hIgG3 core hinge of 90–100 Å has been proposed on the basis of the small-angle X-ray scattering data for some hIgG3 paraproteins [13] and the electron microscopy study of chimeric m/hAb’s with the hIgG3 constant region [14]. For a few other hIgG3 paraproteins, the same methods have
44
V.M. Tischenko et al. / Immunology Letters 90 (2003) 43–47
demonstrated a globule-like form of the hinge [15–18]. The electron microscopy and hydrodynamic studies revealed the transformation of a globule-like form of the hIgG3 Kus hinge into a rod-like shape under non-denaturing perturbations [16,17]. The recent DSC and CD studies have shown that the secondary structure of the hIgG3 Kus core hinge consists of four independent co-operative blocks of the extremely stable double PLP helix separated by sites of non-co-operative structure [19]. These sites permit the hinge to fold into a compact globule-like structure. The hIgG3 subclass represents 5–10% of the common IgG level in sera of blood of the healthy persons. The hIgG3 hinge region confers to the Ab’s enhanced HIV-neutralizing ability [20]. Therefore, of doubtless interest are the functional role of the extreme length of the hIgG3 hinge and the puzzling situation with its conformational features. Furthermore, from an applied point of view the rapidly growing number of humanized and total hIgG Ab’s for therapy of different diseases [21] increases the urgency of such studies. The aim of this work is to study the process of transformation of the globule-like form of the hIgG3 Kus hinge into a rod-like shape using the DSC and hydrodynamic methods. It was demonstrated that the DSC method has sensitivity to the domain organization both globular and fibrillar proteins [22–24]. 2. Materials and methods 2.1. Preparation of purified hIgG3 and isolation of Fab, Fc and Fch fragments The hIgG3 Kuc was obtained from myeloma serum as was described earlier [25]. The isolated protein had the MW of 170 kDa and specifically reacted with the anti-sera to hIgG3. The protein did not display reactivity with staphylococcal protein A. The Fc was prepared by digesting hIgG3 Kuc with trypsin according to reference [26]. The Fab was prepared by the same manner but the time of digestion was 1 h. It was purified using DEAE-Sephadex [27]. The F(ab )2 and Fch, which contain the Fc region and a part of the hinge were prepared as was described earlier [8]. The fragments were isolated according to [28]. The preparations were often slightly contaminated with Fc that is the terminal product of the Fch digestion with papain. The fractions were further purified by affinity chromatography on a column with immobilized Ab’s against the hinge. According to the data of immunoelectrophoresis [29] and SDS-PAGE by Laemmli [30], the hIgG3 samples and their fragments were homogenous. 2.2. Production of anti-sera and purification of antibodies and their Fv fragments against the hIgG3 hinge region, Fab and Fc fragments, CH 2 and CH 3 domains Anti-serum to the hIgG3 hinge was raised in rabbit according to [31]. F(ab )2 was used as Ag. Anti-hinge Ab’s purification was performed using the immunosorbent tech-
nique [32]. Anti-sera to the hIgG3 Fab and Fc fragments, CH 2 and CH 3 domains were raised in rabbit, using the Fab, Fc and pFc fragments as Ag’s. Their purification was performed using the immunosorbent technique. The Fv fragments of Ab’s were obtained according to the earlier described approach [33]. 2.3. Biophysical studies The MW was determined by two equilibrium centrifugation methods [34,35] using a MOM (Hungary) and a Beckman Model E (USA) ultracentrifuges with an interference optical system at 0.3 mg/ml concentration of the proteins. The sedimentation velocity measurements were made using Beckman ultracentrifuges in the protein concentration range of 2–8 mg/ml (Schlieren optics) and of 0.15–0.3 mg/ml (absorption optics). The DSC experiments were performed using a computer driven version of a DASM-4A microcalorimeter [24] with a cell volume of 0.47 ml at a heating rate of 0.5–2 K/min. Gel-filtration on an ACA-34 Ultragel column equilibrated with a corresponding buffer was used prior to the measurements of the samples. The protein concentrations in the experiments varied in the range of 0.5–5.0 mg/ml. The Cp , Hcal and Heff were calculated from the calorimetric data as described previously [24,36]. The solution viscosity was measured in an Ostwald viscosimeter with a water flow time of about 200 s at 20 ◦ C. The protein intrinsic viscosity was determined by extrapolation to zero concentration of the reduced viscosity measured at the corresponding temperature. Extrapolation was done from 5 to 6 points obtained at different concentrations in the range from 1 to 8 mg/ml. The model of the hIgG3 core hinge conformation was built using the DeepView-Swiss-PdbViewer software (GlaxoSmithKline Research and Development S.A., Geneva).
3. Results The hIgG3 Kuc purified in gentle conditions at a neutral pH area exists in a state characterized by S20,w of 6.0 S [16]. The transition in a state with a lower S20,w value (5.1 S) is induced by incubation at an acidic pH area (4.0) or heating to 55 ◦ C, which does not unfold the co-operative structure of globular domains [16,19,25,33,37,38]. In accordance with the electron microscopy data [16], these changes are caused by the transformation of the compact globule-like form of the hinge into an extended rod-like shape. A prolonged incubation at acidic pH area makes the transformation into the extended state nonreversible. Therefore, it is possible to compare the melting of the hIgG3 Kus extended and compact forms at neutral pH area. The solid curve in Fig. 1 shows the melting of the hIgG3 Kus extended form at pH 7.5. When the hinge has a rod-like shape, the Fab and Fc subunits are disposed at a distance
V.M. Tischenko et al. / Immunology Letters 90 (2003) 43–47
Fig. 1. Temperature dependence of the molar heat capacity of the hIgG3 Kus extended (solid curve) and compact (dashed curve) forms at pH 7.5. The dotted curve shows the melting of the compact form in the complex with the Fv fragment of Ab’s against the hinge.
more than 100 Å [6,14,16]. Therefore, even in the intact hIgG3 molecule they represent independent subsystems of globular domains. Consequently, the melting curve of the intact hIgG3 Kus molecule with a rod-like hinge can be obtained as a result of summarising the curves of melting of the Fab and Fc fragments [19,25,33]. This allows relating each of the observed peaks at the hIgG3 Kus melting curve to the co-operative melting of definite globular domains in the protein using the results of analysis for the isolated Fab and Fc fragments [19,25,33,38]. The melting curve of the hIgG3 Kus compact form (Fig. 1, dashed curve) is significantly different from that obtained for the extended form. First, the low-temperature heat absorption peak appears. Second, the heat absorption peak relating to the CH 1, CL and CH 2 domains melting is bisected. The common enthalpy of the hIgG3 Kuc compact form melting is significantly higher than that of the extended form. This indicates that the melting of a compact form is accompanied by disruption of a greater number of non-covalent bonds than that of the extended form. Since the Tm and the intensity of all of the observed peaks do not depend on the concentration of the protein studied, one can conclude that they indicate only disruption of intra-molecular bonds. The question arises: What are the hIgG3 structures melting in two additional peaks of heat absorption? To answer this question, we used Fv fragments of Ab’s against different portions of the hIgG Kus. An interaction of the Fv fragment with a definite domain induces an increase of thermal stability of the co-operative block formed by this domain. The data obtained by this approach coincide with the results of identification of the co-operative blocks in the extended form of hIgG3 Kus using its fragments. The application of this approach to the hIgG3 Kus compact form allowed relating the first low-temperature peak of heat absorption to the melting of a compact globule-like form of the hinge. The second peak was related to the melting of CH 2 domains. The further confirmation of the correct identification of the first low-temperature peak of heat absorption was obtained
45
Fig. 2. Temperature dependence of intrinsic viscosity (circles) and a fraction of the hIgG3 Kus hinge denatured state according to the DSC data (solid curve).
with the study of temperature dependence of the hIgG3 Kuc intrinsic viscosity. Fig. 2 shows that the increase of temperature up to 55 ◦ C causes an increase of this parameter in parallel with the increase of a fraction of the denaturing state of the hinge according to the calorimetric data. According to the electron microscopy data, just in this temperature range the transition from the compact globule-like form to the rod-like shape of the hinge was observed [16]. According to the CD measurements, the transition is not accompanied by any changes in the secondary structure that is a PLP helix [17]. The co-operative melting of the PLP helix accompanied by characteristic changes in the CD spectra at 220–230 nm was observed for the hIgG3 Kus in a significantly higher temperature range [19]. Therefore, one can relate the changes of intrinsic viscosity to the co-operative melting of the tertiary structure of the hinge. The Heff value, calculated from the half-width of transition, coincides with Hcal (210 and 202 kJ mol−1 , respectively). Thus, one can describe the transition by a ‘two-state’ model accepted for small globular proteins [22]. The temperature dependence of Hcal (Fig. 3) is also typical for small globular proteins [22]. The stability of the tertiary structure of the
Fig. 3. Temperature dependence of Hcal of the hIgG3 Kus hinge melting.
46
V.M. Tischenko et al. / Immunology Letters 90 (2003) 43–47
hinge is decreasing in the narrow pH range. The melting of the tertiary structure of the hinge was observed earlier at pH 5.5 [25] as an abnormal pre-denaturing change of the heat capacity, but it was already absent at pH 5.3.
4. Discussion The obtained data indicate that a compact form of the hIgG3 Kus hinge exhibits the features typical for a small globular protein [22]. Thus, at physiological conditions the hIgG3 Kuc hinge may be considered as the thirteenth globular domain. But there are some differences between the thermodynamic properties of the globule-like structure of the hinge and the small globular protein or typical globular domain, in particular, any other IgG globular domain [33,38–42]. The tertiary structure of the hinge should be formed of the co-operative blocks of the double PLP helix, which is the main secondary structure of the core hinge [6,17,19,43,44]. The sites of the non-co-operative structure separate the PLP helices [19]. Potentially, these sites can form turns and, consequently, permit the folding of four double PLP helices into a globule-like structure. According to the CD measurements, the unfolding of the hinge tertiary structure is not accompanied by any change in the secondary structure that is a double PLP helix [17]. The preservation of the main secondary structure of the core hinge after the melting of tertiary structure may explain why the free energy of its stabilization is a low as 13 kJ mol−1 . This value is significantly lower than the averaged 50 ± 20 kJ mol−1 for most of typical globular proteins [22]. This indicates that relatively small perturbations can accent the equilibrium between the globular structure and the rod-like shape of the hinge, e.g. acidification of the medium in the inflammation sites, which is usually accompanied by an increase of the body temperature. The Ag binding by hIgG3 Ab’s also may induce the transition [14] because the average S20,w value for the intact m/hIgG3 Ab’s is 6.09 S, which practically coincides with the S20,w value of 6.0 for the hIgG3 Kus compact form [16]. The globular form of the hinge affects the conformation of CH 2 domains (see Fig. 1) that correlates with less effective inhibition of the complement activation by the hIgG3 compact form [18]. Thus, the 13th globular domain modulates the hIgG3 effector functions. Co-operative unfolding of this domain increases the fraction of molecules with higher biological activity. As was mentioned in Section 1, the core hinge of hIgG3 consists of four sequences that, like hIgG1, may form potentially four double PLP helices. The PLP domains are separated by three identical hydrophilic sequences, in which a few residues are statistically preferable for turns. Fig. 4 shows the model of compact packing of four double PLP helices of the hIgG3 core hinge into the tertiary structure. Two identical turns between adjoining double PLP helices are connected in the middle by S–S bond (Fig. 4A). Consequently, two adjoining double PLP helices can fold
Fig. 4. Model of the tertiary globule-like structure of the hIgG3 core hinge. (A and B) Two different projections of the structure, in which the segments of poly-l-proline helix are shown by cylinders and the side chains of cystines are shown by spherical models. Sulphur atoms are displayed as dark shaded spheres. The letters N and C indicate the N- and C-termini of the hinge sequence. (C) The molecular surface model of the structure. The figures were generated using PyMOL (DeLano Scientific LLC).
in the compact tertiary structure in the symmetrical antiparallel position to each other (Fig. 4A). As a result, the tertiary structure similar to ‘a folded camp bed’ is formed. The hydrophobic core of the structure is composed of eight cystines (Fig. 4B), which are the constituents of the double PLP helices. Fig. 4C demonstrates that the tertiary structure formed by the double PLP helices is dense packed. Fig. 5 shows the compact and extended states of the hIgG3 molecule. These models are in agreement with the images of
V.M. Tischenko et al. / Immunology Letters 90 (2003) 43–47
Fig. 5. The compact (A) and extended (B) states of the hIgG3 molecule, which are shown by the molecular surface models. The models of the Fab and Fc subunits and the lower hinge were built using the PDB atomic coordinate file 1HZH for hIgG1 b12 [4]. The figures were generated using PyMOL (DeLano Scientific LLC).
the compact and extended forms of the hIgG3 Kus molecule visualized by the electron microscopy studies [16]. Acknowledgements This work was carried out under Contract No. 403662-AK2 with the Pacific Northwest National Laboratory of the US Department of Energy and was also supported by the Russian Foundation for Basic Research (RFBRr2001moscow region 01-04-97010). References [1] R. Nezlin, Adv. Immunol. 48 (1990) 1–40. [2] D.R. Burton, J.M. Woof, Adv. Immunol. 55 (1992) 1–84.
47
[3] O.H. Brekke, T.E. Michaelsen, I. Sandlie, Immunol. Today 16 (1995) 85–90. [4] E.O. Saphire, R.L. Stanfield, M.D. Crispin, P.W. Parren, P.M. Rudd, R.A. Dwek, D.R. Burton, I.A. Wilson, J. Mol. Biol. 319 (2002) 9–18. [5] J.L. Dangl, T.G. Wensel, S.L. Morrison, L. Stryer, L.A. Herzenberg, V.T. Oi, EMBO J. 7 (1988) 1989–1994. [6] M. Marquart, J. Deisenhofer, R. Huber, W. Palm, J. Mol. Biol. 141 (1980) 369–392. [7] T.E. Michaelsen, J.B. Natvig, FEBS Lett. 28 (1972) 121–124. [8] T.E. Michaelsen, B. Frangione, E.C. Franklin, J. Biol. Chem. 252 (1977) 883–889. [9] S. Huck, P. Fort, D.H. Crawford, M.-P. Lefranc, G. Lefranc, Nucl. Acid Res. 14 (1986) 1779–1789. [10] S. Radaev, P. Sun, Mol. Immunol. 38 (2001) 1073–1083. [11] C.M. Venkatachalam, Biopolymers 6 (1968) 1425–1436. [12] J.L. Crawford, W.N. Lipscomb, C.G. Schellman, Proc. Natl. Acad. Sci. U.S.A. 70 (1973) 538–542. [13] L. Gregory, K.G. Davis, B. Sheth, J. Boyd, R. Jefferis, C. Naves, D.R. Burton, Mol. Immunol. 24 (1987) 821–829. [14] M.L. Phillips, M.-H. Tao, S.L. Morrison, V.N. Schumaker, Mol. Immunol. 31 (1994) 1201–1210. [15] F. Kilar, I. Simon, S. Lakatos, F. Vonderviszt, G.A. Medgyesi, P. Zavodsky, Eur. J. Biochem. 147 (1985) 17–25. [16] S. Ryazantsev, V. Tishchenko, V. Vasiliev, V. Zav’yalov, V. Abramov, Eur. J. Biochem. 190 (1990) 393–399. [17] V.M. Tischenko, Biochemistry (Moscow) 65 (2000) 1227–1230. [18] V.M. Tischenko, Biochemistry (Moscow) 66 (2001) 1352–1355. [19] V.M. Tischenko, V.P. Zav’yalov, Immunol. Lett. 86 (2003) 281–285. [20] O. Scharf, H. Golding, L.R. King, N. Eller, D. Frazier, B. Golding, D.E. Scott, J. Virol. 75 (2001) 6558–6565. [21] M.A. Van Dijk, J.G.J. Van de Winke, Curr. Opin. Chem. Biol. 5 (2001) 368–374. [22] P.L. Privalov, Adv. Protein Chem. 33 (1979) 167–241. [23] P.L. Privalov, Adv. Protein Chem. 35 (1982) 1–104. [24] P.L. Privalov, S.A. Potekhin, Methods Enzymol. 131 (1986) 4–51. [25] A.I. Denesyuk, V.M. Tishchenko, V.M. Abramov, V.P. Zav’yalov, Mol. Biol. (Moscow) 17 (1983) 1262–1271. [26] B. Sjoberg, E. Rosenqvist, T. Michaelsen, S. Pap, R. Osterberg, Biochim. Biophys. Acta 625 (1980) 10–17. [27] B. Frangione, E.C. Franklin, H.H. Fudenberg, M.E. Koshland, J. Exp. Med. 124 (1966) 715–732. [28] T. Michaelsen, J.B. Natvig, Scand. J. Immunol. 2 (1973) 299–312. [29] A. Jounstone, R. Thorpe, Immunochemistry in Practice. second ed., Blackwell Scientific Publication, Oxford, 1987. [30] U.K. Laemmli, Nature 277 (1970) 680–683. [31] T. Michaelsen, J.B. Natvig, Immunochemistry 8 (1971) 235–242. [32] T. Michaelsen, J.B. Natvig, Scand. J. Immunol. 1 (1972) 255–263. [33] V.P. Zav’yalov, V.M. Tishchenko, Scand. J. Immunol. 33 (1991) 755–762. [34] K.E. Van Holde, R.L. Baldwin, J. Phys. Chem. 62 (1958) 734–743. [35] D.A. Yphantis, Biochemistry 3 (1964) 297–317. [36] V.M. Tischenko, A.M. Ichtchenko, C.V. Andreyev, A.V. Kajava, J. Mol. Biol. 234 (1993) 654–660. [37] A. Schmidt-Kessen, A. Lustig, Hoppe Seylers Z. Physiol. Chem. 362 (1981) 18–19. [38] V.M. Tischenko, V.M. Abramov, V.P. Zav’yalov, Biochemistry 37 (1998) 5576–5581. [39] V.P. Zav’yalov, G.V. Troitsky, N.N. Khechinashvili, P.L. Privalov, Biochim. Biophys. Acta 492 (1977) 102–111. [40] V.M. Tischenko, V.P. Zav’yalov, G.A. Medgyesi, S.A. Potekhin, P.L. Privalov, Eur. J. Biochem. 126 (1982) 517–521. [41] O.I. Loseva, V.M. Tischenko, Z. Olsovska, F. Franek, V.P. Zav’yalov, Mol. Immunol. 23 (1986) 743–746. [42] V.M. Tischenko, V.P. Zav’yalov, Immunol. Lett. 84 (2002) 241–245. [43] P.M. Johnson, T.E. Michaelsen, P.M. Scopes, Scand. J. Immunol. 4 (1975) 113–119. [44] V.M. Tischenko, Mol. Biol. 34 (2000) 95–100.