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Increase in the conformational flexibility of ₂-microglobulin upon copper binding: A possible role for copper in dialysis-related amyloidosis

¹ Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan
² Department of Pathology, Fukui Medical University, Matsuoka, Fukui 910-1193, Japan
³ CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan

Reprint requests to: Yuji Goto, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan; e-mail: ygoto{at}protein.osaka-u.ac.jp; fax: 081-6-6879-8616.

(RECEIVED September 19, 2003; FINAL REVISION October 24, 2003; ACCEPTED November 10, 2003)

	Abstract

TOP Abstract Introduction Results Discussion Materials and methods Electronic supplemental material References

 
A key pathological event in dialysis-related amyloidosis is the fibril formation of ₂-microglobulin (2-m). Because 2-m does not form fibrils in vitro, except under acidic conditions, predisposing factors that may drive fibril formation at physiological pH have been the focus of much attention. One factor that may be implicated is Cu²⁺ binding, which destabilizes the native state of 2-m and thus stabilizes the amyloid precursor. To address the Cu²⁺-induced destabilization of 2-m at the atomic level, we studied changes in the conformational dynamics of 2-m upon Cu²⁺ binding. Titration of 2-m with Cu²⁺ monitored by heteronuclear NMR showed that three out of four histidines (His13, His31, and His51) are involved in the binding at pH 7.0. ¹H-¹⁵N heteronuclear NOE suggested increased backbone dynamics for the residues Val49 to Ser55, implying that the Cu²⁺ binding at His51 increased the local dynamics of -strand D. Hydrogen/deuterium exchange of amide protons showed increased flexibility of the core residues upon Cu²⁺ binding. Taken together, it is likely that Cu²⁺ binding increases the pico- to nanosecond fluctuation of the -strand D on which His51 exists, which is propagated to the core of the molecule, thus promoting the global and slow fluctuations. This may contribute to the overall destabilization of the molecule, increasing the equilibrium population of the amyloidogenic intermediate.

Keywords: Amyloid fibrils; copper binding; dialysis-related amyloidosis; heteronuclear NMR; hydrogen/deuterium exchange; ₂-microglobulin; protein folding/misfolding

Abbreviations: 2-m, ₂-microglobulin • CD, circular dichroism • H/D, hydrogen/deuterium • HSQC, heteronuclear single quantum coherence • NMR, nuclear magnetic resonance • NOE, , nuclear Overhauser effect • R₁, longitudinal relaxation rates • TOCSY, total correlation spectroscopy

Supplemental material: See www.proteinscience.org

⁴ These authors contributed equally to this work.

⁵ Present address: Institute of Chemistry, University of the Philippines, Diliman, Quezon City, Philippines.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03445704.

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods Electronic supplemental material References

 
A growing number of proteins having the propensity to misfold and form amyloid fibrils have been recognized to be associated with the pathology of some important human diseases (Kelly 1998; Rochet and Lansbury Jr. 2000). These fibrils are characterized by a cross- structure where -strands are perpendicularly oriented to the axis of the polymeric fibril (Sunde et al. 1997; López et al. 2002). One such disease is dialysis-related amyloidosis, a debilitating complication acquired by patients undergoing long-term hemodialysis characterized by the deposition of ₂-microglobulin (2-m) amyloid fibrils in the synovial tissue, joint cartilage, and bone tissue (Gejyo et al. 1985; Geyjo and Arakawa 1990). 2-m is the light chain constituent of the class I major histocompatibility (MHC) complex (Bjorkman et al. 1987). It is a 12-kD protein composed of 99 amino acids that forms a typical immunoglobulin domain fold, that is, seven -strands arranged in a -sandwich and stabilized by hydrophobic interactions as well as a single disulfide bond between Cys25 and Cys80 (Bjorkman et al. 1987; Trinh et al. 2002; Verdone et al. 2002).

The formation of 2-m amyloid fibrils in vivo and in vitro has been extensively studied (Naiki et al. 1997; McParland et al. 2000; Chiti et al. 2001a,b; Heegaard et al. 2001; Kad et al. 2001, 2003). We analyzed the amyloid fibrils prepared by the seed-dependent extension reaction by using a novel procedure that combines hydrogen/deuterium (H/D) exchange of amide protons, dissolution of exchanged fibrils by dimethylsulfoxide, and 2D NMR analysis (Hoshino et al. 2002). It is noted that a similar procedure was independently developed by others, and was applied to the model amyloid fibrils formed by cold shock protein A (Alexandrescu 2001) or amyloidogenic fragment of Alzheimer -peptide (Ippel et al. 2002). Our results with 2-m amyloid fibrils indicated that most of the residues in the middle region of the molecule, including the loop regions in the native structure, form a -sheet core, explaining the marked rigidity of amyloid fibrils (Hoshino et al. 2002).

Several studies have offered some hints as to how these fibrils are formed from 2-m in the native state. A partially unfolded conformation of 2-m, having a higher propensity to aggregate compared to the native state in an environment close to physiological conditions, has been isolated using capillary electrophoresis (Chiti et al. 2001a; Heegaard et al. 2001). 2-m, lacking six residues in the N terminus, has been shown to increase amyloidogenicity, and has been found ex vivo together with other truncated species (Esposito et al. 2000). McParland et al. (2002) reported that an amyloid precursor that accumulated at pH 3.6 retains a stable structure in five (B, C, D, E, and F) of the seven -strands that comprise the hydrophobic core of the native protein. They suggested that this stable region is important for amyloid fibril formation. Because the optimal pH for amyloid fibril formation by the seed-dependent extension reaction is 2.5, where the protein is more unfolded, we proposed that the more disordered structure is important for amyloid fibril formation (Katou et al. 2002). We identified the minimal peptide fragment (residues 20–41) responsible for the amyloidogenic property of 2-m, which is substantially unfolded by itself (Kozhukh et al. 2002). Recently, we showed that an 11-residue peptide, Gln21-H31, also forms amyloid fibrils (Hasegawa et al. 2003). On the other hand, Jones et al. (2003) have identified that another peptide, Asp59–Thr71, form amyloid fibrils. More recently, studying the kinetics of spontaneous assembly of amyloid fibrils of 2-m at pH 2.5, Kad et al. (2003) suggested that various kinds of protein aggregates may act as nucleation sites.

One of the factors that could play a significant role in triggering 2-m amyloid fibril formation in vivo is interaction with transition metals, particularly Cu²⁺. As described by Morgan et al. (2001), although the majority of Cu²⁺ is tightly bound to plasma proteins, the dialysis procedure has two potential ways of allowing interaction of 2-m with free Cu²⁺: one is with Cu²⁺ in dialysate, and the other is with Cu²⁺ in cellulose membranes. Using urea and thermal denaturation as assessed by changes in intrinsic fluorescence, Morgan et al. (2001) have proven that the native 2-m is destabilized in the presence of Cu²⁺ at pH 7.4. They estimated a dissociation constant of 2.7 µM assuming an equimolar stoichiometry of Cu²⁺ for 2-m. Cu²⁺ binding has been shown to be specific compared with the nonspecific binding of Ca²⁺ and Zn²⁺ with 2-m. Eakin et al. (2002) further examined the effect of several divalent metal ions on the stability of 2-m, showing that 2-m is specifically destabilized by Cu²⁺. Out of the four histidine residues in 2-m (His13, His31, His51 and His84), Verdone et al. (2002) identified His31 and His13 as Cu²⁺ binding sites by performing Cu²⁺ titrations of 2-m using ¹H -2D TOCSY at pH 6.6. They suggested that Cu²⁺ binding may easily perturb the hydrophilic/hydrophobic balance, leading to the formation of a partially unfolded intermediate. With mutagenesis of potential coordinating histidine residues for Cu²⁺, Eakin et al. (2002) reported that, although 2-m binds Cu²⁺ specifically at His31 in the native state at pH 7.0–7.4, the binding of Cu²⁺ by nonnative states of 2-m at His13, His51, and His84 is responsible for overall destabilization.

The medical implications of the involvement of Cu²⁺ in 2-m amyloidosis are important because about 30% of the commercially available ultrafiltration membranes used in dialysis therapy contain Cu²⁺, which could easily leak out to the plasma, thus promoting fibril formation (Morgan et al. 2001). In a wider context, Cu²⁺ and other transition metals have also been associated with other amyloidogenic proteins. Zn²⁺ and Cu²⁺ in particular have been shown to strongly induce A aggregation in a pH-dependent manner (Atwood et al. 1998; Liu et al. 1999; Miura et al. 2000). Numerous studies have also been done on the binding of Cu²⁺ with prion protein (Stockel et al. 1998; Kramer et al. 2001). Synthetic peptides corresponding to the octapeptide repeat region of prion protein have been shown to bind Cu²⁺ with three sites available at pH 6.0 and four to five Cu²⁺-binding sites at pH 7.4. Cu²⁺ has also been associated with fibril formation of the pathogenic immunoglobulin light chain (Davis et al. 2001).

In this study, using heteronuclear NMR techniques, we have probed changes in the conformational dynamics of 2-m upon Cu²⁺ binding that may provide insight into its role in dialysis-related amyloidosis. First, to verify that Cu²⁺ destabilizes the native state, thermal denaturation using CD was performed. Effects of Cu²⁺ on the conformational dynamics of the 2-m structure were followed using ¹H-¹⁵N 2D NMR to identify perturbed sites. Steady-state {¹H}-¹⁵N heteronuclear NOE was used to monitor restricted backbone motions in the pico- to nanosecond time scale after considering the paramagnetic contribution of Cu²⁺ ion. Backbone amide H/D exchange was carried out to examine the effects of Cu²⁺ on the flexibility of the core residues.

	Results

TOP Abstract Introduction Results Discussion Materials and methods Electronic supplemental material References

 
Thermal denaturation
The heat stability of the native 2-m is nearly constant in the pH range of 6.2–8.0, as judged by thermal denaturation experiments followed by CD (Fig. 1, G_U~23–24 kJ/mole at 25°C). The protein and metal concentrations for the study of the effect of Cu²⁺ on the stability of 2-m were chosen to get a similar extent of saturation of Cu²⁺ as in the NMR measurements: 5 µM 2-m and 50 µM Cu²⁺ for the CD measurements versus 0.5 mM 2-m and 0.5 mM Cu²⁺ for the NMR measurements, resulting a saturation of ~0.94 for both measurements, estimated from a dissociation constant of 2.7 µM of Cu²⁺ on 2-m (Morgan et al. 2001). Destabilization of 2-m in the presence of Cu²⁺ at pH 7.0 was evident from the denaturation curves monitored by ellipticity at 220 nm (Fig. 1, lines 1,3), confirming previous results using tryptophan fluorescence (Morgan et al. 2001; Eakin et al. 2002). Although the validity of a two-state transition is ambiguous, especially in the presence of Cu²⁺, it will be useful to compare quantitatively the effects of Cu²⁺ on the stability of 2-m. The curve-fitting assuming a two-state transition indicated that T_m in the presence of Cu²⁺ is 55.4°C as opposed to 63.0°C in the absence of Cu²⁺, showing a difference of 7°C. G_U values in the absence and presence of Cu²⁺ at 25°C were estimated to be 23.3 kJ/mole and 13.4 kJ/mole, respectively. A previous study (Morgan et al. 2001) reported the G_U values in the absence and presence of 100 µM Cu²⁺ at pH 7.4 and 25°C to be 27.9 kJ/mole and 9.4 kJ/mole, respectively. For comparison, G_U at pH 8.5 and 5°C was estimated to be 34 kJ/mole from the unfolding transition induced by guanidine hydrochloride (Ohhashi et al. 2002).

View larger version (30K): [in this window] [in a new window]   Figure 1. Thermal denaturation of 2-m followed by ellipticity at 220 nm in the absence of Cu2+ at pH 7.0 (solid line, 1) and pH 6.5 (dots, 2) and in the presence of Cu2+ at pH 7.0 (solid line, 3) and pH 6.5 (dots, 4). Note that in the absence of Cu2+, the two transition curves obtained at pH 7.0 and pH 6.5 are overlapping. The protein and Cu2+ concentrations were 5 µM and 50 µM CuCl2, respectively, where the Cu2+ concentration is high enough to form the complex (Morgan et al. 2001). The fitted theoretical lines on the basis of standard thermodynamic equations (Nishii et al. 1995) are also plotted. In the curve fittings, Cp,U was assumed to be 5.6 kJ/mole/K. In the absence of Cu2+, Tm, Hm and GU(25°C) were 63.0°C, 317 kJ/mole and 23.3 kJ/mole, respectively, at pH 7.0, and 62.8°C, 319 kJ/mole and 23.5 kJ/mole, respectively, at pH 6.5. In the presence of Cu2+, the values were 55.4°C, 232 kJ/mole and 13.4 kJ/mole, respectively, at pH 7.0, and 60.7°C, 271 kJ/mole and 17.9 kJ/mole, respectively, at pH 6.5.

Copper binding sites
Assignments for the backbone peak resonances have been reported previously (Katou et al. 2002), where 95 of 97 expected residues have been unambiguously assigned (Fig. 2A). In the presence of 0.17 mM Cu²⁺ at 0.37 mM 2-m, peak intensities of many residues decreased, whereas others remained the same without a notable change in the chemical shift values (Fig. 2B). Although only a small proportion of the molecules were complexed with Cu²⁺, the fast-exchange averaging of the paramagnetic metal ion probably produced notable perturbations. Line broadening and a loss of peak intensity could be observed for residues close to Cu²⁺ due to its large electron relaxation rate, the effect of which diminishes according to the inverse-sixth power of the interatomic distance.

View larger version (28K): [in this window] [in a new window]   Figure 2. (A) 1H-15N HSQC spectra of 2-m at pH 7.0 and 25°C. Protein concentration was 0.37 mM. The backbone assignments are indicated by amino acid name and residue number. The blue labels indicate the residues notably affected by the addition of Cu2+. Green lines indicate the pairs of resonances from Gln/Asn side chain. (B) 1H-15N HSQC spectra of 2-m in the presence of Cu2+ at pH 7.0 and 25°C. Protein and Cu2+ concentrations were 0.37 mM and 0.17 mM, respectively. The four histidine residues are indicated. The completely disappeared histidine (His31) are indicated by red label and circle. (C) Overlay of the NMR spectra at pH 6.2 (red), pH 6.5 (green), and pH 7.0 (blue), showing the pH-dependent shift of the His51 peak. The four histidine peaks are labeled accordingly. These spectra were recorded at 37°C.

View larger version (66K): [in this window] [in a new window]   Figure 3. Ratios of peak intensities of 1H-15N HSQC spectra in the presence and absence of Cu2+ at pH 6.5 (A,B) and at pH 7.0 (C,D). Higher ratios (A,C) indicate less effect on the residues, while low values indicate larger perturbations (B,D). The following 16 residues were not used because of the absence of amide protein or low resolution of the peaks: I1, T4, P5, P14, S20, V27, P32, I35, K58, W60, F62, Y66, P72, S88, P90, and K91. The protein concentration was 0.33 mM and Cu2+ concentration was 0.1 µM for A and C and 0.8 mM for B and D. Titration was done at 25°C. In B and D, the locations of secondary structures obtained from the X-ray structure (7) and His residues are indicated.

The residues significantly affected by the addition of Cu²⁺ at pH 7.0 are indicated in the 3D structure of 2-m, where the locations of four histidine residues are also given (Fig. 4A). It can be seen that most of the affected residues are located near to the Cu²⁺ binding sites, for instance, His13, His31, and His51.

View larger version (35K): [in this window] [in a new window]   Figure 4. Structures of 2-m showing residues perturbed upon Cu2+ binding. (A) The residues that showed a significant decrease in intensity in the presence of Cu2+. The residues observed in the absence of Cu2+ but disappeared in Figure 3D are labeled in red. Other residues present in Figure 3D are labeled in white. (B) The residues with the decreased value of x R1 in the presence of Cu2+. The residues with the decrease larger than 0.1 in Figure 5C are labeled in red, and those with the decrease less than 0.1 are labeled in white. (C) The residues with an increased H/D exchange rate in the presence of Cu2+. The residues with the ratio higher than 1.5 are labeled in red, and those with the ratio less than 1.5 are labeled in white. The residues not used for the analysis are labeled in gray, and the two cysteine residues (Cys25 and Cys80) are labeled in yellow. The side chains of histidine residues are indicated. The 3D structures were drawn by MOLMOL (Koradi et al. 1996).

View larger version (49K): [in this window] [in a new window]   Figure 5. The effects of Cu2+ binding monitored based on longitudinal relaxation time R1 (A), backbone {1H}-15N NOE ( = (Isat - Ieq)/Ieq) (B), the product of R1 and NOE ( x R1) (C), and amide proton H/D exchange protection factors (D), at pH 7.0. Protein and Cu2+ concentrations were 0.5 mM. {1H}-15N steady-state NOE spectra in the presence (blue) and absence (red) of Cu2+ were obtained at 25°C. The locations of seven -strands (bars), His (empty circles), and Cys (filled circles) residues are indicated. In (D), protection factors (P) in the presence (filled bars) and absence (empty bars) of Cu2+ are shown. The ordinate on the right indicates GU estimated assuming the EX2 mechanism. The red dotted line indicates the overall GU value estimated from Gdn-HCl denaturation at 10°C (Ohhashi et al. 2002).

Amide H/D exchange
To address the effect of Cu²⁺ on the protected elements of the secondary structure, H/D exchange experiments were done at 25°C and a pD_r of 7.0. After 40 min of exchange in the absence of Cu²⁺, only 28 residues out of 95 were visible in the ¹⁵N-¹H HSQC spectrum, showing that not many residues are highly protected from exchange (Fig. 6A). After 22 h, several highly protected residues were still clearly seen (Fig. 6B). For each of the 28 protected residues, kinetics of H/D exchange were represented by plotting peak intensities against time (Fig. 7). The kinetics were fitted to a single exponential curve to obtain the apparent exchange rate (k_app). Exchange rates for residues His84, Thr86, and Asp98 were not determined as these residues disappeared quickly in the time course of the experiment. Residues with signal overlap such as Gln8, Ala15, Leu23, His51, Tyr63, and Tyr66 were also precluded from the analysis. The protection factor (P) was defined by k_int/k_ex = P, where k_int is the exchange rate in the random coil conformation (Bai et al. 1993; Connelly et al. 1993). The P values determined for all the residues available (Supplemental Material: Table 1) are plotted against the residue number (Fig. 5D).

View larger version (21K): [in this window] [in a new window]   Figure 6. H/D exchange of the backbone amide protons of 2-m in the absence of Cu2+ at 25°C. Protein concentrations was 0.5 mM. Spectra (A) was obtained 40 min after dissolving the protein in D2O in deuterated phosphate buffer at pDr 7.0. Only 35 core residues remained after 40 min of exchange. Spectrum (B) was obtained after 22 h.

View larger version (35K): [in this window] [in a new window]   Figure 7. Representative examples of peak intensity decay during H/D exchange in the absence (empty circles) and presence (filled circles) of Cu2+. The decay kinetics were fitted to a single exponential curve to obtain the backbone proton exchange rate constant (kEX).

In the presence of Cu²⁺, the exchange rates increased by two- to threefold for residues Val24, Tyr26, Glu36, Asp38, Leu40, Lys41, Ala79, Val82, and Val93 (Figs. 5D and 7, and Supplemental Material: Table 1). Although the magnitude was not large, the promotion of exchange by Cu²⁺ was solid for these residues. Most of these affected residues are located in the highly protected -strands B, C, and F. For residues Phe22, Ser28, Glu44, Leu64, Leu65, Tyr67, Thr68, Leu87, and Lys91, k_app was not significantly changed by Cu²⁺ binding. As reflected in the change of protection factors, at physiological pH and at 25°C, Cu²⁺ increased the H/D exchange rate of most of the core residues (Figs. 4C, 5D). Thus, measured by H/D exchange, the effects of Cu²⁺ binding were transmitted to the core of 2-m.

The apparent decay of H signals could also be explained by the molecular association, as it was reported that fibril formation is enhanced in the presence of Cu²⁺ and low concentration of urea (Morgan et al. 2001). However, this is unlikely under our conditions, judging from the constant line shape of aliphatic signals in the 1D spectra recorded during the time course of H/D exchange reaction.

Mass spectrometry
The observed changes in the protein’s dynamics and conformation could be due to protein oxidation caused by Cu²⁺, a well-known issue with this cation (Schoneich and Williams 2002). We examined the oxidation of proteins by ESI-MS using a Q-TOF mass spectrometer fitted with a Z-spray nanoflow electrospray ion source (Micromass), which has a very high resolution to be able to detect the modification of the whole 2-m molecule without fragmentation. The protein samples were incubated under the conditions of 0.5 mM 2-m, 50 mM sodium phosphate buffer at pH 7.0 in the absence or presence of 0.5 mM CuCl₂ for 3 days at 25°C. The mass spectra clearly showed a small amount of oxidized species with increase of the mass of 16 in both samples incubated in the absence and presence of Cu²⁺ (data not shown). Although the exact oxidation site is not clear, the C-terminal Met99 is most probable site, considering the fact that 2-m has one Met and four His residues. However, these modified species were independent of Cu²⁺, demonstrating that the observed conformational changes are not due to protein oxidation, but the direct effects caused by binding of Cu²⁺ to His residues.

	Discussion

TOP Abstract Introduction Results Discussion Materials and methods Electronic supplemental material References

 
Copper-induced destabilization of 2-m
The results presented here reveal at the amino acid residue level the conformational and dynamic changes that are produced by the interaction of 2-m with Cu²⁺. Although the binding of metal ions with proteins would generally tend to lower the free energy in the native state, thus stabilizing the native form (Werner et al. 2000), the binding of Cu²⁺ to His residues induces a slightly modified native state of 2-m with increased flexibility and decreased stability (Fig. 1). This conformational change, coupled with the binding of Cu²⁺, can be described in an alternative way that the preferential binding of Cu²⁺ to the destabilized native state shifts the equilibrium between protein substates toward the destabilized state, as represented by the Wyman linkage function (Wyman Jr. 1964). Similar but more drastic conformational change coupled with ligand binding has been reported recently for the binding of Congo red to an amyloidogenic immunoglobulin light chain, SMA, where Congo red binding populates partially unfolded states to enhance aggregation and amyloid fibril formation (Kim et al. 2003).

Heteronuclear NMR enabled the residue-based analysis of Cu²⁺ binding. First, we showed that three histidine residues—that is, His13, His31, and His51—are capable of binding Cu²⁺ at pH 7.0 and two—that is, His13 and His31—at pH 6.5 (Fig. 3). The difference in the binding sites at pH 6.5 and pH 7.0 can be explained by the change in the protonation state of His51 between the two pHs. Because the deprotonation of the His side chain occurs at neutral pH, a slight shift in the pH value will result in a large change in binding behavior. A link between the deprotonation of histidines and Cu²⁺ binding was also proposed for the Alzheimer -amyloid and the prion protein (Atwood et al. 1998; Stockel et al. 1998).

Among the three His residues (His13, His31, and His51) responsible for the Cu²⁺ binding at pH 7.0, only the residues near to His51 (i.e., Asp49 and Ser55) revealed the clear increase of backbone dynamics (Fig. 5). Although scarce data points makes solid interpretation difficult, the results suggest that the Cu²⁺ binding at His51 increased pico- to nanosecond time scale backbone dynamics of -strand D. His51 is located at the middle of -strand D, whereas others are in the loop or at the end of -strand. This might be related to the fact that the increase of backbone dynamics is observed for the residues near to His51.

Trinh et al. (2002) reported the crystal structure of a monomeric 2-m that reveals remarkable structural changes in -strand D relative to the MHC-bound 2-m. Although -strand D in the MHC-bound form is separated by a -bulge, the -bulge is lost in the crystal structure of monomeric 2-m, so that the intermolecular hydrogen bonds are formed with adjacent molecules within the lattice. The conformational change of -strand D is accompanied by a 180° rotation of His51. They proposed that this drastic structural change observed in -strand D is a key event leading to amyloid fibril formation. Although such drastic change was not evident in the solution structure studied by NMR (Katou et al. 2002; Verdone et al. 2002), -strand D is the most unstable strand, as shown by the absence of protected amide protons (Fig. 5D). In other words, it is likely that the backbone dynamics of -strand D is easily affected by the ligand binding to His51. Thus, the binding of His51 with Cu²⁺ may perturb -strand D, consequently destabilizing these regions by breaking H-bonds between strands D and E. Increase of backbone dynamics upon ligand binding was also reported for mouse major urinary protein with a hydrophobic pheromone 2-sec-butyl-4,5-dihydrothiazole (Zídek et al. 1999).

A possible mechanism of Cu²⁺-induced destabilization of 2-m can be summarized by Figure 4. Cu²⁺ binds to three histidines (His13, His31, and His51) of the native conformation of 2-m at pH 7.0, as monitored by the decrease in peak intensity (Fig. 4A). The product of R₁ and NOE values, which is independent of the paramagnetic contributions of Cu²⁺, reflects exclusively the dynamical motion (Fig. 5C; see below). Thus, we suggest that, as monitored by ¹H-¹⁵N NOE, Cu²⁺ binding to His51 increases the pico- to nanosecond time scale backbone dynamics of the residues on -strand D (Fig. 4B). These local and rapid fluctuations are linked to the increase of the global and slow fluctuations of 2-m core residues, as monitored by H/D exchange (Fig. 4C). In other words, the specific effects on the residues close to the Cu²⁺ binding sites, in particular at His51, may be cooperatively propagated as these residues increase mobility in the adjacent regions by weakening the hydrogen bonds along the backbone and by subtly altering hydrophobic interactions. In addition, the increased dynamics at the N-terminal residues (Fig. 5C) suggests a mobility increase of -strand A. It is intriguing to consider that the mobility increase of -strand A reflects the unpairing of strand A, leading to the amyloidogenic partially unfolded intermediates as suggested by Verdone et al. (2002).

On the other hand, Eakin et al. (2002) reported that an increased affinity of Cu²⁺ to the nonnative states compared to the native state gives rise to the overall destabilization. The present results strongly suggest that the Cu²⁺ binding to the native state contributes to destabilizing 2-m, even if the binding to the nonnative state might also be involved.

Paramagnetic effects
Although the decrease of the magnitude of NOE suggested the increase in pico- to nanosecond time scale backbone dynamics on D strand on which His51 exists (Fig. 5B), an alternative possibility of the paramagnetic effects of the Cu²⁺ ions on NOE should be considered carefully. It has long been known that the magnetic interaction between an electronic spin and a nuclear spin causes nuclear relaxation. This so-called paramagnetic effect, can cause drastic increases in both the longitudinal and traverse relaxation rates, resulting in line-broadening and loss of NMR signal, as clearly observed in the Cu²⁺ titration experiment (Fig. 3) and the R₁ measurement (Fig. 5A). Here, let us consider the paramagnetic effect on the heteronuclear NOE.

The heteronuclear NOE between amide H-N pair is given by

(1)

in which I_sat and I_eq are the peak intensities when ¹H is saturated and at thermal equilibrium, respectively, _H and _N are the gyromagnetic ratios for ¹H and ¹⁵N, respectively, R_Nz is the ¹⁵N longitudinal relaxation rate, and R_HzNz is the cross-relaxation rate. In the presence of paramagnetic metals, ¹⁵N longitudinal relaxation rate is given by the sum of diamagnetic () and paramagnetic () contributions (Caffrey et al. 1995):

(2)

The diamagnetic terms consist of dipole–dipole coupling and chemical shift anisotropy components, and described by

(3)

(4)

where is Plank’s constant divided by 2, µ₀ is the permeability of free space, _H and _N are the gyromagnetic ratios of ¹H and ¹⁵N, _H and _N are the Larmor frequencies of ¹H and ¹⁵N, r_NH is the N—H bond length, and is the chemical shift anisotropy value. J() is the spectral density function, defined as

(5)

where _c is the correlation time.

The paramagnetic contribution to the longitudinal relaxation rate is essentially dominated by the dipole–dipole coupling between the electron spin and the nuclear spin, and is expressed by

(6)

(7)

where S is the electron spin quantum number, _S is the gyromagnetic ratio for the electron, r_NS is the nuclear-electron distance, _S is the Larmor frequency of electron spin, and _S is the correlation time of the electron. The second equality in equation 6 was obtained because _S >> _N.

By substituting equations 2–7 into equation 1, the heteronuclear NOE could be simulated as a function of rotational correlation time, , at different nuclear-electron distances (Fig. 8A). In the absence of paramagnetic effect (r_NS 15Å), the heteronuclear NOE exhibits the strong dependence on the rotational correlation time, ranging from a minimum of approximately -4 at the correlation time less than 0.1 nsec to a maximum approximately -0.14 at the correlation time larger than 10 nsec. In the presence of paramagnetic effect, this pronounced NOE at longer correlation time strongly reduced (approaches to zero), depending on the nuclear–electron distance. The rotational correlation time of 2-m (11.7 kD) is estimated to be 5.5 nsec using the Stokes-Einstein equation. In this region, the NOE is originally close to 0, and the paramagnetic effect is relatively weak. Most importantly, the paramagnetic effect is always positive, increasing the NOE (i.e., bringing the NOE close to 0).

View larger version (46K): [in this window] [in a new window]   Figure 8. (A) 1H-15N heteronuclear NOE simulated as a function of correlation time with different 15N-electron distance (rNS). Dashed lines show two extreme conditions (rNS = 1 Å or ). (B–D) The effect of intramolecular motions and paramagnetic contribution. The values of R1 (B), NOE (C), and the product of R1 and NOE (D) was plotted as a function of the effective correlation time for internal motions with different generalized order parameter values. In (B), the values of S2 are 1.0, 0.9, 0.8, 0.7, and 0.6 from the top. The solid lines are obtained in the absence of Cu2+, while the dotted lines are in the presence of paramagnetic effect (rNS = 8 Å and c = 5.5 nsec). Note that in (D), both lines are overlapped. The values used for simulation were = 1.05 x 10-34 J•s, µ0 = 4 x 10-7 T2•J-1•m3, H = 2.6752 x 108 T-1•sec-1, N = -2.712 x 107 T-1•sec-1, H = 800.33 MHz, N = 81.13 MHz, rNH = 1.02 Å, = -160 ppm, S = 3/2, S = -1.76 x 1011 T-1•sec-1, S = 526.5 GHz, and S = 0.05 nsec.

(8)

where ^-1 = _m^-1 + _e^-1, S² is the square of the generalized order parameter, _m is the overall rotational correlation time, and _e is the effective correlation time for internal motions. By using equation 8 instead of equation 5, the R₁ and NOE are simulated as a function of effective correlation time (Fig. 8B,C). With this formula, it is clear that the NOE is highly dependent on both the S² (amplitude of the internal motions) and _e (correlation time of the internal motions). Those simulations also show clearly that both R₁ and NOE are always increased if a paramagnetic contribution is present. Conversely, the product of R₁ and NOE does not involve the paramagnetic contribution, as predicted from equations 1–3. Indeed, the simulated value of the product of R₁ and NOE does not depend on the presence of a paramagnetic effect, but rather exclusively depends on the internal dynamic motions (Fig. 8D). Although the quantitative determination of the S² and _e needs comprehensive measurements of the other relaxation parameters, that is, R₁ and R₂ at various magnetic field strengths, the significant decrease in the NOE strongly suggests the Cu²⁺-induced increase of the mobility in the pico- to nanosecond time scale.

Protected core of 2-m
H/D exchange clarified the protected core of 2-m. Protection factors obtained from the H/D exchange experiment can be related to the parameters of protein unfolding at each amide site assuming a two-process model between the folded (N) and unfolded (U) states and the exchanged state (X):

(Scheme 1)

where k_U and k_R represent the unfolding and refolding rate constants, respectively, and k_EX is the intrinsic exchange rate (Kim et al. 1993; Englander 2000). The equilibrium constant (K_U) between N and U is related to the microscopic rate constants by K_U = k_U/k_R. Under conditions where the conformational equilibrium is much faster than the intrinsic rate of exchange (EX2 limit), the apparent exchange rate is represented by K_Uk_EX. Here, the protection factor (P) corresponds to 1/K_U and G_U is estimated by G_U = RT ln P. Under the other extreme condition where k_EX is much faster than the refolding rate constant k_R, the apparent rate constant represents k_U (EX1 limit). Under intermediate conditions where k_R is comparable to k_EX, G _U estimated on the basis of EX2 limit would be larger than the true value.

We converted the protection factors into G_U assuming the EX2 limit (Fig. 5D, right ordinate). The G_U values for the highly protected residues are 30–45 kJ/mole. It is generally considered that, while the largest G_U values obtained from the amide H/D exchange experiment represent those of global unfolding, the local fluctuation or penetration of water molecules into the protein interior decreases the G_U value, thus causing the wide distribution of observed G_U (Kim et al. 1993; Englander 2000). In this context, the G_U value for global unfolding of 2-m at 25°C extrapolated from the thermal denaturation is 23 kJ/mole and G_U at 10°C obtained from the Gdn-HCl denaturation is 34 kJ/mole (Ohhashi et al. 2002). The G_U values for the highly protected amide protons are larger than those for global unfolding. However, the exact comparison of these values is difficult because of the probable deviation from a two-state mechanism, in particular in the presence of Cu²⁺. Although further study is necessary, it is likely that the mechanism of H/D exchange also deviates from the EX2 mechanism. Alternatively, the denatured form of 2-m under physiological conditions retains the native-like core made of -strands B, C, and F, which prevents rapid H/D exchange. The maximal decrease in G_U upon Cu²⁺ binding estimated from the H/D exchange experiment is about 2 kJ/mole, less than that obtained for the thermal denaturation (10 kJ/mole). This is partly explained by the fact that, under the present conditions of H/D exchange at pD_r 7.0, not all of the three histidine residues were fully complexed with Cu²⁺. Moreover, the difference suggests the contribution of the Cu²⁺ binding to the denatured state as indicated by Eakin et al. (2002).

In conclusion, in light of its role in the amyloid fibril formation of 2-m in long-term hemodialysis, Cu²⁺ would act by making the thermodynamics and the kinetics more favorable for the formation of the amyloidogenic 2-m intermediate needed for fibril formation. Cu²⁺ binding are likely to increase the local and rapid fluctuations of the binding site at His51 as observed by {¹H}-¹⁵N NOE measurement, which are then propagated to the core of the molecule, leading to the global and slow fluctuations of the entire molecule as indicated by H/D exchange. This contributes to the Cu²⁺-induced overall destabilization of the 2-m molecule, probably increasing the equilibrium population of amyloidogenic intermediate. The increased flexibility of -strand A may expose the minimal amyloidogenic region found in -strands B and C, identified by Khozukh et al. (2002), which may trigger fibril formation, as suggested by Verdone et al. (2002). Additional binding of Cu²⁺ ions to the denatured state and preformed amyloid fibrils, not examined here, would further promote the fibril formation. Detailed characterization of the amyloid fibril formation in the presence of Cu²⁺ ions will be important to clarify the connection between a destabilization effect of Cu²⁺ ions and amyloid fibril formation.

	Materials and methods

TOP Abstract Introduction Results Discussion Materials and methods Electronic supplemental material References

 
Recombinant 2-m
Recombinant human 2-m with four additional residues at the N terminus (Glu(-4)-Ala(-3)-Tyr(-2)-Val(-1)-Ile(1)) was expressed in methylotrophic yeast Picihia pastoris and purified as described previously (Kozhukh et al. 2002; Ohhashi et al. 2002). It has been confirmed that recombinant 2-m is indistinguishable from 2-m derived from patients with respect to amyloid fibril formation. Uniformly ¹⁵N-labeled 2-m was obtained using ¹⁵N-ammonium hydroxide as the sole nitrogen source.

CD measurements
CD measurements were done with a Jasco spectropolarimeter Model J-720. The results are expressed as mean residue ellipticity. Thermal denaturation was carried out in 10 mM sodium phosphate buffer (pH 6.5 and pH 7.0) containing 300 mM KCl. The protein and CuCl₂ concentrations were 5 µM and 50 µM, respectively. Heating was performed at 0.5°C/min up to 85°C. Thermal denaturation was reversible in both the presence and absence of CuCl₂ judging from the signal intensity obtained after decreasing the temperature. It is noted that, in the presence of CuCl₂ at pH 6.5, a high concentration of salt (i.e., 300 mM KCl) was necessary to prevent the aggregation at high temperature. The transitions in the absence of CuCl₂ at 300 mM KCl were the same as those at 100 mM KCl at both pH 6.5 and pH 7.0. The unfolding curves were analyzed on the basis of a two-state transition between the native state (N) and the unfolded state (U): N U, with the standard thermodynamic equations (Nishii et al. 1995). The observed unfolding curves were fitted to the theoretical curve to obtain the following thermodynamic parameters: the midpoint temperature of unfolding (T_m), the enthalpy change at T_m (H_m), and the free energy change of unfolding at 25°C (G_U [25°C]). It is noted that a constant value (5.6 kJ/mole/K) was assumed for the heat capacity change of unfolding (C_p,U), which was obtained based on the pH-dependence of the unfolding transition curves monitored by CD (J. Kardos and Y. Goto, unpubl.).

NMR measurements
The spectra were recorded mainly on a 500 MHz spectrometer (Bruker DRX 500) as described before (Katou et al. 2002). For the relaxation measurements (R₁ and NOE), a 800 MHz spectrometer (Bruker DRX 800) was used. Processing of the data acquired was done using nmrDraw and PIPP (Garrett et al. 1991; Delaglio et al. 1995). For the titration experiments, a ¹⁵N-labeled sample (0.47 mM) was titrated with CuCl₂ and the ¹H-¹⁵N HSQC spectra were measured at 25°C. The buffer used was 50 mM sodium phosphate containing 100 mM KCl at pH 7.0 or pH 6.5.

The measurement of steady-state heteronuclear {¹H}-¹⁵N NOE was carried out at 25°C according to the procedure described by Farrow et al. (1994). The NOE values were calculated as the ratios of peak heights from spectra obtained with and without a ¹H saturation period. The protein and CuCl₂ concentrations were 0.5 mM, in 50 mM sodium phosphate buffer (pH 7.0). It is noted that because NMR spectra were measured at high protein concentrations in comparison with those used for CD measurements, a low molar ratio of Cu²⁺ over protein concentration is enough to obtain the stoichiometric binding.

The measurement of R₁ was carried out as described by Farrow et al. (1994) at 25°C with relaxation times of 20, 100, 200, 300, 400, 600, 800, and 1000 msec. The peak intensity as a function of relaxation time was analyzed on the basis of a single exponential decay. The same protein solutions as used for NOE measurements were used.

H/D exchange in the presence and absence of Cu²⁺ was performed at 25°C and pD_r 7.0, where pD_r is the pH meter reading without correction for the isotope effect. Both the protein and the CuCl₂ concentrations were 0.5 mM. The amide proton decay was followed by measuring peak intensities in the ¹H-¹⁵N HSQC spectra. The decay curves were fitted to a single exponential curve to obtain the apparent rate constant of exchange (k_app).

	Electronic supplemental material

TOP Abstract Introduction Results Discussion Materials and methods Electronic supplemental material References

 
Supplemental material is presented for H/²H exchange protection factors of 2-microglobulin in the absence and presence of CuCl₂ (Table 1).

	Acknowledgments

 
We thank Dr. Gennady Khozhuk for the preparation of the ¹⁵N-labeled 2-m, Dr. Yoshinori Satomi and Professor Takafumi Takao for mass spectroscopy, and Professor Takahisa Ikegami for helpful suggestions. This work was supported in part by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports and Culture. J.K. is supported by Japan Society for the Promotion of Science, and J.V. was supported by the COE Foreign Fellowship Grant from the Osaka University.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

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