Allosteric modulation of myristate and Mn(III)heme binding to human serum albumin Optical and NMR spectroscopy characterization Gabriella Fanali1, Riccardo Fesce1, Cristina Agrati1, Paolo Ascenzi2,3 and Mauro Fasano1 ` Dipartimento di Biologia Strutturale e Funzionale, and Centro di Neuroscienze, Universita dell’Insubria, Busto Arsizio (VA), Italy ` Dipartimento di Biologia, and Laboratorio Interdisciplinare di Microscopia Elettronica, Universita ‘Roma Tre’, Italy Istituto Nazionale per le Malattie Infettive I.R.C.C.S ‘Lazzaro Spallanzani’, Roma, Italy Keywords allostery; fatty acid binding; heme binding; human serum albumin; NMR relaxation Correspondence M Fasano, Dipartimento di Biologia ` Strutturale e Funzionale, Universita dell’Insubria, Via Alberto da Giussano 12, I-21052 Busto Arsizio (VA), Italy Fax: +39 0331 339459 Tel: +39 0331 339450 E-mail: mauro.fasano@uninsubria.it Website: http://fisio.dipbsf.uninsubria.it/cns/ fasano (Received 21 April 2005, revised 25 July 2005, accepted 26 July 2005) doi:10.1111/j.1742-4658.2005.04883.x Human serum albumin (HSA) is best known for its extraordinary ligand binding capacity HSA has a high affinity for heme and is responsible for the transport of medium and long chain fatty acids Here, we report myristate binding to the N and B conformational states of Mn(III)heme–HSA (i.e at pH 7.0 and 10.0, respectively) as investigated by optical absorbance and NMR spectroscopy At pH 7.0, Mn(III)heme binds to HSA with lower affinity than Fe(III)heme, and displays a water molecule coordinated to the metal Myristate binding to a secondary site FAx, allosterically coupled to the heme site, not only increases optical absorbance of Mn(III)heme-bound HSA by a factor of approximately three, but also increases the Mn(III)heme affinity for the fatty acid binding site FA1 by 10–500-fold Cooperative binding appears to occur at FAx and accessory myristate binding sites The conformational changes of the Mn(III)heme–HSA tertiary structure allosterically induced by myristate are associated with a noticeable change in both optical absorbance and NMR spectroscopic properties of Mn(III)heme–HSA, allowing the Mn(III)-coordinated water molecule to exchange with the solvent bulk At pH ¼ 10.0 both myristate affinity for FAx and allosteric modulation of FA1 are reduced, whereas cooperation of accessory sites and FAx is almost unaffected Moreover, Mn(III)heme binds to HSA with higher affinity than at pH 7.0 even in the absence of myristate, and the metal-coordinated water molecule is displaced As a whole, these results suggest that FA binding promotes conformational changes reminiscent of N to B state HSA transition, and appear of general significance for a deeper understanding of the allosteric modulation of ligand binding properties of HSA Human serum albumin (HSA) is the most prominent protein in plasma, but it is also found in tissues and secretions throughout the body HSA abundance (its concentration being 45 mgỈmL)1 in the serum of human adults) contributes significantly to colloidosmotic blood pressure HSA, best known for its extraordinary ligand binding capacity, is constituted by a single nonglycosylated all-a chain of 65 kDa containing three homologous domains (labelled I, II, and III), each composed of two (A and B) subdomains The three domains have different binding capacity for a broad variety of ligands such as aminoacids (Trp and Cys), hormones, metal ions, and bilirubin Moreover, HSA has a high affinity for heme and is Abbreviations FA, fatty acid; HSA, human serum albumin; MSE, mean square error; NMRD, nuclear magnetic relaxation dispersion 4672 FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS G Fanali et al responsible for the transport of lipophilic compounds and drugs and of medium and long chain fatty acids; among them, myristic acid is a stereotypic ligand to investigate fatty acid binding and transport properties of HSA [1–8] Fatty acids (FAs) are required for the synthesis of membrane lipids, hormones and second messengers, and serve as an important source of metabolic energy Although the binding of fatty acids to human and bovine serum albumin has been thoroughly investigated over many years, their binding mode and thermodynamics are still objects of debate By combining biochemical and biophysical approaches, a common consensus view has been reached on there being three high-affinity fatty acid binding sites, and at least three further low affinity sites have been envisaged NMR studies on tryptic and peptic fragments of bovine serum albumin have localized two high affinity sites in domain III and one in the N-terminal half of the protein Structural X-ray diffraction studies have demonstrated that HSA is able to bind up to seven equivalents of long chain FAs at multiple binding sites (labelled FA1 to FA7; Fig 1) with different affinity In sites FA1–5 the carboxylate moiety of fatty acids is anchored by electrostatic ⁄ polar interactions; on the contrary, sites FA6–7 not display a clear evidence of polar interactions that keep in place the carboxylate head of the fatty acid, thus Fig Ribbon representation of the heart-shaped structure of HSA with the seven fatty acid binding sites labeled (FA1 to FA7); sites are occupied by myristate anions rendered with red sticks N- and C-termini of the polypeptide chain are labeled accordingly Atomic coordinates are taken from [6,8,13,14] The figure was drawn using the SWISS PDB viewer (http://www.expasy.org/spdbv/) FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS Myristate and Mn(III)heme binding to HSA–heme suggesting that sites FA6–7 are low-affinity fatty acid binding sites [1,6,7,9–17] The fatty acid binding site FA1, located in subdomain IB (Fig 1), acts as the heme binding site as well, with the tetrapyrrole ring arranged in a d-shaped cavity limited by two tyrosine residues (Tyr138 and Tyr161) that provide p-p stacking interaction with the porphyrin and supply a donor oxygen (from Tyr161) for the ferric heme iron Ferric heme is secured by the long IA-IB connecting loop that fits into the cleft opening Heme propionates point toward the interface between domains I and III and are stabilized by salt bridges with His146 and Lys190 residues [6,8] HSA undergoes pH- and allosteric effector-dependent reversible conformational isomerization(s) Between pH 2.7 and 4.3, HSA shows a fast (F) form, characterized by a dramatic increase in viscosity, much lower solubility, and a significant loss in helical content Between pH 4.3 and 8, in the absence of allosteric effectors, HSA displays the normal (N) form that is characterized by heart-shaped structure Between pH 4.3 and 8, in the presence of allosteric effectors, and at pH greater than 8, in the absence of ligands, HSA changes conformation to the basic (B) form with loss of a-helix and an increased affinity for some ligands, such as warfarin [5,18–23] Fatty acids are effective in allosterically regulating ligand binding to Sudlow’s site I and to the heme cleft Myristate regulates HSA binding properties in a complex manner, involving both competitive and allosteric mechanisms The structural changes associated with FAs binding can essentially be regarded as relative domain rearrangements to the I-II and II-III interfaces This allosteric regulation is not observed for short FAs (e.g octanoate) that preferably bind to Sudlow’s site II and displace the specific ligands (e.g ibuprofen) without inducing HSA allosteric rearrangement(s) This indicates that the hydrophobic interactions between the long FA polymethylenic tail and HSA drives allosteric rearrangements In turn, Sudlow’s site I ligands (e.g warfarin) displace FA7, while Sudlow’s site II ligands (e.g ibuprofen) displace FA3 and FA4 Moreover, heme binding to HSA displaces FA1 [6,8,13,16,23–26] Heme binding to HSA endows this protein with peculiar optical absorbance and magnetic spectroscopic properties that can be used to follow ligand- and pHdependent conformational transition(s) [19–22,27] In particular, Mn(III)heme can be used instead of Fe(III)heme in order to increase the strength of the dipolar interaction with water protons when their NMR relaxation rate is measured [19,20] Although an even stronger dipolar interaction could be obtained using 4673 Myristate and Mn(III)heme binding to HSA–heme G Fanali et al Mn(II)heme, the metal undergoes oxidation under aerobic conditions in porphyrin complexes [28] Heme regulates allosterically drug binding to Sudlow’s site I In fact, heme affinity for HSA decreases by about one order of magnitude upon warfarin binding Reciprocally, heme binding to HSA decreases warfarin affinity by the same extent [19] Fe(III)heme allosterically inhibits ligand binding to Sudlow’s site I, possibly by stabilizing the neutral (N) state of HSA Vice versa, ligand binding to Sudlow’s site I impairs Fe(III)heme–HSA formation, possibly by stabilizing the basic (B) state of HSA [5,18,23,29–31] Here, we report the spectroscopic analysis of the myristate-dependent conformational changes of the N and B states of Mn(III)heme–HSA, by optical absorbance spectroscopy and NMR spectroscopy, that show allosteric interaction(s) between FAs and Mn(III)heme with HSA Interestingly, FAs increase Mn(III)heme affinity to HSA, whereas warfarin and FA7 ligands were reported to behave in the opposite way with respect to ferric heme binding to HSA [19,21,31] Additionally, the affinity of Mn(III)heme for HSA and the spectroscopic properties of the Mn(III)heme–HSA adduct in the presence of myristate are similar to those of the B conformational state of HSA, suggesting that myristate binding to one or more modulatory sites possibly drives the N to B state HSA transition Results In the absence of myristate, at pH 7.0 (i.e HSA in the N conformational state), Mn(III)heme binds to fatty acid-free HSA with a dissociation constant KH % 2.0 · 10)5 m (Fig 2A) Although the binding curve does not reach saturation and therefore the KH value should be considered as a lower limit, it is worth to note that it is two order of magnitude larger than that measured for Fe(III)heme [32] In the presence of 1.0 · 10)4 m myristate, the optical absorbance spectrum of Mn(III)heme–HSA displays a characteristic shoulder at 440 nm with well-defined isosbestic points (Fig 2B) In the presence of myristate, the expression for HSA-bound Mn(III)heme concentration could not be solved analytically Four major features are evident in optical absorbance difference (DA) curves: (a) at low HSA concentrations, the curves are depressed by myristate, indicating that myristate affinity for FA1 is higher than that of Mn(III)heme in the absence of myristate, and Mn(III)heme binding to FA1 is precluded by competition equilibrium (left column of Scheme 1) (b) Maximal DA values are clearly increased in the presence of myristate, thereby indica4674 Fig (A) Binding isotherms for Mn(III)heme binding to fatty acidfree HSA and to the HSA–myristate complexes, at pH 7.0 and 25.0 °C; open triangles: no myristate; solid triangles: 5.0 · 10)6 M myristate; open circles: 1.0 · 10)5 M myristate; solid circles: 2.5 · 10)5 M myristate; crossed diamonds: 5.0 · 10)5 M myristate; open diamonds: 7.5 · 10)5 M myristate; solid diamonds: 1.0 · 10)4 M myristate The continuous lines were obtained by numerical fitting of the data Values of the dissociation equilibrium constants obtained according to Scheme are given in Table (B) UV-visible spectral changes observed for a solution of 1.0 · 10)5 M Mn(III)heme titrated with HSA (0–3.0 · 10)5 M) in the presence of 1.0 · 10)4 M myristate, at pH 7.0 and 25.0 °C The arrows indicate the increase of HSA concentration ting that binding of myristate to a modulatory site FAx increases the signal yield of the complex The DA value for 1.0 · 10)5 m Mn(III)heme–HSA–myristate complex can be estimated about A10* ¼ 0.33, by normalizing the value observed at 1.0 · 10)4 m myristate and 30 lm HSA (0.255) to full Mn(III)heme binding, based on the molar fraction of the Mn(III)heme–HSA adduct that gave similar spectral data at pH 10.0 (see below) Furthermore, (c) at intermediate HSA concentration the binding curves rapidly rise and appear to FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS Myristate and Mn(III)heme binding to HSA–heme KH (Hem)P(…)–(…) KMM (…)P(Myr)–(…) KHM KMSn ↔ KMH ↔ (Myr)P(Myr)–(Myr)n KM (…)P(Myr)–(Myr)n KHM ↔ ↔ ↔ (…)P(…)–(…) KM* ↔ KM (Myr)P(Myr)–(…) KMSn ↔ ↔ ↔ (Myr)P(…)–(…) KM* ↔ ↔ G Fanali et al (Hem)P(Myr)–(…) KMSn ↔ (Hem)P(Myr)–(Myr)n Scheme Allosteric and competition equilibria involving Mn(III)heme and myristate binding to HSA Binding sites are indicated with the notation (FA1)P(FAx)–(FAS), where FA1 is the heme binding site, acting as myristate binding site as well, FAx is a different myristate binding site allosterically coupled to FA1, and FAS are n secondary myristate binding sites, with different affinities, allosterically uncoupled to FA1 P ¼ protein, HSA; Myr ¼ myristate; Hem ¼ Mn(III)heme Values of the dissociation equilibrium constants are given in Table and in the text The framed transition is associated with a change in the optical absorption spectrum (see text) reach saturation for HSA concentrations well lower than in the absence of myristate This indicates that binding of myristate to the modulatory site also increases the affinity of Mn(III)heme for FA1 (Scheme 1, central column) Finally, (d) at high HSA concentration and intermediate myristate concentrations (1.0– 5.0 · 10)5 m) the binding curves decline, suggesting that unbinding of myristate from FAx occurs, according to equilibrium of the framed reaction in Scheme A kinetic model was set up to numerically fit the optical absorbance data reported in Fig 2A The minimal core of the model was based on the competition between Mn(III)heme and myristate for binding to FA1 (defined by the parameters KH and KM) and on the allosteric modulation of FA1 properties by myristate binding to FAx (defined by the parameters KM*, K H M , and possibly K MM1 KM, if myristate binding to FA1 is also modulated; see Experimental procedures for explanation of the notations for the equilibrium constants) However, binding of myristate to additional FA sites must also be considered, to take into account the decrease in free myristate concentration at increasing concentrations of HSA; this requires the further set of parameters K MS1 to K MS5 For the sake of simplicity, these constants were bound to a fixed affinity ratio series, with K MS1 as a free parameter and K MSn ¼ K MS1 ⁄ 10(n)1) ⁄ 2, n ¼ 2–5; this is in general agreement with the estimates reported in the literature [1,9,10,33] Two further free parameters (in addition to KH, KM, K HM , KM* and K MS1 ) completed the model: the asymptotic absorbance in the absence of myristate (A10) and the absorbance of 1.0 · 10)5 m Mn(III)heme–HSA–myristate complex (A10*) However, this simplified model did not adequately fit the experimental data (MSE ¼ 4.9 · 10)5); in particular, it could not FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS reproduce the peak followed by partial decline observed at intermediate myristate concentrations, particularly evident for 1.0–5.0 · 10)4 m myristate, and in general the right part of the curves (at high [HSA]) In order to qualitatively reproduce this feature, positive cooperation must be introduced between at least one of the additional FA binding sites and FAx, so that the Mn(III)heme–HSA–myristate adduct releases myristate from FAx, as free myristate concentration vanishes, and the optical absorbance signal declines Several sets of parameters gave good fits to the experimental data, yielding almost identical curves (MSE ¼ 3.0 ± 0.1 · 10)5): an example of a set of fitting curves is displayed as continuous lines in Fig 2A All these solutions indicate a value for K MS1 in the range between 1.5 · 10)6 and 3.0 · 10)6 m (and thus values of the dissociation constants for the additional FA sites ranging from · 10)6 to · 10)8 m) and suggest that the affinity of myristate for FAx is modulated by additional site no or 4, with dissociation constant in the order of 8.0 · 10)8 to 1.3 · 10)7 m and a 50–200fold decrease in FAx affinity when the coupled site releases myristate Very similar fits were obtained, whether or not the affinity of FA1 for myristate was assumed to change when FAx is occupied The strength of cooperative coupling between accessory sites and FAx could also change over a wide extent (10–500-fold decrease in KM* when additional FA site no or releases myristate) producing equally good fits However, the set of estimated dissociation constants for FA1 and FAx changed quite markedly depending on the assumptions regarding cooperativity among FA binding sites The best fitting values for the parameters of the model (Scheme 1) are reported in Table for two nicely 4675 Myristate and Mn(III)heme binding to HSA–heme G Fanali et al Table Values of the thermodynamic dissociation constants (M) for myristate and Mn(III)heme binding to HSA at pH 7.0 and 10.0 (Scheme and see text) Assumptions: a KMM ¼ KM KM* · 100 for unoccupied FAS3 b K MM ⁄ KM ¼ K HM ⁄ KH KM* · 100 for unoccupied FAS3 pH Constant 7.0a 10.0a 7.0b 10.0b KH (FA1 + Heme) KM (FA1 + Myr) K HM [FA1 + Heme (FAx bound)] K MM [FA1 + Myr (FAx bound)] KM* (FAx + Myr) K MS3 (FAS3 + Myr) A10 [Asympt DA (no Myr)] A10* [Asympt DA (+ Myr)] Mean square error 1.5 · 10)5 3.4 · 10)7 8.2 · 10)7 3.4 · 10)7 3.2 · 10)7 9.2 · 10)8 0.011 0.030 2.9 · 10)5 1.0 · 10)6 1.1 · 10)6 9.3 · 10)8 1.1 · 10)6 1.2 · 10)5 9.2 · 10)8 0.027 0.028 9.2 · 10)6 1.7 · 10)5 7.2 · 10)6 8.5 · 10)8 3.7 · 10)7 5.7 · 10)7 1.4 · 10)7 0.012 0.030 2.8 · 10)5 1.1 · 10)6 1.3 · 10)5 7.9 · 10)7 9.4 · 10)6 2.6 · 10)6 1.4 · 10)7 0.027 0.028 8.8 · 10)6 fitting models: no change in FA1 affinity for myristate, depending on FAx binding (K MM ¼ KM), or a similar change in FA1 affinity for both Mn(III)heme and myristate (K MM ⁄ KM ¼ K HM ⁄ KH); in both cases 100-fold decrease was assumed in KM* when additional FA site no releases myristate By inspection of the model parameters (Table 1) it is clear that the assumptions strongly affect the estimated affinity of FAx for myristate (KM*) and, as a consequence, the magnitude of the allosteric modulation of FA1 (K HM ⁄ KH ¼ 5.4 · 10)2 vs 5.0 · 10)3) The estimate of KM also differs by about one order of magnitude, but the difference is smaller for the estimate of K MM , i.e FA1 affinity for myristate with occupied FAx, which presumably is the relevant dissociation constant for competition between Mn(III)heme and myristate with the latter in excess The same model was also applied to data obtained at pH 10.0 (Fig 3) The model is over-defined, and several sets of parameters give comparable fits; the results obtained by fixing K MS3 to the value observed at pH 7.0 are displayed in Table The consistent aspects, relatively independent of the model assumptions, are the following: (a) FA1 affinity for Mn(III)heme (KH) increases by at least one order of magnitude with respect to pH 7.0, but both FAx affinity and allosteric modulation of FA1 are reduced (b) Cooperation of accessory sites and FAx is almost unaffected Finally, (c) the asymptotic absorbance of the Mn(III)heme–HSA complex (A10) becomes comparable to that of the Mn(III)heme–HSA–(FAx + myristate) complex (A10*), and the latter is not altered by the change in pH Again, the occurrence of well-defined isosbestic points indicate that the binding equilibrium occurs through only two forms, the HSA-free and the HSA-bound Mn(III)heme (Fig 3B) 4676 Fig (A) Binding isotherms for Mn(III)heme binding to fatty acidfree HSA and to the HSA–myristate complex, at pH 10.0 and 25.0 °C; open triangles: no myristate; solid diamonds: 1.0 · 10)4 M myristate The continuous lines were obtained by numerical fitting of the data Values of the dissociation equilibrium constants obtained according to Scheme are given in Table (B) UV-visible spectral changes observed for a solution of 1.0 · 10)5 M Mn(III)heme titrated with HSA (0–3.0 · 10)5 M) in the presence of 1.0 · 10)4 M myristate, at pH 10.0 and 25.0 °C The arrows indicate the increase of HSA concentration FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS G Fanali et al Myristate and Mn(III)heme binding to HSA–heme Fig Change of the relaxivity measured at 10 MHz of a 1.0 · 10)3 M solution of Mn(III)heme–HSA as a function of myristate concentration Data were obtained at pH 7.0 and 25.0 °C Fig (A) Absorbance change measured at 440 nm for a solution of 1.0 · 10)5 M Mn(III)heme–HSA as a function of myristate concentration Data were obtained at pH 7.0 and 25.0 °C (B) UV-visible absorption spectra of a solution of 1.0 · 10)5 M Mn(III)heme–HSA in the absence (continuous line) and in the presence of 4.5 · 10)5 M (dotted line) and 1.0 · 10)4 M myristate (dashed line) Mn(III)heme–HSA was titrated with myristate in order to follow the conformational transition(s) associated to the fatty acid binding As shown in Fig 4, binding of myristate to Mn(III)heme HSA causes the appearance of a shoulder at 440 nm that disappears on increasing myristate concentration due to the displacement of Mn(III)heme from FA1 Here, the equilibrium occurs through three different forms, Mn(III)heme– HSA in the absence of myristate, Mn(III)heme–HSA with myristate bound to site(s) other than FA1 (spectrum with the shoulder at 440 nm), and free Mn(III)heme; therefore, no isosbestic points are observed A consistent behavior has been observed by measuring the paramagnetic contribution of Mn(III)heme to the solvent water proton NMR relaxation rate (Eqn in Experimental procedures) Figure shows the relaxivity of fatty acid-free Mn(III)heme–HSA observed at 10 MHz, 25.0 °C, as a function of the myristate concentration The relaxation rate increases, apparently FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS with a varying slope, up to sevenfold molar excess of myristate, while it starts to decrease when myristate concentration is further increased An overview of the conformational changes due to both fatty acid binding and pH may be obtained by plotting 1H-NMR relaxation rate data vs pH for the different Mn(III)heme ⁄ HSA ⁄ fatty acid ratios Figure shows the pH dependence curves of the observed relaxation rate measured at 10 MHz, where this parameter is most affected, for Mn(III)heme–HSA and Mn(III)heme– HSA-myristate at : : 3, : : 4.5, and : : molar ratios Values of pK for the three titration steps, obtained at the different Mn(III)heme–HSA-myristate ratios, have been determined using Eqn (2) (Experimental procedures; Table 2) Fig Water proton relaxation rates measured at 10 MHz and 25.0 °C, as functions of pH, for fatty acid-free Mn(III)heme–HSA (solid squares), Mn(III)heme–HSA–myristate at : : (solid triangles), : : 4.5 (open diamonds), and : : molar ratios (open circles) The continuous lines were calculated according to Eqn (2) Results of the fitting are given in Table Under all the experimental conditions, the Mn(III)heme–HSA concentration was 1.0 · 10)3 M 4677 Myristate and Mn(III)heme binding to HSA–heme G Fanali et al Table pK values of pH-dependent water proton relaxation rates measured at 10 MHz and 25.0 °C of fatty acid-free Mn(III)heme– HSA and of Mn(III)heme–HSA-myristate pK values were obtained by fitting data in Fig according to Eqn (2) Mn(III)heme ⁄ HSA ⁄ fatty acid ratio pK1 1 1 6.60 6.30 5.43 5.40 : : : : 1 1 : : : : 4.5 pK2 ± ± ± ± 0.04 0.01 0.01 0.01 9.40 8.00 7.32 7.40 pK3 ± ± ± ± 0.16 0.02 0.01 0.02 11.80 12.10 11.40 11.50 ± ± ± ± 0.05 0.03 0.02 0.03 Contributions to relaxation differ, depending on the conformational state of HSA and on the occupancy of the myristate binding sites The relaxation rate change is highest between pH 5.5 and 8.0, where HSA is in the native form (N state) The relaxivity of the Mn(III)heme–HSA complex increases with myristate concentration It should be noticed that at pH lower than 5.5, myristate is expected to be in the protonated form that is not able to bind HSA [34] Conversely, between pH 8.3 and 11.9 (i.e where HSA is in the B form), the contribution of Mn(III)heme–HSA-myristate to paramagnetic relaxation does not differ significantly from that of fatty acid-free Mn(III)heme–HSA As myristate binding appears to enhance the relaxivity of Mn(III)heme–HSA, we attempted to gain more information from the analysis of NMRD profiles at various myristate concentrations Figure shows NMRD profiles of fatty acid-free Mn(III)heme–HSA and of Mn(III)heme–HSA-myristate obtained at : : 3, : : 4.5, and : : molar ratios at pH 7.0 Note that NMRD profiles are significantly different in the high field region whereas at the low frequency limit they are almost coincident NMRD profiles of Mn(III)heme–HSA as a function of myristate concentration were also measured at pH 10.0 in order to check whether any change occurred for the B state of HSA as well As shown in Fig 7, the NMRD profiles of Mn(III)heme–HSA at pH 10.0 not appear to be affected by myristate Optical absorbance spectra are suggestive of different coordination modes of Mn(III)heme in the different conformational states of HSA [35], therefore we measured the paramagnetic contribution to the 17O-NMR linewidth at pH 7.0 and 10.0 as a function of myristate concentration (Fig 8) For paramagnetic metalloproteins, the width of the 17O NMR resonance is affected by the presence of the paramagnetic metal through the exchange of water molecules directly coordinated to the metal center, according to Eqn (3) (Experimental procedures) [20] Unlike protons, 17O nuclei are negligibly affected by dipolar coupling with nearby unpaired 4678 Fig NMRD profiles of fatty acid-free Mn(III)heme–HSA (solid squares) and of Mn(III)heme–HSA–myristate at : : (solid triangles), : : 4.5 (open diamonds), and : : molar ratios (open circles) at pH 7.0 (A) and at pH 10.0 (B) Under all the experimental conditions, Mn(III)heme–HSA concentration was 1.0 · 10)3 M Data were obtained at 25.0 °C Fig Paramagnetic contribution to the linewidth of the 17O water resonance of 1.6 · 10)3 M solution of Mn(III)heme–HSA as a function of myristate concentration Solid squares: pH 7.0, HSA N state; open circles: pH 10.0, HSA B state Data were obtained at 25.0 °C electrons, and the paramagnetic broadening of the 17O resonance is diagnostic of the occurrence of a direct coordination bond between water and Mn(III) [20,36] FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS G Fanali et al As shown in Fig 8, the linewidth change is significant (about 20 Hz) for the protein in the N state, and becomes larger (about 40 Hz) in the presence of saturating concentration of myristate On the other hand, this contribution is almost negligible for HSA in the B state, and starts to increase to about 20 Hz in the presence of high myristate concentration Discussion Myristate binding to HSA affects the Mn(III)heme binding properties The results presented here indicate that current views – seven FA binding sites, FA1 involved in ipsosterical competition with heme binding, FAx allosterically coupled to FA1, a scale of affinity ratios of about half a decade among FA sites, with dissociation constants in the range 10)6)10)8 m, and several possible allosteric cross interactions among FA sites [1,3,6,9–16,33] – allow us to numerically model the experimental results with good accuracy In particular, modeling indicates that binding of myristate to FAx not only increases optical absorbance of Mn(III)hemebound HSA by a factor of % 3, but also increases FA1 affinity for Mn(III)heme by 10–500-fold (depending on the assumptions about possible similar changes in affinity of FA1 for myristate) This brings the value of HSA affinity for Mn(III)heme, with myristate bound to FAx, in the range of HSA affinity for Fe(III)heme [32] Furthermore, modeling indicates that positive cooperation between an accessory FA site (with affinity 0.8– 1.5 · 10)7 m for myristate) and FAx is needed to account for the shape of the DA curves (Fig 2A) At pH 10.0 (i.e where HSA is in the B state), Mn(III)heme binds more strongly to HSA than at pH 7.0 (i.e where HSA is in the N state) even in the absence of myristate, with KH % 10)6 m (Fig 3) Moreover, in the presence of saturating concentrations of myristate, the tendency of the curve to become sigmoidal is much attenuated, suggesting a substantial impairment of allosteric modulation by myristate binding to FAx Numerical analysis of the data, using the same models that fit the data at pH 7.0, indicate that, independently of the model assumptions, both FAx affinity for myristate and allosteric modulation of FA1 are reduced, whereas cooperation of accessory sites and FAx is almost unaffected Furthermore, the asymptotic absorbance of the Mn(III)heme–HSA adduct (A10) becomes comparable to that of the Mn(III)heme–HSA-(FAx+myristate) complex (A10*), whereas the latter is not altered by the change in pH Taken together, these observations strongly suggest that the conformational changes produced by changing the pH from 7.0 to 10.0 (i.e shifting the HSA conforFEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS Myristate and Mn(III)heme binding to HSA–heme mation from the N to the B state) is very similar to that induced by myristate binding to site FAx Indeed, the HSA affinity for Mn(III)heme and the absorbance of Mn(III)heme–HSA increase by factors of about 10 and 3, respectively, and myristate effects become much attenuated Still, the same interaction(s) that at pH ¼ 7.0 produces marked differences among absorbance curves at various myristate concentrations appear to fully account for the small reshaping of the curve produced by 1.0 · 10)4 m myristate at pH ¼ 10.0 Myristate binding to HSA determines conformational changes that open the FA1 cavity allowing Mn(III)heme binding and consequently myristate displacement Actually, addition of up to three moles of long-chain FAs is reported to enhance the binding of Sudlow’s site I (i.e FA7) ligands, and this behaviour is usually explained by a cooperative effect established by FA binding to domain III (i.e to FA4 and FA5) [26,37–39] On the other hand, myristate bound at the limit of subdomain IA (i.e to FA2) was suggested to be functionally linked to Sudlow’s site I [25] It should be noticed that binding of more than three equivalents of myristate decreases warfarin affinity for Sudlow’s site I, as Fe(III)heme does [19,21,31,40] Sudlow’s site II (i.e FA4) ligands not appear to be effective in modulating Sudlow’s site I ligands and heme binding properties [21,26] The marked variation in the optical absorbance spectrum of Mn(III)heme–HSA induced by myristate binding at pH 7.0 might be explained in terms of a change in the coordination sphere of Mn(III) [35] Although structural data for Mn(III)heme–HSA are not available yet, evidence for a Mn(III)-coordinated water molecule was gained by 17O-NMR linewidth measurements, that showed a transverse relaxation rate different from Fe(III)heme–HSA, where no Fe(III)coordinated water molecule(s) were observed [20] On the other hand, both X-ray structures deposited in PDB so far for Fe(III)heme–HSA display the Tyr161 residue as the only axial ligand for Fe(III) [6,8] In the absence of myristate, Mn(III)heme–HSA in the N state has a water molecule coordinated to the metal (Fig 8) that could provide a source for paramagnetic relaxation of the solvent water bulk This is at difference with Fe(III)heme, due to the different affinity of the metals for phenolic oxygen ligands [6,8] Nevertheless, this contribution is not evident from the NMRD profile, which is almost superimposable to that of Mn(III)heme–HSA in the B state This finding could indicate that there is a water molecule coordinated at both pH but that its exchange is limiting the relaxivity Therefore, the binding of myristate seems to markedly increase the exchange rate and induce a relaxivity 4679 Myristate and Mn(III)heme binding to HSA–heme enhancement, although at pH 10.0 the possible increase in the exchange rate by myristate is incapable to induce a significant increase of the relaxivity At pH 10.0 (i.e when HSA is in the B state), 17 O-NMR linewidth measurements show no evidence of water molecules coordinated to Mn(III)heme, as already observed in the case of Fe(III)heme–HSA Two hypotheses should be taken into consideration: either the absence of water molecules in the coordination sphere of the metal ion, or the presence of one water molecule with a very slow exchange rate Myristate binding to HSA might increase the exchange rate, thereby producing a small broadening, but this is only observed at pH 7.0 The structural similarity of Mn(III)heme vs Fe(III)heme and the structural evidence of a pentacoordinated Fe(III) atom, with no water molecules coordinated to it, favour the first hypothesis: in this case, upon deprotonation at pH 10.0 the phenolic Tyr161 oxygen becomes more nucleophylic and displaces the Mn(III)-coordinated water molecule with the consequent quenching of the paramagnetic relaxation Conclusions The conformational transition(s) driven by myristate binding to HSA may be efficiently monitored by taking advantage of the optical and relaxometric properties of the Mn(III)heme label Mn(III)heme binds to FA1 in the fatty acid-free HSA with KH % 2.0 · 10)5 m; myristate not only competitively binds to FA1, but also binds to a different site(s) and induces conformational changes that lowers the equilibrium constant for Mn(III)heme binding to the FA1 site by a factor of 10–500 (depending on possible modulation of myristate binding to FA1) This conformational change(s) also favours the exchange of the Mn(III)-coordinated water molecule with the solvent bulk At pH ¼ 10.0, Mn(III)heme binds to HSA with higher affinity even in the absence of myristate, releasing the metal-coordinated water molecule As a general remark, NMRD data prove a valuable complement to X-ray crystallography to add dynamic information to structural data, and to provide thermodynamic description of the binding equilibria As an addition to conventional optical methods, NMRD provides a useful hint to follow environment changes that involve the coordination sphere of the paramagnetic metal Experimental procedures All reagents were purchased from Sigma-Aldrich (St Louis, MO, USA), were of highest purity available, and were used 4680 G Fanali et al without further purification HSA was essentially fatty acidfree according to the charcoal delipidation protocol [41–43] and used without any further purification Absence of significant amounts of covalent dimers was checked by MALDI-TOF mass spectrometry Mn(III)heme was prepared as previously reported [28] The actual concentration of the Mn(III)heme stock solution was checked as bisimidazolate complex in sodium dodecyl sulfate micelles with an extinction coefficient of 10.3 cm)1Ỉmm)1 (at 556 nm) [44] Mn(III)heme–HSA was prepared by adding the appropriate volume of 3.0 · 10)2 m Mn(III)heme dissolved in 1.0 · 10)1 m NaOH to a 1.0 · 10)3 m HSA solution in NaCl ⁄ Pi (1.0 · 10)2 m phosphate buffer, 0.15 m NaCl) The final solution of Mn(III)heme–HSA was 1.0 · 10)3 m Under all the experimental conditions, no free Mn(III)heme was present in the reaction mixtures The actual concentration of the HSA stock solution was determined by using the Bradford method [45] The sodium myristate 0.1 m solution was prepared by adding 0.1 m fatty acid to NaOH 0.1 m The solution was heated to 100 °C and stirred to dissolve the fatty acid The sodium myristate solution was then mixed with 1.0 · 10)3 m Mn(III)heme–HSA (fatty acid free) to achieve the desired fatty acid to protein molar ratio The Mn(III)heme–HSA-myristate complex was incubated for one hour at room temperature with continuous stirring [6] Sample pH was changed by adding a few lL of 0.1 m HCl or NaOH solutions Binding experiments of Mn(III)heme to HSA-myristate and titrations of Mn(III)heme–HSA with myristate were investigated spectrophotometrically using an optical cell with 1.0-cm path length on a Cary 50 Bio spectrophotometer (Varian Inc., Palo Alto, CA, USA) In a typical experiment, a small amount of a solution of Mn(III)heme in NaOH (about 3.0 · 10)3 m) was diluted in the optical cell with a solution of 1.0 · 10)4 m sodium myristate in a solvent mixture of DMSO-aqueous 0.1 m phosphate buffer pH 7.0 to a final chromophore concentration of 1.0 · 10)5 m This solution was titrated with HSA by adding small amounts of a 1.0 · 10)3 m protein solution in the aqueous buffer and recording the spectrum after incubation for a few after each addition Difference spectra with respect to Mn(III)heme were taken and the binding isotherm was analyzed by plotting the difference of absorbance between the maximum and the minimum of the two-signed difference spectra against the protein concentration [27] Data have been numerically analyzed using the matlab language (The MathWorks, Natick, MA, USA) according to Scheme 1, with the following dissociation equilibrium constants: KH for Mn(III)heme binding to site FA1; KM for myristate binding to site FA1 and competing ipsosterically with Mn(III)heme; KM* for myristate binding to site FAx, allosterically coupled to FA1; K HM for Mn(III)heme binding to the HSA–myristate complex, with myristate bound FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS G Fanali et al Myristate and Mn(III)heme binding to HSA–heme to FAx Accordingly, the dissociation constant for myristate binding to FAx with FA1 occupied by Mn(III)heme was set to K MH ¼ (KM* · K HM ) ⁄ KH [46] Five additional dissociation constants K MS1 to K MS5 have been introduced in the model to take into account subtraction of myristate by additional binding sites no to essentially uncoupled to FA1 and ⁄ or FAx Fatty acid binding sites (FA1 to FA7) are numbered according to literature [6,7,14] Water proton T1 measurements at 10 MHz, at 25.0 °C and at variable pH were obtained on a Stelar SpinmasterFFC fast field cycling relaxometer (Stelar, Mede, Italy) with 16 experiments in four scans The reproducibility in T1 measurements was ± 0.5% H nuclear magnetic relaxation dispersion (NMRD) profiles were recorded at variable concentration of myristate by measuring water proton longitudinal relaxation rates (R1obs ) at magnetic field strengths in the range from 2.4 · 10)4 to 0.235 T (corresponding to 0.01–10 MHz proton Larmor frequencies) with the field cycling relaxometer described above The R1p relaxivity values (i.e paramagnetic contributions to the solvent water longitudinal relaxation rate referenced to a 1.0 mm concentration of paramagnetic agent) were determined by subtracting from the observed relaxation rate (R1obs ) the blank relaxation rate value (R1dia ) measured for the buffer at the experimental temperature, divided by the concentration of the paramagnetic species For 1H nuclei, R1p values are mostly affected by dipolar interaction with unpaired electrons of the paramagnetic center Unbound water protons relax by means of diffusion– controlled dipolar interaction (outer sphere contribution, R1os – see Eqn 1), whereas for water molecules coordinated to the metal ion or bound to the protein in close proximity of the paramagnetic center the dipolar interaction is modulated by the reorientation of the macromolecule with respect to the applied magnetic field The latter term is described by Eqn (1): ẵMq 1ị R1p ẳ R1os ỵ 55:56 T1M þ sM where sM is the exchange lifetime and q is the number of water molecules close to the metal centre [M] is the concentration of the paramagnetic metal ion, and T1M is the longitudinal relaxation time of localized water protons [20,36] Relaxivity of Mn(III)heme–HSA solutions at 25.0 °C as a function of pH was analyzed according to Eqn (2): R1p ẳ C0 ỵ X i Ci ỵ ẵHỵ Š=Ki ð2Þ where C0 is the R1p value at the low pH limit, Ki is the thermodynamic constant of the i-th titration, and Ci is the R1p change associated to the i-th titration 17 O-NMR linewidth measurements at 25.0 °C were recorded at 7.0 T on a Bruker Avance 300 spectrometer (Bruker Biospin, Rheinstetten, Germany), equipped with a mm FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS inner diameter tunable broadband probehead, by using a D2O external lock Sample solutions were supplemented with enriched H217 O (Cortec Ltd, Paris, France) to an isotopic abundance of 2% Experimental settings: spectral width 6.0 kHz, 90° pulse 16 ls, acquisition time 0.47 s, 128 scans, no sample spinning [20] Paramagnetic contributions to the 17O-NMR linewidth (DW) were obtained by subtracting the width of the H217 O signal in the presence of HSA from the width of the H217 O signal in the presence of Mn(III)heme–HSA at different myristate concentrations DW values are related to the transverse relaxation time of the directly coordinated water oxygen (TO2M) by Eqn (3): DW ẳ ẵMq O 55:56 T2M ỵ sM 3ị where sM is the exchange lifetime of the metal-coordinated water molecule, [M] is the 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