Solution NMR structure and dynamics of human apo-S100A1 protein

Journal of Structural Biology 174 (2011) 391–399

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Solution NMR structure and dynamics of human apo-S100A1 protein Michał Nowakowski a, Łukasz Jaremko a,b, Mariusz Jaremko a, Igor Zhukov a,c,d, Agnieszka Belczyk a, Andrzej Bierzyn´ski a, Andrzej Ejchart a,⇑ a

´ skiego 5A, 02-106 Warsaw, Poland Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawin Faculty of Chemistry, Warsaw University, Pasteura 1, 02-093 Warsaw, Poland c Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, P.O.B. 660, SI-1001 Ljubljana, Slovenia d EN-FIST Center of Excellence, Dunajska 156, SI-1000 Ljubljana, Slovenia b

a r t i c l e

i n f o

Article history: Received 3 December 2010 Received in revised form 28 January 2011 Accepted 28 January 2011 Available online 3 February 2011 Keywords: Calcium binding protein, Human apoS100A1 NMR Backbone dynamics Protein structure

a b s t r a c t S100A1 belongs to the EF-hand superfamily of calcium binding proteins. It is a representative of the S100 protein family based on amino acid sequence, three-dimensional structure, and biological function as a calcium signal transmitter. It is a homodimer of noncovalently bound subunits. S100A1, like most of other members of the S100 protein family, is a multifunctional, regulatory protein involved in a large variety of biological processes and closely associated with several human diseases. The three-dimensional structure of human apo-(i.e. calcium free)-S100A1 protein was determined by NMR spectroscopy (PDB 2L0P) and its backbone dynamics established by 15N magnetic relaxation. Comparison of these results with the structure and backbone dynamics previously determined for bovine apo-S100A1 protein modified by disulfide formation with b-mercaptoethanol at Cys 85 revealed that the secondary structure of both these proteins was almost identical, whereas the global structure of the latter was much more mobile than that of human apo-S100 protein. Differences between the structures of human and rat apo-S100A1 are also discussed. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction S100 proteins play a crucial role in many biological processes regulated by calcium in the cells of vertebrate species (Santamaria-Kisiel et al., 2006). S100A1 protein can be treated as an ideal representative of the S100 protein family because of its amino acid sequence, three-dimensional structure, and biological function as a calcium signal transmitter. It is a homodimer of noncovalently bound subunits. Each of which contains two EF-hand structural motifs formed by two ahelices flanking a calcium-binding loop. These motifs are covalently connected through a short linker and noncovalently by interhelical hydrophobic interactions and a hydrogen bond(s)

Abbreviations: CPMG, Carr–Purcell–Meiboom–Gill; DSS-d4, 3-trimethylsilyl2,2,3,3-tetradeuteropropionic acid sodium salt; EDTA, ethylenediaminetetraacetic acid; HSQC, heteronuclear single quantum coherence; HPLC, high performance liquid chromatography; NMR, nuclear magnetic resonance; NOE, nuclear overhauser effect; S100A1, homodimeric S100A1 with noncovalent interactions at the dimer interface; S100A1-bME, mixed disulfide of S100A1 protein with b-mercaptoethanol; TRIS-d11, perdeuterated 2-amino-2-(hydroxymethyl)1,3-propanediol. ⇑ Corresponding author. Fax: +48 22 5922038. E-mail addresses: [email protected] (M. Nowakowski), [email protected] (Ł. Jaremko), [email protected] (M. Jaremko), [email protected] (I. Zhukov), [email protected] (A. Belczyk), [email protected] (A. Bierzyn´ski), aejchart@ibb. waw.pl (A. Ejchart). 1047-8477/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2011.01.011

between the two Ca2+-binding loops that stabilizes a short, antiparallel b-sheet. Such a structure, determined for rat S100A1 protein using NMR methods (Rustandi et al., 2002; Wright et al., 2005), closely corresponds to other known structures of S100 proteins (Marenholtz et al., 2004). S100A1, like other members of the S100 protein family (with rare exceptions) is a multifunctional, regulatory protein involved in a large variety of biological processes. It is closely associated with several human diseases including cardiomyopathy, neurodegenerative disorders, and cancer (Heizmann et al., 2004). Recently, we have shown that chemical modifications of the unique SH group within the protein (that of Cys 85) by the covalent S–S bonding to small thiols, such as glutathione, cysteine or even b-mercaptoethanol, results in an enormous increase (a couple of orders of magnitude) of the protein affinity for calcium (Goch et al., 2005). Therefore, it is highly probable that the protein can act as a linker between the redox- and calcium-signal pathways that, in a number of biological processes, were proven to be coupled with each other (Dalle-Donne et al., 2008), although very little is known about the molecular mechanism of this coupling. It seems, therefore, to be interesting to find out how the S100A1 structure is changed by chemical modifications of its Cys 85 residue. We tried to answer this question by determining the structure of bovine apo-S100A1 modified by b-mercaptoethanol and comparing it with the only structure of apo-S100A1 determined so

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far: that of rat protein (Rustandi et al., 2002). The results of that work were presented and discussed in our previous paper (Zhukov et al., 2008). Nevertheless, we were aware that the conclusions presented in it were perhaps arguable. The observed structural differences between the both proteins were small. They could arise, at least in part, from the difference of their sequences or, even worse, they simply could be an artifact, because experimental procedures and data analysis methods used to determine these structures had not been the same. Therefore, we decided to determine and compare the structures of the same S100A1 molecule, native and modified at Cys 85 in its apo and holo states. Human S100A1 protein has been chosen as the object of our studies and in this paper its structure and backbone dynamics in the apo state are presented. 2. Materials and methods 2.1. Sample preparation 15

N-labeled and 13C, 15N-double labeled S100A1 protein was obtained as previously described (Bolewska et al., 1997). The synthetic gene encoding for human S100A1 was cloned into pET-30a+ plasmid and expressed in E. coli utilizing the T7 expression system. The bacteria were grown in M9 media containing (15NH4)2SO4 as the sole nitrogen source and either unlabeled or 13C labeled glucose as the sole carbon source. Expression products were isolated using the classical method of ammonium sulfate precipitation (Dixon and Webb, 1961; Falconer et al., 1953), purified by reversed-phase HPLC on a semipreparative Vydac C18 column, and identified by electrospray ionization mass spectrometry using a Macromass Q-Tof spectrometer. Two forms of the protein, one with the sequence strictly corresponding to its gene sequence and another one, with the additional initiator methionine at the N-terminus, were obtained from HPLC as partly overlapping peaks. NMR measurements indicated that structural differences between both forms are small and restricted to the close proximity of the N-terminal Met residue (Baldisseri et al., 1999). Therefore, a mixture of both forms of proteins was used in all NMR measurements. 650 lL samples contained 1 mM protein solution (monomer concentration) in 90%/10% H2O/D2O, 50 mM TRIS-d11, 1 mM EDTA, 0.1 mM NaN3 and 50 mM NaCl with pH adjusted to 6.8 (uncorrected value). In case of HC detected experiments 100% deuterated buffer was used. 2.2. NMR spectroscopy All NMR measurements were performed at the temperature carefully adjusted to 37 °C checked by an ethylene glycol reference sample. Time domain data were acquired using the States-TPPI quadrature detection (Marion et al., 1989) followed by the sensitivity enhanced detection introduced by Kay et al. (1992a). 1.6 s recycling delay was used, if not stated otherwise. All chemical shifts in 1 H NMR spectra were reported with respect to external DSS-d4. Chemical shifts of 13C and 15N signals were referenced indirectly using the 0.251449530 and 0.101329118 frequency ratios for 13 1 C/ H and 15N/1H, respectively (Wishart et al., 1995). Experimental data were processed using the NMRPipe software package (Delaglio et al., 1995). Zero filling and 90° shifted squared sine-bell filter were performed prior to the Fourier transformation. Processed spectra were analyzed with both the CARA (Keller, 2004) and the SPARKY (Goddard and Kneller) software. The sequence-specific assignment was performed using a uniformly 13C,15N-double labeled sample of apo-S100A1. 3D heteronuclear HNCO (Muhandiram and Kay, 1994), HNCA (Ikura et al., 1990), HN(CO)CA (Ikura et al., 1991), HNCACB (Wittekind and

Mueller, 1993), CBCA(CO)NH (Grzesiek and Bax, 1992) and (HCA)CO(CA)NH (Lohr and Ruterjans, 1995) spectra were used to obtain assignments of the backbone 1H, 13C and 15N resonances. The assignment was additionally confirmed by analysis of sequential and medium-range NOEs in the 3D 15N-edited NOESY-HSQC experiment (Zhang et al., 1994). Aliphatic side chain 1H and 13C resonances were assigned from the analysis of the 1H–13C HSQC, C(CO)NH (Grzesiek et al., 1993), HBHA(CBCACO)NH (Grzesiek and Bax, 1993), HCCH-TOCSY (Bax et al., 1990) and 13C-edited NOESY-HSQC spectra (Muhandiram et al., 1993). Aromatic side chain resonances were fully assigned from the (HB)CBHD, (HB)CBHE (Yamazaki et al., 1993), 1H–13C HSQC and the 13C-edited NOESY-HSQC spectra recorded with the offset, spectral widths and 13 C–1H coupling constants tuned to aromatic carbons. Distance constraints were obtained from the 15N-edited NOESY-HSQC spectrum and the 13C-edited NOESY-HSQC spectra separately tuned to aliphatic and aromatic carbons. The detailed information about all experiments is summarized in Supplementary Table A.1. Relaxation measurements were performed at three magnetic fields: 9.4, 11.7 and 16.4 T, with 15N-labeled sample. Longitudinal (R1) and transverse (R2) relaxation rates were measured using the sensitivity enhanced 1H–15N HSQC pulse sequence (Kay et al., 1992a) with the option of either R1 or R2 measurements of 15N nuclei (Farrow et al., 1994). The R2 relaxation rate measurements were performed with the CPMG pulse train. A refocusing time of 650 ls was used during the evolution delays. The acquisition parameters for R1 and R2 measurements on each spectrometer were identical with the exception of the delay between p (1H) pulses used for the cross-correlation effect suppression (Kay et al., 1992b). Five and ten ms delays were used in the R1 and R2 measurements, respectively. Delays between the scans of 1.8 s were employed in both types of experiments. {1H}–15N NOEs were measured with the pulse sequence included in the ProteinPACK Varian Inc. (Palo Alto, CA) software. Details and experimental parameters of relaxation spectra are gathered in Supplementary Table A.2. Resonance intensities were used in calculating relaxation rates and NOE values. All experiments were repeated twice. Experimental errors of relaxation rates were obtained from appropriate elements of the variance–covariance matrix. Experimental errors in NOE values were evaluated based on signal-to-noise ratios obtained for corresponding signals in spectra with and without NOE (Fushman, 2003). Consistency of measurements performed at different magnetic field strengths was confirmed as proposed by Morin and Gagne (2009). The 1H, 13C and 15N resonance assignments and 15N magnetic relaxation data have been deposited in the BioMagResBank with the accession code 16360. 2.3. Structure calculation The NOESY interproton distance constraints were derived from the 3D heteronuclear 15N- and 13C-edited NOESY-HSQC experiments. The initial structure calculations were performed with the CYANA 3.0 software (Guentert et al., 1997). The dimeric interface between the protein subunits linked by a chain of dummy atoms, was defined basing on structural similarity to the known structure of rat S100A1 protein (PDB entry 1K2H). The automatic NOESY assignment procedure (Herrmann et al., 2002) provided 754 short, 434 medium and 272 long range intrasubunit distance constraints and 132 intersubunit distance constrains, defining their mutual orientation. Additionally, 134 restraints for backbone u and w torsion angles for Ser 2–His 18, Leu 28–Glu 63, and Val 69–Thr 82 were predicted from the chemical shifts using the PREDITOR server (Berjanskii et al., 2006). For the less defined protein regions (like Ser 19–Lys 27 and Asn 64–Glu 68 calcium-binding loop fragments

M. Nowakowski et al. / Journal of Structural Biology 174 (2011) 391–399

or the C-terminus of helix IV starting from Val 83) the dihedral angle constraints were not applied. Stereospecific assignments for 122 side chain chiral groups in each subunit were generated by the program GLOMSA (Guentert et al., 1991) that is included in the CYANA software. Before the final structure calculations, 184 constraints defined as rHN-O = 1.5–2.5 Å and rN-O = 2.5–3.5 Å for 92 hydrogen bonds were added based on geometric criteria. If a given hydrogen bond existed in more than 75% of structures in the ensemble, then it was selected for the final refinement. The final structure refinement was performed with the XPLOR-NIH 2.2.5 program (Schwieters et al., 2003) using the symmetry equivalence constraints between two subunits. Evaluation of the obtained human apo-S100A1 structure quality was done with the PROCHECK-NMR (Laskowski et al., 1996) and the What-If (Vriend, 1990) programs.

2.4. Analysis of

15

model-free approach with anisotropic overall tumbling (Woessner, 1962). Three Euler angles relate the directions of the principal axes of the asymmetric diffusion tensor to the molecule fixed coordinate system. Because of the symmetry requirement one of the principal axes of the diffusion tensor of homodimeric S100A1 protein is collinear with the protein symmetry axis. This condition reduces the number of Euler angles from three to one. The coefficients describing orientation of H–N vectors into the molecule fixed coordinate system have been calculated using the atomic coordinates of the NMR-derived structure. The vibrationally averaged N–H distance was assumed to be 0.104 nm (Ottiger and Bax, 1998) and chemical shift anisotropy of axially symmetric 15N chemical shift tensor was taken to be 170 ppm. The least-squares procedure used to optimize the model parameters consisted of minimization through a grid-search of the target function v given by:

N relaxation data

Methodology for analyzing 15N relaxation data measured at multiple magnetic fields has been described in our previous papers (Zhukov et al., 2007, 2008). There are, however, two differences between the method utilized earlier and that used in this paper. First, the extended model-free approach (Clore et al., 1990) has been applied rather than the genuine one (Lipari and Szabo, 1982). The original model often fails to represent experimental data accurately, especially the calculated NOE values tend to be too small. The extended model includes five parameters; rotational diffusion correlation time sR, two order parameters, Sf and Ss, and two effective correlation times, sf and ss, associated with fast and slow internal motions. One can expect that local motions of residues located in calcium-binding loops and a linker will be more adequately described with two internal motions taking place on significantly different time scales. Second, the approximation of the axially symmetric overall motion has been replaced with a fully asymmetric one. It has been checked with Fisher–Snedecor statistics (F test) that the fully asymmetric model is significantly better at the probability 0.01 (details of the model selection are given in Supplementary Table A.3). Appropriate spectral density functions can be derived (Baber et al., 2001; Tjandra et al., 1995) combining the extended

393

v¼

N X i¼1

Ci ¼

N X M X ½ðPij;exp  Pij;calc Þ2 =r2ij  i¼1

j¼1

where the sum was over M relaxation parameters for each of N residues, and Pij,calc were the appropriate relaxation parameters calculated from the assumed model. The rij values were the corresponding standard deviations of experimentally derived Pij,exp. The minimization procedure delivered four global parameters (three diffusion coefficients and one Euler angle) and N sets of local, residue specific parameters comprising Sf, Ss, sf, ss, and Rex. The residues with incomplete experimental data (5 6 M < 9) were excluded from the procedure at this stage of calculations. Their local parameters were determined separately for each residue by minimizing partial target function Ci and using known global parameters obtained earlier. The residues of both flexible termini were also excluded from the calculation delivering the overall diffusion parameters. Those unstructured, flexible terminal segments cannot be regarded as a part of the rigid rotor and description of their motions in terms of a single overall correlation time does not seem to be appropriate (Alexandrescu and Shortle, 1994; Brutscher et al., 1997). Model parameter uncertainties derived in the minimization of target functions v and Ci were obtained as standard deviations from 200 Monte Carlo simulations (Press et al., 1986).

Fig. 1. 1H–15N HSQC spectrum of human apo-S100A1 protein. The terminal NH2 groups of Asn and Gln residues are not labeled.

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twenty two pairs of prochiral groups have been stereoassigned in each monomer unit.

3. Results 3.1. Sequence-specific resonance assignment The 2D 1H-15N HSQC spectrum of homodimeric S100A1 protein displayed relatively good dispersion of amide correlation signals (Fig. 1). Cross peaks for all residues could be assigned, except that of Asn 87, which was not observed due to line broadening most possibly caused by an exchange process. The sequence-specific backbone and side-chain assignments were completed using 2D and 3D NMR experiments. The 13Cb chemical shifts for the unique cysteine residue (Cys 85) showed that its thiol group was in the reduced state (Martin et al., 2010; Sharma and Rajarathnam, 2000). All amide 15N resonances except that of Asn 87, all resonances of 13 C nuclei with attached protons and all 1HC and amide 1HN resonances have been assigned unequivocally. One hundred and

3.2. Three-dimensional structure of human apo-S100A1 The three-dimensional structure of the protein was calculated based on NMR data (1460 intrasubunit distance constraints and 63 dihedral restraints for each subunit together with 132 intersubunit NOE-based distance constraints). 98.8 % of all residues are located in the most favored (86.9%) or additionally allowed regions (11.9%) of the Ramachandran plot giving an estimated precision of the structure comparable to the resolution of 1.7 Å of X-raybased structure. The statistics for the ensemble of 20 most favorable structures is given in Table 1. A ribbon diagram for the lowest energy structure is shown in Fig. 2. The atomic coordinates for all

Table 1 NMR-derived constraints and statistic for the ensemble of 20 structuresa. NOE distance constraints within subunitb Intraresidual & sequential (|i-j| P 1) Medium-range (1 < |i-j| < 5) Long-range (|i-j| P 5) Intersubunit NOE distance constraints per subunit Hydrogen bonds constraints Restraints per residue Torsion angle constraints

1460 754 434 272 132 184 19.1

Backbone (u/w) Side chains (v1, v2) Mean r.m.s.d. from experimental restraints (±s.d.)

67/67 0/0

NOE (Å) Dihedral angles (deg) r.m.s.d. from idealized covalent geometry (region 1.93) (±s.d.)

0.0055 ± 0.0003 0.2168 ± 0.0002

Bonds (Å) Angles (deg) Impropers (deg) Ramachandran plot (1.93)c residues in the most favored regions (%) Residues in additional allowed regions (%) Residues in generously allowed regions (%) Residues in disallowed regions (%) Ramachandran plot (5.85)

0.0012 ± 0.0001 0.3711 ± 0.0009 0.2007 ± 0.0010 86.8 ± 1.0

Residues in the most favored regions (%) Residues in additional allowed regions (%) Residues in generously allowed regions (%) Residues in disallowed regions (%) r.m.s.d. to the mean subunit structured Ordered backbone atoms (1.93) (Å)

87.4 ± 1.0 11.5 ± 1.0 0.9 ± 0.1 0.1 ± 0.1

Ordered heavy atoms (1.93) (Å) r.m.s.d. to the mean subunit structure Ordered backbone atoms (5.85) (Å) Ordered heavy atoms (5.85) (Å) r.m.s.d. to the mean structure of the whole dimer (both subunits) Ordered backbone atoms (1.93 A and B) (Å) Ordered heavy atoms (1.93 A and B) (Å) r.m.s.d. to the mean structure of the whole dimer (A and B subunit) Ordered backbone atoms (5.85 A and B) (Å) Ordered heavy atoms (5.85 A and B) (Å) Structure Z-scorese 1st generation packing quality 2nd generation packing quality Ramachandran plot appearance v1 and v2 rotamer normality Backbone conformation Equivalent X-ray resolution of Ramachandran plot (1..93)c Equivalent X-ray resolution of Ramachandran plot (5..85) Equivalent X-ray resolution of v1 and v2 (1.93)

1.50 ± 0.24

11.9 ± 1.0 1.0 ± 0.1 0.1 ± 0.1

0.92 ± 0.21

0.46 ± 0.07 0.97 ± 0.09 0.98 ± 0.22 1.54 ± 0.24 0.53 ± 0.08 1.01 ± 0.10

0.728 ± 0.192 3.250 ± 0.198 4.183 ± 0.286 3.296 ± 0.611 0.024 ± 0.412 1.7 Å 1.6 Å 1.3 Å/1.0 Å

a The ensemble of 20 lowest energy structures (out from 130 submitted to calculations) of human apo-S100A1 homodimer conformers calculated with XPLOR-NIH 2.2.5 (Schwieters et al., 2003). b None of the 20 structures has a distance violation exceeding 0.2 Å and a dihedral angle violations greater than 5o. c Quality of the 20 protein structures was checked by PROCHECK-NMR (v.3.4) (Laskowski et al., 1996). d The r.m.s.d. values were calculated with MOLMOL (Koradi et al., 1996). e Ensemble of structures was validated and analyzed using the WhatIf program (Vriend, 1990).

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Each subunit contains four a-helices formed by residues Glu 3– His 18, Lys 30–Leu 41, Val 51–Asn 64 and Phe 71–Asn 86, two short 310-helical segments: Ser 42–Ala 47 and Asn 87–Phe 89 and one short antiparallel b-sheet formed by Leu 28, Ser 29, Glu 68, and Val 69. The existence of helices, especially of the second short 310-helix at the C-terminus was additionally confirmed by short and medium-range NOEs from the 15N-edited NOESY-HSQC spectrum. For residues Glu 5–Cys 85 within one subunit, the RMSD for the backbone atoms (N, Ca, C0 ) equals 0.46 ± 0.07 Å and for all heavy atoms 0.97 ± 0.09 Å, while for both subunits in the dimer, the corresponding values are 0.53 ± 0.08 Å and 1.01 ± 0.10 Å, respectively. Almost identical RMSD values obtained for the single subunit and the homodimer prove that the interface between subunits is unequivocally determined and the resulting ensemble of structures represents a well defined homodimeric protein. The dimer interface is located mainly between helices I and IV0 and helices IV and I0 . On average 4–5 long-range distance restraints are observed for residues involved in the dimer formation. 3.3. Protein dynamics

Fig. 2. Ribbon diagram of the lowest energy structure of human apo-S100A1 protein.

20 structures of human apo-S100A1 have been deposited in the Protein Data Bank with the accession code 2L0P.

The lowest energy S100A1 conformer was used to calculate the molecular inertia moment tensor and the geometric coefficients Ak. The ratios of the inertia tensor principal values were equal to Ix:Iy:Iz = 1.13:1.00:1.54 with the z axis collinear with the molecular symmetry axis. Full relaxation data were available for 60 out of 92 backbone amides and they were used for the simultaneous determination of four global parameters and 60 sets of local parameters. Incomplete relaxation data for four additional residues (Leu 4, Ser 19, Leu 28, Gln 48) were sufficient to obtain the local parameters. The remaining 28 residues, mostly located at both

Fig. 3. Experimental data (vertical bars) and predicted values from TALOS+(Shen et al., 2009) (full circles) for order parameter values S2 (upper part) and exchange terms Rex at 9.4 T (lower part) with corresponding error bars for amino acid residues of human apo-S100A1 protein.

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Fig. 4. Weighted means of order parameter values hS2i (upper part) and exchange terms hRexiat 9.4 T (lower part) with corresponding error bars for amino acid residues in various structural elements of human apo-S100A1 and bovine apoS100A1-bME proteins. The experimental 15N relaxation data for bovine apoS100A1-bME protein (Zhukov et al., 2008) were reprocessed as described in the Analysis of 15N relaxation data section, i.e. assuming fully anisotropic molecular reorientation and extended model-free approach.

Table 2 Angles (in degrees) between helices I, II, and IV in three apo-S100A1 structuresa. Helices

Rat apo-S100A1b

Human apo-S100A1c

Bovine apoS100A1-bMEd

I–II I–IV II–IV I–I0 IV–IV0

120 ± 3 107 ± 2 46 ± 1 165 ± 3 176 ± 2

133 ± 1 117 ± 1 34 ± 1 144 ± 2 151 ± 1

113 ± 2 124 ± 2 43 ± 3 170 ± 3 143 ± 5

a Interhelical Toronto). b Taken from c Taken from d Taken from

angles were calculated using interhelix (K. Yap, University of NMR structure (PDB entry 1K2H) of Rustandi et al. (2002). NMR structure (PDB entry 2L0P), this paper. NMR structure (PDB entry 2JPT) of Zhukov et al. (2008).

termini and in helices I and IV, displayed partially superposed 1 H/15N correlations in the 2D spectra (see Fig. 1). Therefore, their precise quantitative analysis was not possible. The principal values of the overall diffusion tensor are equal to: Dx = (1.94 ± 0.02) 107 s1, Dy = (2.02 ± 0.02) 107 s1, and Dz = (1.83 ± 0.01) 107 s1. The angle between the directions of Ix and Dx axes was found to be equal to 26 ± 7 deg. The averaged isotropic rotational correlation time, sR = (2Dx + 2Dy + 2Dz)1 = 8.63 ± 0.08 ns is typical for globular proteins of about 20 kDa size and corresponds well to the results obtained for other proteins from the S100 family (Bertini et al., 2009; Dutta et al., 2008; Inmam et al., 2001; Zhukov et al., 2008).

Out of the five residue specific parameters describing the local mobility of backbone amide N–H vectors within the frame of the extended model-free approach, S2 = Sf2Ss2 and Rex are of special importance. The former describes restrictions imposed on motions much faster than the overall protein diffusion (the ps time scale) and the latter allows the detection of much slower motions, but fast enough to average chemical shifts of the exchanging sites (usually on the ls–ms time scale). The S2 values of the residues located in both calcium-binding loops and in the linker indicate that the mobility of these protein segments is higher than those situated in the helices. On the other hand, residues involved in chemical exchange processes are predominantly located in the N-terminal calcium-binding loop (Fig. 3). The obtained model parameters reproduce well input experimental data. The plot of calculated vs experimental R2/R1 ratios is given in Supplementary Fig. S.1. Since the local motion analysis was not possible for all backbone NH amide groups, the S2 values were also predicted from the chemical shifts using TALOS + software (Shen et al., 2009). As can be seen in the upper part of Fig. 3 the predicted S2 values reasonably well reproduce the experimentally determined ones. Differences in mobility of the protein structural elements become much more evident when the weighted means of S2 and Rex values determined for the residues building them are compared. The data presented in Fig. 4 indicate that the outer helices I and IV are more rigid than the inner ones: II and III. It seems to be a common feature of apo-S100 protein structures as shown by relaxation studies of S100B (Inmam et al., 2001), S100A4 (Dutta et al., 2008), and S100A5 (Bertini et al., 2009) proteins. It is also visible that small but significant Rex values appear in the C binding loop. Summing up, comparison of the dynamics of the structural elements of the protein reveals that, relative to helices, the binding loops display increased mobility on both, ps and ls–ms time scales, whereas the linker is more mobile only within the range the ps time scale. It is worth to mention here that the Rex values determined by us for 15N nuclei are by no means correlated with their chemical shift changes induced in S100A1 proteins by calcium binding as calculated from the data published for apo (Rustandi et al., 2002; BMRB accession no. 4285) and holo (Wright et al., 2005; BMRB accession no. 6583) states of rat S100A1 and fully confirmed by our preliminary studies of human holo protein (to be published). The most striking examples corroborating this statement are as follows. The largest shift of 15N signal induced by calcium binding is observed for Asp 63: as much as 10.8 ppm. Yet, its Rex value is very low: 0.16 s1. On the other hand, the highest Rex (3.7 s1) has been found for Glu 22 amide nitrogen (see Fig. 3) which signal position does not change considerably upon Ca2+ coordination (Dd = 0.9 ppm).

4. Discussion The complete resonance assignment of aromatic residues allowed us to determine the precise positions and orientations of aromatic side chain groups, especially that of Trp 90, which displayed two unequivocal NOE contacts to Tyr 260 located in the N loop of neighboring subunit: He1(W90)/Hd1(Y260 ) and HN(W90)/ Hd1(Y260 ). The three 1H resonances involved in these contacts are well separated from others. Together with another unequivocal NOE contact, Hb(N87)/He1(Y260 ), they are decisive for the determination of the structure terminating helix IV in the both protein subunits as a short 310-helix comprising Asn 87, Phe 88 and Phe 89 residues. Such a tightening of the final a-helix turn is not unusual (Barlow and Thornton, 1988). This structure is less precisely defined than the a-helical ones. The lower number of NOEs per

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Fig. 5. Ribbon representations of helices IV and C-terminal segments (residues 70–93) in three structures of apo-S100A1 proteins: (a) rat, (b) human, (c) bovine modified with b-mercaptoethanol at Cys 85. A hydrogen bonded turn may be recognized for residues N87–W90 in the rat protein. (Color reproduction on the web and in print).

residue for the Asn 87–Phe 89 region (10.8 NOE/res. in comparison with 34.3 NOE/res. for Phe 71–Asn 86 helix IV) is accompanied by the lack of Asn 87 NH cross peak in the 2D 1H-15N HSQC spectrum suggesting the involvement of this region in an exchange process. Up till now there are three available structures of apo-S100A1 proteins: rat (1K2H) (Rustandi et al., 2002), human (2L0P) [this paper], and bovine modified at Cys 85 by b-mercaptoethanol (2JPT) (Zhukov et al., 2008). All of them have been determined by NMR methods. The positions of secondary structure elements as well as mutual orientations of helices (Table 2), are very similar in all of the structures. Nevertheless, there are a few small, but important, structural differences between these proteins. Differences between the interhelical angles listed in Table 2 considerably exceed the estimated error limits. They can be explained in two ways: either the method commonly used to determine the a-helix orientation and/or error estimation in such a structure analysis is untrustworthy, or even very small changes in the protein sequence or its slight chemical modification can result in noticeable structural changes. Both explanations should be taken into account. Nevertheless, a dramatic increase of Ca+2-binding affinity of bovine S100A1 protein induced by its Cys 85 chemical modification strongly suggests that at least the latter is correct. From our point of view (see below) the most important structural fact is the absence, in the structure of rat apo-S100A1 protein (Rustandi et al., 2002) of the helical turn formed by residues 87–89 found in both human and modified bovine proteins as can be seen in Fig. 5. All these structures are based on solid evidence provided by NOE spectra. He1(W90)/Hd1(Y260 ) and HN(W90)/Hd1(Y260 ) cross peaks identified in the human protein spectrum, decisive for determination of the 87–89 helix, were not observed in rat S100A1. On the other hand, Ha(W90)/Hb(D46) and Hb(W90)/Hd(K49) signals, incompatible with the 87–89 helical structure, are present only in the rat protein spectrum but not in that of human apo-S100A1. Nevertheless, the conformational differences between the Ctermini of both proteins can be much less dramatic than it looks at first glance. First of all, medium range NOE signals: Ha(N87)/ HN(W90) and HN(F88)/HN(W90), characteristic for a helical arrangement or a turn of the involved residues have been observed by us in human apo-S100A1 and also by Rustandi et al. (2002) in the rat protein. In fact, a hydrogen bonded turn comprising residues N87–W90 may be identified in this structure (Fig. 5a). Besides, the predicted S2 values (Fig. 3) clearly show that 87–89 helical structure in human apo-S100A1 is quite labile, so that this

protein segment can easily unwind and take some other, nonhelical conformation. Therefore, it seems that we deal here with an equilibrium between the helical and non-helical states which are differently populated in both proteins. This point is discussed here at length because of its importance for our studies whose main goal is to find out why chemical modification of the Cys 85 SH group affects, in a dramatic way, the calcium binding ability of S100A1 protein (Goch et al., 2005). We tried to solve this problem by comparing the structure of bovine apoS100A1 modified at Cys 85 by b-mercaptoethanol (Zhukov et al., 2008) with the only corresponding structure of S100A1 published at the time – that of the rat protein (Rustandi et al., 2002). This comparison led us to the conclusion that the primary effect of Cys 85 modification by thiol molecules is the elongation of helix IV (Fig. 5c). The results presented in this paper do not confirm this conclusion: in unmodified human S100A1 protein amino acid residues 87–89 participate in the a-helical conformation. Even if Cys 85 thionylation stabilizes this structure, such an effect cannot explain the dramatic increase of the protein affinity for calcium ions. Further, meticulous studies (see Section 1) are needed to solve this problem. Nevertheless, comparison of modified bovine and native human apo-S100A1 can be very instructive. Their structures were determined using very similar, though not identical, experimental procedures and data analysis methods. Their sequences are much more similar to each other than those of rat and human proteins: only two mutations: (A51?V and D64?N) convert the sequence of bovine protein studied by us previously into that of human S100A1 presented in this paper, whereas as many as six mutations are needed in the case of rat protein (D35 ? E, S43 ? G, V47 ? A, A51 ? V, I57 ? V, and F74 ? Y). Moreover, the dynamics of modified bovine and native human apo-S100A1 proteins have been measured and analyzed using the same theoretical model. Within error, the secondary structure of both these proteins is almost identical. This statement, based on structural studies, is strongly supported by comparison of S2 parameters (see Fig. 4) characterizing fast movements within a protein, which, in turn, are mainly dependent on its secondary structure. Nevertheless, the tertiary/quaternary structure is different. The Rex values for helices II and IV, the linker and the C-terminal loop are considerably greater in the modified bovine protein that indicates that its global structure is much more mobile than that of human apoS100A1 protein. The results of dynamics measurements are

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Fig. 6. The differences of the chemical shift combination, Ddtot, for backbone nuclei between S100A1 protein (BMRB accession code: 16360) and its thionylated derivative (BMRB accession code: 4982). The formula proposed by Ayed et al. (2001)) is given as: Ddtot = [(DdHN)2 + (0.154DdN)2 + (0.341DdCO)2 + (0.276DdCa)2]1/2. The horizontal line corresponds to the mean value of Ddtot.

strongly supported by chemical shift differences of backbone nuclei signals of both proteins calculated according to Ayed et al. (2001) and presented in Fig. 6. These observations lead to the conclusion that Cys 85 modification in S100A1 protein does not provoke its structural transition to any different, well defined, rigid structure, but rather it has a general destabilizing effect. Acknowledgments This work was supported by a grant from the Ministry of Science and Higher Education (N301 031234) and the EU Social Fund Project (no. POKL.04.01.01-00-072/09 (to Ł.J.). Authors thank dr Patric Groves for the help during preparation of manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jsb.2011.01.011. References Alexandrescu, A.T., Shortle, D., 1994. Backbone dynamics of a highly disordered 131 residue fragment of staphylococcal nuclease. J. Mol. Biol. 242, 527–546. Ayed, A., Mulder, F.A.A., Yi, G.S., Lu, Y., Kay, L.E., Arrowsmith, C.H., 2001. Latent and active p53 are identical in conformation. Nature Struct. Biol. 8, 756–760. Baber, J.L., Szabo, A., Tjandra, N., 2001. Analysis of slow interdomain motion of macromolecules using NMR relaxation data. J. Am. Chem. Soc. 123, 3953–3959. Baldisseri, D.M., Rustandi, R.R., Zhang, Z., Tang, C., Bair, C., Landar, L., Zimmer, D.B., Weber, D.J., 1999. 1H, 13C, and 15N NMR sequence specific resonance assignments for rat apo-S100A1(aa). J. Biomol. NMR 14, 91–92. Barlow, D.J., Thornton, J.M., 1988. Helix geometry in proteins. J. Mol. Biol. 201, 601– 619. Bax, A., Clore, G.M., Gronenborn, A.M., 1990. 1H–1H correlation via isotropic mixing of 13C magnetization: a new three-dimensional approach for assigning 1H and 13 C spectra of 13C-enriched proteins. J. Magn. Reson. 88, 425–431. Berjanskii, M.V., Neal, S., Wishart, D.S., 2006. PREDITOR: a web server for predicting protein torsion angle restraints. Nucl. Acids Res. 34, W63–W69. Bertini, I., Gupta, S.D., Hu, X., Karavelas, T., Luchinat, C., Parigi, G., Yuan, J., 2009. Solution structure and dynamics of S100A5 in the apo and Ca2+-bound states. J. Biol. Inorg. Chem. 14, 1097–1107. Bolewska, K., Kozłowska, H., Goch, G., Mikołajek, B., Bierzyn´ski, A., 1997. Molecular cloning and expression in Escherichia coli of a gene coding for bovine S100A1 protein and its Glu32–Gln and Glu73–Gln mutants. Acta Biochim. Polon. 44, 275–284. Brutscher, B., Brüschweiler, R., Ernst, R.R., 1997. Backbone dynamics and structural characterization of the partially folded A state of ubiquitin by 1H, 13C, and 15N nuclear magnetic resonance spectroscopy. Biochemistry 36, 13043–13053.

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