Backbone dynamics of oxidised and reduced forms of human atrial natriuretic peptide

Journal of

Structural Biology Journal of Structural Biology 148 (2004) 214–225 www.elsevier.com/locate/yjsbi

Backbone dynamics of oxidised and reduced forms of human atrial natriuretic peptideq Heather Peto,a Katherine Stott,b Margaret Sunde,c and R. William Broadhurstb,* a Centre for Protein Engineering, MRC Centre, Hills Road Cambridge CB2 2QH, UK Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK c School of Molecular and Microbial Biosciences, University of Sydney, New South Wales 2006, Australia

b

Received 12 March 2004, and in revised form 12 May 2004 Available online 19 June 2004

Abstract The backbone dynamics of the 28 residue 15 N-labelled human atrial natriuretic peptide have been examined by 15 N NMR methods. 15 N R1 ; R2 and {1 H}–15 N NOE values were determined for the oxidised and reduced forms of the peptide (ANPox and ANPrd , respectively), and analysed using reduced spectral density mapping and an extended model-free approach. The two forms possessed correlation times for overall molecular motion of 4.7 ns and were highly flexible, with substantial contributions to relaxation processes from internal motions on picosecond to nanosecond time scales. Reduction of the Cys7–Cys23 disulphide bond to form ANPrd produced a very dynamic linear peptide with a mean overall order parameter of 0.2; the intramolecular cross-link in ANPox increased this to a mean value of 0.4. A simple model for segmental backbone motion accounted for the R2 values of both species using only two variable parameters, indicating that relaxation is dominated by interactions with sites <7 residues distant in the covalent network and that changes in the conformation of the disulphide bond lead to significant chemical exchange broadening in ANPox . The contributions of backbone dynamics to configurational entropy were determined and accounted for the different receptor binding affinities of cyclised and linear natriuretic peptides. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Atrial natriuretic peptide; Backbone dynamics; Configurational entropy; Disulphide bridge; Segmental motion

1. Introduction Atrial natriuretic peptide (ANP)1 belongs to a family of polypeptide hormones that maintain blood pressure and volume and also play roles in cellular proliferation q Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jsb.2004.05.002. * Corresponding author. Fax: +44-1223-766002. E-mail address: [email protected] (R.W. Broadhurst). 1 Abbreviations used: ANP, atrial natriuretic peptide; cGMP, cyclic guanosine monophosphate; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; GTP, guanosine triphosphate; GST, glutathione-S-transferase; HPLC, high performance liquid chromatography; HSQC, heteronuclear single quantum correlation spectroscopy; IPTG; isopropyl-b-D -thiogalactopyranoside; ITC, isothermal titration calorimetry; MALDI, matrix assisted laser desorption ionisation; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; NPR, natriuretic peptide receptor; TOCSY, total correlation spectroscopy.

1047-8477/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2004.05.002

and differentiation (Kuhn, 2004). All natriuretic peptides (including the B- and C-type natriuretic peptides, BNP and CNP) possess a highly conserved amino acid sequence encompassing a 17 residue ring closed by a disulphide bridge, XCFGXXXDRIGXXSGLGC (Mimeault et al., 1995). After secretion by the heart in response to blood volume expansion, ANP exerts most of its physiological activity by binding to a single span transmembrane receptor, NPRA, which triggers a signalling cascade by converting GTP to the second messenger cGMP. In mouse models, targeted deletion of ANP or NPRA leads to severe, chronic arterial hypertension, cardiac hypertrophy, and sudden death (Kuhn, 2004). Natriuretic peptide receptors occur as dimers, with the transmembrane region connecting an extracellular ligand-binding domain to an intracellular portion that comprises a protein kinase homology domain, a coiled–coil hinge region and (in the case of NPRA) a cyclase-homology domain that synthesises cGMP.

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Crystallographic studies show that the extracellular domains of the related receptors NPRA and NPRC form 2:1 complexes with ANP, the hormone binding in a ‘‘disk’’ conformation along the centre of the receptor dimer axis (He et al., 2001; Qui et al., 2004). On binding ANP, both C-terminal extracellular domains of the receptor dimer close around the ligand, thereby repositioning the N-terminal domains into the correct conformation for hormone-induced signalling. The consequent rearrangement of the intervening linker region requires the breaking of several H-bonds, but this unfavourable process is offset by the highly favourable enthalpy of binding to ANP (He et al., 2001). The structures of ANP in complex with the extracellular domains of NPRA and NPRC are very similar (Qui et al., 2004). According to solution NMR spectroscopy, native natriuretic peptides appear to be very flexible and are predominantly unstructured (Mimeault et al., 1995; Weber et al., 1994). Higher degrees of structure have been reported in the presence of organic solvents or detergent micelles (Carpenter et al., 1997; Kobayashi et al., 1988), but with conflicting conclusions about the location and nature of any new features. Aqueous solution studies of a rigidified ANP variant (Fairbrother et al., 1994) and a C-type natriuretic peptide from platypus venom (Torres et al., 2002) indicated that Phe8, Leu12, Ile15, Leu21, and Phe26 can form a hydrophobic cluster, but in the crystal structure of the NPRC/CNP complex the side chains of these residues do not interact (He et al., 2001). Rather than reflecting the receptor-bound structure, the conformations sampled by ANP in solution represent a compromise between the stability and flexibility necessary for potency, specificity, and resistance to degradation. In fact, the relatively high net charge and low mean hydrophobicity of ANP suggest that the hormone could be classified as being ‘‘natively unfolded’’ (Uversky, 2002). ANP could exploit intrinsic disorder for gaining high specificity coupled with low affinity, the ability to bind to multiple partners because of structural plasticity, and the creation of a large interface region with the dimeric receptor. In this context, we studied the 15 N relaxation properties of the oxidised and reduced forms of ANP to gain a probe of backbone dynamics and assess the functional differences between cyclic and linear forms of the hormone.

2. Materials and methods 2.1. Protein expression and purification Previous attempts to express ANP were too inefficient to produce sufficient quantities for NMR studies (Lennick et al., 1987; Qin et al., 1991; Saito et al., 1987). Our strategy was to increase yield and facilitate purification by expressing ANP as a C-terminal fusion to glutathi-

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one-S-transferase (GST). The lack of glutamic acid in the sequence of ANP was exploited by inserting an extra Glu residue after the GST linker sequence in order to release the desired product using endoproteinase Glu-C, thereby avoiding the introduction of unwanted residues at the N-terminus of the peptide. The GST–ANP fusion was expressed in Escherichia coli (BL21 DE3 pLysS) using a pGEX-4T3 expression vector under control of the T7 lac promotor (Peto, 2003). Optimum soluble expression occurred after induction with 0.1 mM IPTG for 4 h at 37 °C. Insoluble material from the cell lysate was removed by centrifugation and the GST–ANP in the soluble phase was purified by affinity chromatography (using glutathione– Sepharose 4B matrix). To avoid sporadic spontaneous cleavage of the fusion protein and unwanted formation of disulphide bonds, the lysate and washing buffers were supplemented with 10 mM EDTA and 2 mM DTT, respectively. When preparing 15 N labelled samples, the cells were grown in minimal media using 15 NH4 Cl as the sole nitrogen source; according to MALDI mass spectrometry the degree of incorporation of 15 N was
100% (molecular weight 3125.0 Da, data not shown). Release of ANP from the fusion protein was essentially completed after exposure to GluC at a ratio of 200:1 by mass in solution with 50 mM NH4 HCO3 and 2 mM DTT for 30 min at 37 °C. Proteolysis was quenched by acidification to pH 3.0, following which ANP was purified by HPLC (C18 reverse phase) using a linear acetonitrile gradient (5–95%), with oxidised peptide (ANPox ) eluting at 29% acetonitrile and the reduced form (ANPrd ) at 30.5% (see Supplementary Material). ANPrd was oxidised by titration to pH 7.8, with sodium phosphate buffer, followed by air oxidation for 72 h and a further round of HPLC. ANPox in phosphate buffer at pH 7, was converted to the reduced form by addition of 2 mM DTT for 1 h and then desalted by HPLC. ANPrd was maintained in a reduced state by including 2 mM DTT in the sample buffer. Analytical ultracentrifugation experiments indicated that in 20 mM phosphate buffer at pH 3.0, 4 °C and peptide concentrations of 1 mM both states of ANP are monomeric (data not shown). 2.2. NMR spectroscopy All NMR samples were prepared containing 1 mM ANP, 20 mM sodium phosphate, 0.05% (w/v) sodium azide, 20 lM 3,3,3-trimethylsilylpropionate, and 10% D2 O, to a final volume of 550 ll in 5 mm Ultra-Imperial grade NMR tubes (Wilmad). All experiments were recorded on a Bruker DRX500 spectrometer equipped with a z-shielded gradient triple resonance probe using standard procedures (Cavanagh et al., 1995). Two dimensional (2D) NOESY and TOCSY experiments, with mixing times of 110 and 72.5 ms, respectively, were collected with 256* and 1024* pairs of complex (*)

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points and acquisition times (smax ) of 26 and 102 ms in the indirectly and directly acquired 1 HN dimensions, respectively. 2D [1 H, 15 N]-HSQC spectra were acquired with 200*  1024* points and smax of 100 and 102 ms in the 15 N and 1 HN dimensions, respectively. 3D 15 N-separated NOESY-HSQC (mixing time 200 ms), TOCSYHSQC (mixing time 82.9 ms) and HNHA experiments were collected with 32*  114*  1024* points and smax of 19, 21, and 102 ms in the 15 N, 1 H, and 1 HN dimensions, respectively. Data processing and analysis were carried out on a Silicon Graphics O2 workstation using the programs AZARA (W. Boucher, unpublished work) and ANSIG 3.3 (Kraulis et al., 1994). Sensitivity enhanced 15 N relaxation experiments (Farrow et al., 1994) were recorded with 150* and 1024* points and acquisition times (smax ) of 168 and 102 ms in the 15 N and 1 HN dimensions, respectively. 15 N longitudinal (R1 ) and transverse (R2 ) relaxation rate experiments used relaxation delays of 10, 30, 60, 100, 180, 280, 400, and 700 ms and of 14.4, 28.8, 43.2, 72.0, 100.8, 144.0, 187.2, 230.4, and 288.0 ms, respectively. 2.3.

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N relaxation data analysis

tween simulated and experimental values (v2 ) by a simple grid search procedure. The first approach assumed that the influence of neighbouring residues on the relaxation rate of residuePi, R2 ðiÞ, decayed exponentially such that R2 ðiÞ ¼ Rint Ni¼1 expðMij =kint Þ, where Rint represents the intrinsic relaxation rate, kint is the persistence length of the polypeptide chain (in number of residues) and N is the total length of the polypepetide (number of residues). The effect of the disulphide bond in ANPox was modelled using a topological distance matrix, Mij , which counts the number of residues along the shortest path from residue i to residue j, treating the distance across the disulphide bridge as one residue. The second approach extended the simulation to include chemical exchange contributions with a common amplitude Rex and persistence length kex (in number of residues) at a number Nex of different residues, laP belledP Xk , such that R2 ðiÞ ¼ Rint Ni¼1 expðMij =kint Þ ex þRex Nk¼1 expðjði  Xk Þj=kex Þ. The change in conformational entropy on conversion from state a to state b was estimated from the order parameters (Sa2 and Sb2 , respectively), according to Eq. (2) of Bracken et al. (1999).

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N R1 and R2 relaxation rates were estimated by fitting crosspeak heights to the two parameter exponential decay profile IðtÞ ¼ I0 expðtR1;2 Þ, where IðtÞ is the intensity after the relaxation delay t and I0 is the intensity at zero time, using a Levenberg–Marquardt non-linear least squares procedure. Values of the steady-state heteronuclear NOE ({1 H}–15 N NOE) were determined according to the formula NOE ¼ ðI 0  Iref Þ=Iref where I 0 is the intensity of a crosspeak in an experiment with 3 s broadband 1 H presaturation and Iref is the intensity in a reference spectrum recorded without presaturation. Reduced spectral density mapping was performed using Eqs. (5)–(7) of Farrow et al. (1995), with the av
and the chemical erage amide bond length set to 1.02 A shift anisotropy of the 15 N nucleus to )172 ppm. Theoretical curves for the dependence of J ð0:87xH Þ and J ðxN Þ on J ð0Þ were calculated for a range of rotational correlation times, s, by modelling the spectral density function J ðxN Þ as a single Lorentzian (Barthe et al., 1999), such that J ðxN Þ ¼ 0:4s=ð1 þ x2 s2 Þ. An extended Lipari–Szabo analysis (Clore et al., 1990) of the 15 N relaxation parameters was performed using version 4.15 of the ModelFree program (Mandel et al., 1995), assuming an isotropic diffusion tensor. Overall order parameters S 2 were interpreted in terms of a motional model of diffusion of the 15 N–1 H bond vector within a cone of semi-angle h using the relation S 2 ¼ ½0:5 cos h  ð1 þ cos hÞ2 (Dayie et al., 1996). The transverse relaxation rates of ANPox and ANPrd were fitted simultaneously using two models derived from Schwalbe et al. (1997) with variable parameters optimised according to the least squares difference be-

3. Results 3.1. Resonance assignments Sequence-specific resonance assignments for the oxidised and reduced forms of ANP at pH 3.0, and 4 °C were derived using a combination of 2D homonuclear and 3D 15 N-separated NOESY and TOCSY experiments (Cavanagh et al., 1995). Although both ANPox and ANPrd possess limited 1 HN chemical shift dispersions (0.73 and 0.71 ppm, respectively), as expected for an unfolded polypeptide, the 27 backbone resonances of each form are clearly resolved in [1 H, 15 N]-HSQC spectra (see Supplementary Material). The similar chemical shift dispersions of backbone 1 HN nuclei in the two states suggest that no major structural changes occur when the sidechains of residues Cys7 and Cys23 are cross-linked by a disulphide bridge. In common with other unstructured peptides, two distinct 1 Hb signals are observed for each cysteine residue in the oxidised form, indicating that each of the prochiral nuclei in these sites experience unique chemical environments as a result of conformational restrictions imposed by the disulphide bridge (Wishart et al., 1995). By contrast, in ANPrd the Hb signals of each cysteine residue are degenerate, presumably due to increased sidechain dynamics on a time scale rapid enough to average the effects of the different environments of the accessible rotameric states. The differences between the chemical shifts of the 1 a 1 N H , H , and 15 N nuclei of the oxidised and reduced forms of ANP are displayed in Fig. 1. The largest

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et al., 1994), and PNP, a natriuretic peptide found in the venom of the snake Pseudocerastes persicus (Amininasab et al., 2004). The absence of stretches of neighbouring residues in either ANPox or ANPrd with 3 JHNHa values greater than 8 Hz or less than 6 Hz implies a lack of extended regions of secondary structure, consistent with a high degree of flexibility in the peptide backbone. This conclusion is supported by a lack of medium and long range NOE connectivities in the NOESY spectra of both states. In addition, sequential dNN and daN NOEs possess similar intensity throughout both forms of ANP, indicating that each residue samples a range of / and w torsion angles that are characteristic of both a- and b-structure. 3.2.

Fig. 1. Histograms describing the chemical shift differences between ANPox and ANPrd as a function of residue number for (A) 1 Ha , (B) 1 N H , and (C) 15 N nuclei. The amino acid sequence of ANP is displayed at the top of the figure.

changes in 1 Ha shifts are seen for Cys7 and Cys23, once again the result of changes in local electronic environments that occur on reduction of the disulphide bond; by comparison the 1 Ha shifts of the other residues show only small variations. The 1 HN and 15 N shift differences are also in general rather small; the largest changes in 15 N shifts are found for Met12 to Ile15 (which could be involved in a turn enforced by the need to form a cysteine bridge in ANPox ) and for Phe8 and Asn24 (residues adjacent to Cys7 and Cys23, respectively, in the primary sequence). A study of the sequence dependence of random coil chemical shifts noted that the 15 N shifts of residues immediately following cysteine require a large correction factor (Schwarzinger et al., 2001); this is consistent with the 15 N nuclei of Phe8 and Asn24 being particularly sensitive to the oxidation state of ANP. By contrast, in flexible peptides 1 Ha chemical shifts depend mostly on the nature of the attached sidechain, with the identity of neighbouring residues in the sequence having only small effects. After changes in solution conditions and amino acid composition are taken into account, the 1 N H and 1 Ha chemical shifts of ANPox differ little from those recorded in earlier studies of a hexamutant ANP variant (Fairbrother et al., 1994) and of more obviously random coil homologues including urodilatin, which has a 4 amino acid extension at the N-terminus (Weber

15

N relaxation parameters

Fig. 2 summarises the 15 N R1 ; R2 , and {1 H}–15 N NOE relaxation parameters of ANPox and ANPrd measured in a sample containing a mixture of both forms (60:40%), the use of which insured that variables such as total peptide concentration, temperature, and viscosity had identical effects on both species. In [1 H, 15 N]-HSQC spectra of the mixture distinct resonances were resolved for each backbone site in both states for all but five residues (Leu2, Arg3, Arg4, Gly16, and Arg17); the relaxation parameters obtained for the coincident signals of these residues were included in the analysis of the oxidised state, as it was present in slight excess. In the 200 ms mixing time, 15 N-separated NOESY spectrum no crosspeaks were detected that could be attributed to chemical exchange between the two species, indicating that any interconversion must occur on timescales slower than 1 s; furthermore, crosspeak intensities in [1 H, 15 N]-HSQC spectra of this sample were unchanged after 2 weeks. Backbone amide sites in ANPox have broadly similar relaxation properties, apart from residues at the N- and C-termini. For Leu2, Arg3, and Tyr28 the values of R1 and R2 are significantly smaller and the {1 H}–15 N NOE much more negative than those of the intervening residues, suggesting that the peptide backbone is highly flexible close to the extrema. Residues Arg4 to Arg27 are less dynamic, possessing mean values for R1 ; R2 and the {1 H}–15 N NOE of 2.2  0.2 s1 , 4.3  0.7 s1 , and )0.63  0.11, respectively. The R2 profile rises to maxima at residues Phe8, Ile15, and Asn24, indicating that transverse relaxation at these sites may be increased by contributions from conformational exchange processes. By contrast, the 24 sites probed by resonances assigned to ANPrd have more uniform relaxation properties and appear to experience a greater degree of internal motion, giving mean values of 1.8  0.1 s1 , 2.8  0.4 s1 , and )0.99  0.15 for R1 ; R2 and the {1 H}–15 N NOE, respectively. These observations support the idea that reduction of the disulphide bond confers a higher degree of flexibility on the entire peptide backbone of ANP.

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Fig. 2. Histograms describing 15 N relaxation parameters measured in a sample containing a mixture of the oxidised (A–C) and reduced (D–F) forms of ANP as a function of residue number: (A and D) R1 ; (B and E) R2 ; and (C and F) {1 H}–15 N NOE.

The mean value of R1 is significantly smaller than that of R2 for both states, indicating that the overall rate of rotational diffusion at 4 °C is slow enough for the peptide to be in the slow tumbling regime. For residues 4–27 of ANPox and residues 5–15 and 18–28 of ANPrd the values of the ratio R2 =R1 predict overall rotational correlation times (sm ) of 3.3  0.6 and 1.5  0.6 ns, respectively (Kay et al., 1989). However, the R2 =R1 ratio is only independent of the effects of internal motion for residues in elements of regular secondary structure, which typically possess {1 H}–15 N NOE values more positive than )0.35. Since none of the backbone amide sites in either species fall into this category (and all are considerably more negative than the threshold of )0.13 expected for zero internal motion) these estimates of sm should be viewed with caution. Nevertheless, both apparent sm values are similar to those determined for

small partially structured peptides under similar conditions, including a 17 residue disulphide cross-linked immunogenic peptide from the receptor binding domain of Pseudomonas aeruginosa, which possessed an overall rotational correlation time of 2.2  0.5 ns at 5 °C (Campbell et al., 2000).

4. Discussion 4.1. Reduced spectral density mapping Protein 15 N relaxation parameters report on the dynamic properties of the polypeptide chain, which are encoded in spectral density functions that describe the range and amplitude of frequencies sampled by reorientation of the 15 N–1 H bond vectors at each backbone

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amide site (Dayie et al., 1996). Spectral density functions are often reconstructed using the approach of Lipari and Szabo (1982), which employs a number of models incorporating fast or slow internal motions, a single overall correlation time or a local correlation time for each residue, and local contributions from conformational exchange. The most appropriate model is identified by its ability to describe the data using the least number of variables. However, for a Lipari–Szabo analysis to be meaningful the time scales of the rapid internal and slower overall motions must differ by more than an order of magnitude. In the case of a highly flexible polypeptide, this criterion may not be met, since the time scales of internal motions would be expected to be significantly longer than those encountered in well structured portions of folded proteins, which are typically <100 ps. In addition, it may be inappropriate to expect a single conformation with a unique overall rotational correlation time to characterise the motional properties of a flexible peptide like ANP. A more realistic picture is that of an ensemble of interconverting conformations (Dyson and Wright, 1998; Yao et al., 2001). An alternative to the Lipari–Szabo approach for analysing relaxation data is to use reduced spectral density mapping, which makes no assumptions about the time scales of molecular motions (Farrow et al., 1995). This method allows the spectral density, J ðxÞ, to be sampled at three frequencies, zero, xN , and 0.87xH , corresponding to relaxation contributions from the motions of 15 N–1 H bond vectors on slow (millisecond– nanosecond), intermediate (nanosecond) and rapid (nanosecond–picosecond) time scales, respectively. Large values of J ð0Þ and small values of J ð0:87xH Þ are consistent with well structured regions of the backbone that possess limited internal mobility and for which the relaxation properties are dominated by the overall rate of rotational diffusion of the molecule. Small values of J ð0Þ and large values of J ð0:87xH Þ indicate substantial contributions from local motions. After reduced spectral density mapping of the oxidised and reduced states of ANP, all backbone sites showed the expected monotonic decrease in J ðxÞ as a function of frequency (see Supplementary Material). The J ð0Þ and J ðxN Þ profiles of ANPox display a bellshaped dependence on sequence position, reproducing the broad features of the profiles observed in the R2 and R1 data, respectively, and suggesting that the peptide backbone is most flexible at the termini. A similar but inverted pattern is reflected in the J ð0:87xH Þ profile, signifying dominant contributions from rapid internal motions at the termini of the peptide. Following the R2 data (Fig. 2B and E), the J ð0Þ profile for ANPrd shows a slight increase for residues in the middle of the primary sequence, but the maxima apparent for residues 8, 15, and 24 in ANPox are clearly

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absent. Several studies have described extensive conformational exchange processes on time scales from 20 ls to 2 ms at sites that are close in space to cysteine bridges, particularly for disulphide cross-links that are exposed to solvent at a protein surface (Grey et al., 2003; Kopple et al., 1988; Otting et al., 1993). Similar line broadening effects, which have been attributed to conformational averaging around the v1 torsion angles of cysteine sidechains coupled to changes in the chirality of the disulphide bond, could be responsible for the large values of J ð0Þ found for residues 8 and 24 in the oxidised state of ANP. Chemical exchange contributions have also been invoked to account for unexpectedly large R2 and J ð0Þ values in turn regions, resulting perhaps from the sampling of several different turn configurations or from the kinetics of water molecules binding to the peptide backbone (Campbell et al., 2003). Motions of this sort may cause the exchange contributions required to account for the larger values of R2 and J ð0Þ observed for residues towards the centre of the sequence in both ANP species. However, these subtle effects are difficult to quantify without acquiring experiments at multiple magnetic field strengths or using sophisticated relaxation dispersion techniques (Grey et al., 2003). Reduced spectral density function data can be further analysed by investigating the dependence of J ðxN Þ and J ð0:87xH Þ on J ð0Þ (Lefevre et al., 1996). As shown in Fig. 3, the J ðxN Þ and J ð0:87xH Þ values of ANPox and ANPrd appear to be linearly correlated to the corresponding values of J ð0Þ for each amide site. The negative slope of J ð0:87xH Þ as a function of J ð0Þ (see Fig. 3B) is a result of the area under the spectral density function J ðxÞ being constant, so that an increased contribution from high frequency motions must be compensated for by a decrease in the value of J ð0Þ. Interestingly, the data points for ANPrd appear to lie on the same straight line as those of the oxidised form in both plots, indicating that both species can be described by the same unique correlation time for overall molecular tumbling. To assist in interpreting the motional information embedded in these correlations, Fig. 3 also displays dashed curves that indicate the reduced spectral density values expected for a simple Lorentzian model of J ðxÞ calculated for a range of rotational correlation times, s (Barthe et al., 1999). The straight line fit to the data in Fig. 3A intersects the theoretical curve twice at J ðxN Þ and J ð0Þ coordinates corresponding to s values of 0.7 ns (intercept 1) and 4.7 ns (intercept 2), which on the basis of magnitude characterise the time constants for a generalised internal motion and the overall tumbling, respectively, of ANP. The motional properties of data points that lie close to the straight line can be accounted for by spectral density functions that consist of a sum of two Lorentzians, with time constants equal to the values at the intercepts and scaled by an order parameter S 2 which varies between extreme values of 0 (for internal

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4.2. Lipari–Szabo analysis

Fig. 3. (A) Plot of the dependence of J ðxN Þ on J ð0Þ for 15 N nuclei in ANPox (open circles) and ANPrd (closed circles). (B) Plot of dependence of J ð0:87xH Þ on J ð0Þ for 15 N nuclei in ANPox (open circles) and ANPrd (filled circles). Dashed lines represent the dependencies predicted for a simple Lorentzian spectral density function on the rotational correlation time. Unbroken straight lines represent least squares fits to the data of both species; intercepts of these lines with the dashed theoretical curve are marked with stars and numbered.

motion only) and 1 (for overall motion only) (Lefevre et al., 1996). The data points for ANPrd are all close to intercept 1, indicating that local motions are dominant, consistent with values of S 2 between 0.1 and 0.3. By contrast, most of the data points for ANPox are located towards the centre of the chord, corresponding to values of S 2 between 0.3 and 0.6; only a few residues near the N- and C-termini exhibit a greater degree of internal motion. In Fig. 3B, the intercepts define motions with s values of 0.1 ns (intercept 3), 2.4 ns (intercept 4), and 3.2 ns (intercept 5); the last two values are similar in magnitude to the time constants for generalised internal motion and overall tumbling obtained above, while the first can be taken to represent an additional, more rapid internal motion. The fact that the two sets of time scales do not agree precisely reflects the higher degree of error in determining J ð0:87xH Þ than J ðxN Þ or J ð0Þ, as well as potential defects of the Lorentzian model used for the spectral density function.

Our analysis of the reduced spectral density functions of ANP suggested that, counter to our expectations, the motional properties of both forms of the peptide can be described by a single overall rotational correlation time with a value between 3.2 and 4.7 ns, along with local motions on two time scales: a rapid motion around 0.1 ns and a slower process between 0.7 and 2.4 ns. However, as the data points for ANPrd possess JðxN Þ and J ð0Þ coordinates that are clustered particularly close to the zero order parameter intercept (intercept 1 in Fig. 3A), their apparent colinearity with the data for ANPox may be only coincidental. When the two data sets are fitted separately the plot of J ðxN Þ against J ð0Þ gives intercepts with the theoretical single Lorentzian curve that corresponded to time constants of 0.7 and 4.6 ns for ANPox but 0.8 and 5.3 ns for ANPrd . It therefore seemed appropriate to analyse the 15 N relaxation parameters of ANP using the Lipari–Szabo approach for two cases: (1) assuming that overall motion can be accounted for by a single correlation time that describes both states; and (2) assuming that the two states require different overall correlation times. The simplest Lipari–Szabo models, which include contributions from a global correlation time (sm ) and a single rapid local motion described by a correlation time (se ) and scaled by an order parameter (S 2 ) for each residue, were not able to give a satisfactory fit to the data in either case. Better agreement was obtained by introducing two internal motions describing local processes that are either fast or slow on the sub-nanosecond time scale, governed by their own order parameters, Sf2 and Ss2 , respectively (Clore et al., 1990). As is usual for this approach, only the time scale of the slower internal motion was estimated. The parameters displayed in Fig. 4 for case (1) were obtained using a common sm of 4.7 ns for both species after a small exchange contribution, Rex of 1.22 s1 had been included for residue Phe8 of ANPox . The analysis for case (2) yielded sm values of 4.4 and 4.9 ns for ANPox and ANPrd , respectively, along with an Rex term of 1.37 s1 for residue Phe8 of ANPox . Both cases resulted in overall correlation times that are larger than those derived from the na€ıve analysis of the R2 =R1 ratios of ANPox and ANPrd described in Section 3.2. This discrepancy is consistent with picosecond– nanosecond time scale internal motions dominating the relaxation mechanisms of 15 N nuclei in both species. In contrast to the trend suggested by the R2 =R1 estimates of sm , the results for case (2) agree with the expectation that the overall correlation time of the expanded reduced state should be larger than that of the cyclised oxidised state. In both cases and for both species of ANP the faster internal motions make only minor contributions to relaxation rates, reflected in relatively uniform values of

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Fig. 4. Histograms describing the results of an extended model free Lipari–Szabo analysis of the 15 N relaxation parameters of ANPox (A–D) and ANPrd (E–H) as a function of residue number under the case (1) assumption (see text): (A and E) S 2 ; (B and F) Ss2 ; (C and G) Sf2 ; and (D and H) se . The results of the case (2) analysis are displayed as filled circles.

Sf2
0:8. The time scales of the slower local motions (se ) are
1.5 ns, which is too close to the time scale of sm for the results of the Lipari–Szabo analysis to be used quantitatively for either case (1) or case (2). In addition, because just three relaxation parameters were measured for each 15 N nucleus, it would only be possible to identify the relative merits of different models that require three or more local variables (such as additional chemical exchange contributions) by acquiring data at multiple magnetic field strengths. However, it is clear that while the effects of nanosecond time scale internal motions on the relaxation properties of each backbone amide site are dominant for both oxidation states, they are in general less significant for ANPox than for ANPrd , as indicated by mean values of the Ss2 order parameter of

0.5 and 0.2, respectively, in the case (2) analysis. This is consistent with the loss of the disulphide bridge allowing more conformation freedom in the peptide backbone. Fig. 4 also shows that the largest differences between cases (1) and (2) are observed for the Ss2 and se parameters of ANPox . If the overall tumbling rates of the two species are considered separately, the Ss2 values obtained for ANPox are larger than in case (1) and the motional time scales smaller, due to compensation between the sm ; se , and Ss2 parameters in the fitting procedure. Both approaches lead to values of the overall order parameter (S 2 ¼ Ss2  Sf2 ) of
0.4 and
0.2 for ANPox and ANPrd , respectively; these are significantly smaller than values typical of structured portions in folded globular proteins (
0.86). Order parameters are derived without reference

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to a specific model for the internal motion, but if this is assumed to be diffusion of a 15 N–1 H bond vector within a cone, then S 2 values of 0.4 and 0.2 corresponds to cone semiangles of 43° and 55°, respectively (Lipari and Szabo, 1982). Local motions with such large amplitudes can only be accommodated by high degrees of backbone flexibility, and the sites probed by 15 N relaxation measurements in ANPrd appear to be generally less restrained than those in ANPox . 4.3. Simulation of R2 data The generally low values of the order parameter determined by the Lipari–Szabo analyses of ANPox and ANPrd imply that the relaxation properties of a given backbone amide site may be determined more by its location in a polypeptide chain of a given topology than by tertiary contacts or the specific nature of its neighbours. For flexible peptides the contributions of neighbouring residues to transverse relaxation rates of backbone 15 N nuclei can be simulated by assuming an exponential dependence on distance from a given site (see Section 2.3). This simple model was used to fit the 15 N R2 values of both ANPox and ANPrd simultaneously, using a single variable (Rint ) and fixing kint at 7 residues (Klein-Seetharaman et al., 2002; Schwalbe et al., 1997; Schwarzinger et al., 2002), as shown in Fig. 5A and B. In the absence of disulphide cross-links,

the model predicts that R2 should have a simple bellshaped dependence on position in the primary sequence (Fig. 5B), which describes the data points obtained for ANPrd quite well. The more complex topology of the oxidised species has a major effect on its relaxation properties (Fig. 5A), leading to R2 values that are larger than those of ANPrd and giving the profile a bilobal form. The two maxima predicted by this approach are, however, shifted to the covalently linked cysteine residues (7 and 23) rather than appearing at Phe8 and Asn24. The broad features of the profiles of both species are successfully reproduced, suggesting that the relaxation rate at each 15 N site is determined mostly by motions of the polypeptide chain involving segments comprised of residues that are less than 7 residues distant in the covalent network (Schwalbe et al., 1997). Studies of protein denatured states have noted that significant chemical exchange contributions are required to account for the 15 N transverse relaxation rates of residues that are in the vicinity of disulphide bridges (Buck et al., 1996). The relaxation model was therefore extended to include exchange terms for residues 8, 15, and 24 (see Section 2.3). With kint fixed at 7 residues and kex at 2 residues, the optimum values of Rint and Rex were 0.25 and 1.75 s1 , respectively, and the fit to the data improved significantly (see Fig. 5C and D). Alternative integer values of kex gave poorer agreement with experiment, so for ANPox in aqueous solution at 4 °C

Fig. 5. Simulations of R2 profiles for ANPox (A and C) and ANPrd (B and D): open circles, experimental values; thick line, simulated values; (A and B) simulations with Rint set to 0.28 s1 and kint fixed at 7 residues, giving a v2 value of 19.9; (C and D) simulations with Rint set to 0.26 s1 , Rex set to 1.75 s1 , kint fixed at 7 residues, and kex fixed at 2 residues, giving a v2 value of 8.6.

H. Peto et al. / Journal of Structural Biology 148 (2004) 214–225

chemical exchange contributions to R2 appear to operate more locally than the Rint mechanism. This observation contrasts with earlier studies of unfolded states of lysozyme at 20 °C in 8 M urea (Schwalbe et al., 1997) and in water (Klein-Seetharaman et al., 2002), which used kex values of 7 and 1–10 residues, respectively, indicating that the magnitude of the line broadening effect may be dependent on temperature or solvent viscosity. In a detailed analysis of the 15 N relaxation properties of apomyoglobin (which lacks disulphide cross-links) in 8 M urea at pH 2.3, and 20 °C, local clusters of hydrophobic sidechains required exchange contributions that affected sites 2–7 residues distant in the amino acid sequence (Schwarzinger et al., 2002). Since ANP has few hydrophobic residues, this suggests that line broadening effects due to hydrophobic clustering may operate over a longer range than those caused by cysteine bridges. 4.4. Estimates of changes in conformational entropy Order parameters calculated from a Lipari–Szabo analysis can be used to calculate the contributions made by motions that reorient 15 N–1 H bond vectors to the conformational entropy of a peptide or protein (Yang and Kay, 1996). The results of the case (1) analysis of the relaxation parameters of ANPox and ANPrd suggest that the backbone conformational entropy of the peptide decreases by approximately 5.5 J mol1 K1 residue1 when the disulphide bond is formed, leading to a total entropy change (DSconf ) of )90 to )150 J mol1 K1 , depending on the number of residues that should be included in the comparison between the two states (i.e., from 17 to 28 residues). Polymer chain theory predicts that the effect of introducing a cross-link on the conformational entropy of a polypeptide is 8:79  1:5R ln n, where n is the number of residues in the loop forming the disulphide bond (Pace et al., 1988); for ANP, n is 17, so DSconf should be )44 J mol1 K1 . The change in conformational entropy derived from the 15 N relaxation analysis ignores the effects of sidechain motions, which should lead to an underestimation of DSconf , but this is probably compensated for by neglecting the possibility of correlated backbone motions or dynamics that are slower than the millisecond time scale, both of which were suggested by simulations of ANP R2 data in Section 4.3. For ANP the greater magnitude of DSconf calculated from 15 N order parameters (S 2 ) may be due to sequence dependent factors, such as the high content of low volume glycine and alanine residues in the disulphide linked loop (35%), which could render the reduced state more flexible than normal (Zhang et al., 1994). This result will also be affected by applying the Lipari–Szabo analysis beyond its appropriate limits. He et al. (2001) used isothermal titration calorimetry (ITC) to show that single molecules of ANPox bind with high affinity to NPRC dimers with a dissociation

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constant (Kd ) of
1 nM and an entropy change for complex formation of )48 J mol1 K1 at 30 °C. By assuming that only residues 6–24 of ANP become rigidified in the complex so S 2 can be set to 0.86, the mean value for well-ordered portions of small globular proteins (Bracken et al., 1999), while all other residues possess the same dynamic properties as the free peptide, the contribution of the backbone motions of the hormone to this process can be estimated. Using the results of the case (1) analysis this approach gives mean DSconf values of )13 and )19 J mol1 K1 residue1 and total DSconf values of )250 and )350 J mol1 K1 for the formation of complexes by ANPox and ANPrd , respectively. The total entropy change for complex formation predicted by the relaxation data for ANPox is fivefold larger than that determined by ITC, which will be modified by contributions from rearrangement of the receptor (negative) and the solvent (positive). The mean conformational entropy changes calculated for both forms of ANP are slightly larger than the )12 J mol1 K1 residue1 value obtained in studies of the binding of the S-peptide to the S-protein fragment of ribonuclease A; this probably reflects the higher degree of residual a-helical structure (
22%) in the free S-peptide under the conditions of the experiment (Alexandrescu et al., 1998). Complex formation with the receptor is less favourable for ANPrd than for ANPox because the task of freezing out the more extensive local motions of the peptide backbone costs more in terms of free energy. If the enthalpy changes for the formation of complexes of ANPox and ANPrd with NPR-C are assumed to be equal, then the difference in the free energy changes of these processes (DDGconf ) will be determined purely by entropic factors (DDSconf ). A value of )100 J mol1 K1 for DDSconf leads to an upper bound estimate of 6  106 for the ratio of dissociation constants Kd ðANPox Þ= Kd ðANPrd Þ; a lower bound of 5  103 can be obtained by setting DDSconf equal to the value predicted by polymer chain theory (Pace et al., 1988). Since Kd ðANPox Þ is
1 nM (He et al., 2001), our calculations place the dissociation constant for binding of ANPrd to NPRC in the range 190 nM–160 lM. As found for linear mimetics of natriuretic peptides (Bovy et al., 1989), ANPrd will have lower affinity for the receptor due to the large increase in conformational entropy that results from cleavage of the cysteine bridge. Our results therefore illustrate a principle long acknowledged in the field of drug design, that reducing the conformational freedom of a ligand can dramatically enhance its affinity for a target (Hruby, 2001). The dissimilarity between the solution structure of a restrained analogue of CNP (Fairbrother et al., 1994) and the extended conformation of the bound form in the NPRC/CNP complex (He et al., 2001) has led some authors to question the relevance of structural studies of unbound flexible peptides

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(Dyson and Wright, 2002). By contrast, the work presented here confirms that the dynamic properties of an unbound ligand have a very important role to play in signal transduction processes.

Acknowledgment M.S. gratefully acknowledges the Royal Society for the award of a Research Fellowship.

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