Solution structure of Ca²⁺-free rat α-parvalbumin

Abstract

Mammals express two parvalbumins—an α isoform and a β isoform. In rat, the α-parvalbumin (α-PV) exhibits superior divalent ion affinity. For example, the standard free energies for Ca²⁺ binding differ by 5.5 kcal/mol in 0.15 M KCl (pH 7.4). High-resolution structures of the Ca²⁺-bound proteins provide little insight into this disparity, prompting a structural analysis of the apo-proteins. A recent analysis of rat β-PV suggested that Ca²⁺ removal provokes substantial conformational changes—reorientation of the C, D, and E helices; reorganization of the hydrophobic core; reduced interdomain contact; and remodeling of the AB domain. The energetic penalty attendant to reversing these changes, it was suggested, could contribute to the attenuated divalent ion-binding signature of that protein. That hypothesis is supported by data presented herein, describing the solution structure and peptide backbone dynamics of Ca²⁺-free rat α-PV. In marked contrast to rat β-PV, the apo- and Ca²⁺-loaded forms of the rat α isoform are quite similar. Significant structural differences appear to be confined to the loop regions of the molecule. This finding implies that the α-PV isoform enjoys elevated divalent ion affinity because the metal ion-binding events do not require major structural rearrangement and the concomitant sacrifice of binding energy.

Parvalbumins are small, vertebrate-specific EF-hand proteins (Heizmann and Kagi 1989; Pauls et al. 1996). In fact, the crystal structure of carp parvalbumin (PV) established the EF-hand paradigm (Kretsinger and Nockolds 1973). The PV family includes α- and β sublineages (Goodman and Pechere 1977; Moncrief et al. 1990), and mammals express one isoform from each (Fohr et al. 1993). Although parvalbumins are believed to function primarily as cytosolic Ca²⁺-buffers, certain isoforms evidently play additional roles. For example, the mammalian β-PV, a.k.a. oncomodulin, is secreted by activated macrophages and functions as a potent nerve growth factor for retinal ganglion cells (Yin et al. 2006).

The PV tertiary structure includes six α-helical segments (A–F) organized into two domains—the N-terminal AB domain (residues 1–38) and the CD-EF domain. The two metal ion-binding sites, the CD and EF sites, are related by an approximate twofold symmetry axis and are physically linked by a short fragment of antiparallel β structure.

Despite 49% sequence identity (Berchtold and Means 1985; Gillen et al. 1987), the rat α- and β-PVs exhibit distinct divalent ion-binding affinities. (Hapak et al. 1989; Cox et al. 1990). In Hepes-buffered saline (pH 7.4), the ΔG°′ for Ca²⁺ binding is 3.6 kcal/mol more favorable for the α isoform. This difference increases to 5.5 kcal/mol when K⁺ replaces Na⁺ as the major solvent cation (Henzl et al. 2004).

High-resolution structures are available for the Ca²⁺-bound α- and β-parvalbumins from rat (Ahmed et al. 1993; McPhalen et al. 1994; Bottoms et al. 2004). However, they provide little insight into the disparate ion-binding properties of the proteins, suggesting that the unliganded state may influence the divalent ion-binding signature. We recently described the solution structure of Ca²⁺-free rat β-PV (Henzl and Tanner 2007). This article presents corresponding data for the Ca²⁺-free α isoform.

Results

Resonance assignments

At 25°C, Ca²⁺-free rat α-PV exhibits a well-resolved ¹H-¹⁵N HSQC spectrum (Fig. 1), which differs perceptibly from that of the Ca²⁺-loaded protein under comparable solution conditions (Supplemental Fig. S1). Except for M2 and D25, all of the expected amide signals are present. The following resonances are substantially overlapped: I5/E62, L6/K12, A14/V43, A20/L63, S36/E108, L67/M86, E81/V99, K83/E100, and E101/A107.

Figure 1.

¹H-¹⁵N HSQC spectrum of Ca²⁺-free rat α-PV in 0.15 M NaCl, 0.01 M Mes, and 0.005 M EDTA (pH 6.0) at 25°C.

Assignments were made using a suite of three-dimensional (3D), triple-resonance experiments. Aliphatic side-chain carbon assignments were complete, excluding the 24 carboxylates (Asp, Glu, and C terminus), the Q31 carboxamide, the R75 guanidinino group, and the methionine methyl groups. Aromatic side-chain assignments were lacking for C^γ and C^ζ in the eight phenylalanine residues. Proton assignments (side-chain and backbone) were 95% complete.

The tabulated chemical shifts served as input for CSI and TALOS. The former returned φ and ψ estimates for 71 residues. The latter provided φ,ψ predictions for 77 residues.

Solution structure of Ca²⁺-free rat α-PV

The tertiary structure of Ca²⁺-free rat α-PV was calculated with distance and dihedral angle restraints (Table 1), using ARIA/CNS (Brunger et al. 1998; Linge et al. 2001). Figure 2 displays an ensemble of 20 low-energy conformers. Relative to the ensemble average, the RMSD is 0.81 Å for the backbone atoms (C^β, C^α, C′, O, and N) and 1.26 Å for all heavy atoms. Table 1 presents additional structural quality statistics for the ensemble. According to PROCHECK, all but 0.3% of the φ,ψ combinations reside in allowed regions of the Ramachandran plot. Additionally, the structures exhibit few distance violations exceeding 0.5 Å or dihedral violations exceeding 5°.

Table 1.

List of restraints and statistical analysis for the apo-rat α-PV solution structure

Figure 2.

Solution structure of Ca²⁺-free rat α-PV. An ensemble of the 20 lowest energy structures determined with ARIA-CNS. This figure and Figures 3, 5, and 6 were produced with PyMOL (DeLano Scientific).

Comparison of the Ca²⁺-bound and Ca²⁺-free α-PV structures

The ensemble-averaged Ca²⁺-free structure (silver) has been superimposed on the crystal structure (blue) of Ca²⁺-bound rat α-PV (Protein Data Bank [PDB] 1RWY) in Figure 3. Although the protein fold is very similar in the presence and absence of Ca²⁺, several local conformational differences are observed. The C^α RMSD values for the apo- and Ca²⁺-loaded forms are plotted in Figure 4A. The largest discrepancies between the two structures occur in the loop between the A and B helices, the CD binding loop, the extended loop joining the D and E helices, and the EF binding loop. The average C^α RMSD is 2.4 Å.

Figure 3.

Stereoview of the superimposed structures of Ca²⁺-free (silver) and Ca²⁺-bound (blue) rat α-PV. The two structures were superimposed so as to minimize the overall RMSD. Coordinates for the Ca²⁺-bound structure were obtained from PDB 1RWY (Bottoms et al. 2004).

Figure 4.

(A) C^α RMSD values, as a function of residue number, for apo- and Ca²⁺-bound rat α-PV. Values represent the average for the 20 conformers in the ensemble. (B) C^α RMSD values, as a function of residue number, for the Ca²⁺-free α- and β-PV isoforms. Coordinates for the Ca²⁺-free β isoform were obtained from PDB 2NLN (Henzl and Tanner 2007). The values represent double-ensemble averages.

Figure 5 displays the CD and EF binding pockets in the Ca²⁺-loaded (panel A) and unliganded (panel B) forms of rat α-PV. The short fragment of antiparallel β structure that links the CD and EF sites is evidently preserved in the apo-protein.

Figure 5.

View of the metal ion-binding loops in Ca²⁺-bound (A) and Ca²⁺-free (B) rat α-PV.

Comparison of Ca²⁺-free rat α- and β-PVs

The representative solution structures of the Ca²⁺-free rat α-PV (silver) and β-PV (magenta) isoforms are compared in Figure 6. The C^α RMSD values are plotted in Figure 4B. In this superposition, the B and F helices are largely coincident, and good agreement is also observed for the E helix. However, the orientations of the remaining three helices differ substantially. The deviation in helix D is particularly noteworthy. In the α isoform, the side chain of F70 remains associated with the hydrophobic core in the absence of Ca²⁺, requiring distortion of the D helix. In the β isoform, however, F70 has withdrawn from protein interior, permitting the D helix to straighten. Smaller, but significant, deviations are also observed in the C and A helices. The ensemble-averaged C^α RMSD is 3.1 Å.

Figure 6.

Stereoview of the superimposed structures of Ca²⁺-free rat α-PV (silver) and Ca²⁺-free rat β-PV (magenta). The coordinates for the latter structure were obtained from PDB 2NLN (Henzl and Tanner 2007). The view in panel B represents an approximate 90° rotation about the vertical axis of the view in panel A.

PV CD- and EF-binding loops are related by an approximate twofold symmetry axis. In the Ca²⁺-bound form, they are physically joined by a segment of antiparallel β structure formed by I58 and I97. Whereas the α-PV retains this motif in the Ca²⁺-free form, as noted above, the β isoform does not.

¹⁵N relaxation data analysis

Relaxation data were collected on Ca²⁺-free rat α-PV at 25°C. The T ₁ and T ₂ data are well accommodated by a two-parameter exponential decay model. Representative decays are displayed in Supplemental Figure S2. R ₁ and R ₂ values were extracted for 88 of 108 amide vectors and are plotted in Figure 7, A and B, respectively. The numerical values are listed in Supplemental Table S1.

Figure 7.

Summary of ¹⁵N relaxation data and internal mobility analysis. (A) Experimental R ₁ values. (B) Experimental R ₂ values. (C) R₂/R₁ ratios. Filled circles (●) denote amide vectors included in the calculation of the mean value. (D) Experimental {¹H}¹⁵N NOE values. (E) Generalized order parameter (S ²) determined by model-free analysis, as described in the text. A subset of the residues required inclusion of a second order parameter (●), corresponding to motion on a slower timescale, to satisfactorily model their relaxation behavior. (F) τ _e (○) and R _ex (●) values for those residues displaying motion on a timescale exceeding 20 psec. The locations of the six helical elements are indicated at the top of the figure.

The overall rotational correlation time (τ _c) was estimated from a subset of amide vectors (Fig. 7C, ●) exhibiting an R ₂/R ₁ ratio within 1 SD of the mean. These data are well accommodated by a spherically symmetric rotational diffusion model, yielding a τ _c value of 6.49 ± 0.03 nsec. Axially symmetric and fully symmetric models yielded insignificant reductions in χ².

The {¹H}¹⁵N NOE values (Fig. 7D) exhibit a high degree of uniformity. Significant reductions in the NOE are restricted to A21, D22, K37, K52, D61, K91, and S109. Excluding these residues, the average NOE value is 0.80.

Internal mobility and model-free analysis

The Lipari-Szabo model-free treatment was used to assess main-chain flexibility in Ca²⁺-free rat α-PV. Relaxation data for 88 amide vectors were analyzed, using a spherically symmetric diffusion model. The results are displayed in Figure 7, E and F, and the model-free parameters are listed in Supplemental Table S2.

The majority of signals (67/88) can be modeled with the overall rotational correlation time (τ _c) and a generalized order parameter (S ²). Five amide vectors require a τ _e term to describe internal motion on the 20-psec to 10-nsec timescale. An additional three require an R _ex term to describe internal motion on the microsecond to millisecond timescale. Three amides (residues 19, 52, and 92) can be accommodated only by inclusion of both τ _e and R _ex terms. Finally, 10 NH vectors (7, 10, 23, 26, 40, 42, 74, 85, 105, 109) exhibit behavior consistent with motion on two timescales shorter than the overall rotational correlation time.

The average order parameter for the apo-protein is 0.92. This value is significantly higher than the value of 0.85 reported for the Ca²⁺-bound protein (Baldellon et al. 1998). However, the latter analysis was performed at 32°C, rather than 25°C, and the order parameter is expected to decrease somewhat with elevated temperature (Bracken et al. 1999). Nevertheless, the implication is that removal of Ca²⁺ does not produce a major increase in backbone motion, at least on the picosecond timescale. Similar behavior was noted previously for the rat β-PV isoform (Henzl et al. 2002; Henzl and Tanner 2007).

Discussion

Although the α- and β-PV isoforms from rat exhibit distinct divalent ion-binding properties, the Ca²⁺-bound structures of rat α- and β-PV are very similar (Ahmed et al. 1993; McPhalen et al. 1994; Bottoms et al. 2004). The average main-chain RMSD value is just 0.8 Å. Thus, inspection of the Ca²⁺-loaded molecules offers few clues as to the source of the apparent 5.5 kcal/mol difference in standard binding free energy observed in 0.15 M KCl (pH 7.4). Because divalent ion affinity reflects the difference in the stabilities of the bound and unliganded forms, the disparate divalent ion-binding behaviors could conceivably be dictated at the level of the apo-proteins. To explore this possibility, we have obtained solution structures for the Ca²⁺-free forms of rat α- and β-PV. The structure of the β isoform (Henzl and Tanner 2007) departs significantly from that of the Ca²⁺-bound form. Ca²⁺ removal provokes substantial reorientation of the C, D, and E helices and reorganization of the hydrophobic core. F70 and F66 have evidently withdrawn from the protein interior, permitting the D helix to straighten. By contrast, F49 and L85 have assumed more interior positions. These changes in the CD-EF domain reduce the area of apolar contact with the AB domain by nearly 10%. In consequence, perhaps, the apolar side chains in the AB region exhibit increased intradomain interactions. It was suggested that the energetic penalty associated with reversing these changes could significantly reduce the net binding free energy, providing a potential explanation for the attenuated divalent ion affinity.

The structural alterations accompanying Ca²⁺ removal are evidently less extensive in the rat α isoform. The largest differences are observed in the CD binding loop and in the extended loop joining the A and B helices. Significant differences are also observed in the loop between the D and E helices and in the EF ion-binding loops. However, the six helical segments display good agreement with the Ca²⁺-loaded form, and the hydrophobic core is largely undisturbed.

These findings are consistent with one-dimensional NMR studies of rat α-PV conducted by Sykes and coworkers two decades ago (Williams et al. 1986). At 37°C, in 0.5 M KCl, they observed evidence for two forms of the apo-protein—an unfolded form and a form having a spectroscopic signature very similar to that of the Ca²⁺-bound protein. No evidence of unfolded protein is observed in the present study, presumably because the NMR data were collected at lower temperature (25°C) and in the presence of 0.15 M NaCl. Whereas Ca²⁺-free α-PV exhibits negligible affinity for K⁺, it binds 1.0 equivalent of Na⁺ with an association constant estimated at 630 M⁻¹ (Henzl et al. 2000, 2004). Binding of the monovalent cation exerts a pronounced stabilizing influence on the protein. For example, whereas Ca²⁺-free rat α-PV denatures at 46 ± 0.2°C in 0.20 M NaCl, it denatures at 35.5 ± 0.3°C in 0.20 M KCl. Even at 25°C, the appearance of the ¹H,¹⁵N HSQC spectrum of rat α-PV is strongly dependent on the identity of the major solvent cation (data not shown).

Although the average S ² values obtained from model-free analysis are essentially identical for the two apo-PVs—0.916 for α, 0.924 for β—their backbone dynamics offer several interesting contrasts. The β isoform displays markedly elevated R ₂ values in the CD binding loop and in the F helix, indicative of motion on the microsecond to millisecond timescale. Consistent with that observation, the majority of those amide vectors require an R _ex term to adequately model their relaxation behavior. By contrast, the R ₂ values measured for rat α-PV in these regions are comparatively uniform, and the relaxation of the amide vectors in question can be satisfactorily modeled without an R _ex contribution. The R ₂ values for the two proteins are compared in Supplemental Figure S3.

Additionally, Ca²⁺-free rat β-PV displays systematically lower NOE values near the C terminus of the CD binding loop and in the EF binding loop, indicative of motion on the nanosecond timescale. Consistent with that observation, the amide vectors in these regions require a τ _e term for satisfactory model-free treatment. Once again, the corresponding NOE values determined for the α isoform are remarkably constant over these regions, and there is no requirement for τ _e in the model-free analysis. The {¹H} ¹⁵N NOE values for the two proteins are compared in Supplemental Figure S3B. The contrast in the R ₂ and NOE profiles suggests that the metal ion-binding loops in the rat β isoform, but not in the α isoform, experience nanosecond to millisecond motion. Recall that the antiparallel β segment linking the CD and EF loops in the Ca²⁺-loaded proteins is evidently retained in the Ca²⁺-free α-PV and may serve to dampen longer timescale motions in the ion-binding loops.

When subjected to model-free analysis, nearly all of the N-H vectors in rat β-PV can be modeled with a single order parameter. The two vectors requiring a second order parameter, corresponding to motion on a slower timescale, reside in the EF-site binding loop (D94, G95). By contrast, there are 10 amide vectors in the Ca²⁺-free α isoform that require the second order parameter. The residues in question are scattered throughout the sequence but are absent from the metal ion-binding loops. Thus, although the average order parameters for the two apo-proteins do not differ appreciably, the model-free analysis suggests that, relative to the β isoform, the peptide backbone of Ca²⁺-free rat α-PV experiences more complex motions on timescales shorter than the overall rotational correlation time.

Under resting-state conditions in vivo, the α-PV resides almost exclusively in the Mg²⁺-loaded form, and the apo-form described here is negligibly populated. However, because the divalent ion affinity depends on the free energies of both the bound and unliganded forms, the apo-protein contributes to the divalent ion-binding signature of the protein. The previous structural analysis of Ca²⁺-free rat β-PV suggested that the atypically low divalent ion affinity displayed by that protein might reflect the requirement for an energetically costly interconversion of the apo- and Ca²⁺-bound conformations. The structural data presented here for the Ca²⁺-free rat α isoform are compatible with that argument. Specifically, the apo- and Ca²⁺-loaded proteins exhibit very similar tertiary structures. Because energy need not be expended to isomerize the protein, the net free energy change that accompanies divalent ion binding is more favorable.

This hypothesis clearly requires additional confirmation. If ultimately shown to be valid, it provides an interesting correlation between structure and energetics. It does not, however, speak to the underlying physical basis for the structural, and functional, differences. The solution of that problem, still tantalizingly out of reach, will require identification of specific amino acid sequence assignments associated with the low- and high-affinity phenotypes.

Materials and Methods

Protein expression and purification

Recombinant rat α-PV was expressed and purified as described previously (Henzl and Graham 1999). Besides ¹³C₆-d-glucose and/or ¹⁵NH₄Cl, the medium consisted of M9 salts supplemented with 2.5% (v/v) Celtone (Spectra Stable Isotopes), 100 μM FeCl₃, trace elements, and ampicillin (300 μg/mL). Each L yielded 20–25 mg of protein, with purity exceeding 98%.

NMR sample preparation

Sufficient protein to yield a 3 mM sample was concentrated to 5 mL by ultrafiltration and then dialyzed at 4°C for 48 h, against 4 L of 0.15 M NaCl, 0.025 M Hepes, and 5.0 mM EDTA (pH 7.4). Dialysis was continued for 48 h against 0.15 M NaCl, 0.01 M Mes, and 5.0 mM EDTA (pH 6.0). After adding 0.1 volume of buffer prepared in D₂O and sodium azide (final concentration 0.1%), the solution was concentrated to 0.35 mL and loaded into a 5-mm Shigemi microcell (Shigemi, Inc.).

NMR spectroscopy

All data were acquired at 25°C with BioPack pulse sequences on a Varian INOVA 600-MHz spectrometer, employing a triple-resonance cryoprobe equipped with pulsed-field z gradient. ¹H chemical shifts were referenced relative to DSS; ¹³C and ¹⁵N shifts were referenced indirectly, employing the ¹H/X frequency ratios. Data were processed with NMRPipe and analyzed with Sparky (T.D. Goddard and D.G. Kneller, University of California, San Francisco).

Resonance assignments

Backbone resonance assignments were made using the following pairs of 3D experiments: HNCA (Ikura et al. 1990) and HN(CO)CA (Bax and Ikura 1991), HNCACB (Kay et al. 1994; Muhandiram and Kay 1994) and CBCA(CO)NH (Grzesiek and Bax 1992), and HNCO (Ikura et al. 1990) and HCACOCANH (Lohr and Ruterjans 2005). Aliphatic ¹³C assignments beyond C^β were collected using the CCONH (Grzesiek et al. 1993) spectrum. Aliphatic side-chain ¹H assignments were made with HCCONH (Grzesiek et al. 1993), ¹⁵N-edited TOCSY-HSQC (Marion et al. 1989a), and HCCH-TOCSY (Kay et al. 1993) experiments. Aromatic side-chain resonances were assigned using the HBCBCGCDHD (Yamazaki et al. 1993) and HBCBCGCDCEHE (Yamazaki et al. 1993) experiments. Stereospecific assignments of the methyl protons in valine and leucine were made by analysis of a fractionally ¹³C-labeled sample (Neri et al. 1989).

Structural restraints

For collection of NOE-based distance restraints, a 3D ¹⁵N-edited NOESY-HSQC (Marion et al. 1989b) data set was collected on ¹⁵N-labeled protein, employing a mixing time of 125 msec. A corresponding ¹³C-edited experiment was performed on ¹³C,¹⁵N-labeled material, using a mixing time of 100 msec. Cross peaks were picked manually and integrated in Sparky. φ,ψ dihedral angle restraints were obtained from TALOS (Cornilescu et al. 1999) and CSI (Wishart and Sykes 1994).

¹⁵N relaxation data were collected on ¹⁵N-labeled protein, employing the T ₁, T ₂, and {¹H}¹⁵N NOE pulse sequences supplied in BioPack. R ₁ data were acquired with relaxation delays of 50, 100, 150, 250, 350, 450, 600, 800, 1000, and 1200 msec. R ₂ data were acquired with delays of 10, 30, 50, 70, 90, 110, 130, 150, 170, and 190 msec. Replicate data were obtained at three delay values for evaluation of experimental uncertainty. For the measurement of steady-state heteronuclear {¹H}¹⁵N-NOEs, HSQC spectra were collected, with or without proton saturation (3.0 sec), employing a total recycle delay period of 5.0 sec. Duplicate experiments were performed to obtain an estimate of the experimental uncertainty.

Structure calculations were performed with ARIA2.0a (Habeck et al. 2004). ARIA (Ambiguous Restraints for Iterative Assignment) integrates automated NOE assignments with CNS-based structure calculations (Linge et al. 2001). In addition to the ¹⁵N- and ¹³C-edited NOESY cross-peak lists, the initial input to ARIA also included dihedral angle restraints (TALOS and CSI). The quality of the final structures was analyzed using PROCHECK (Laskowski et al. 1993).

¹⁵N relaxation analysis

Peak intensities in the T ₁, T ₂, and {¹H}¹⁵N NOE data sets were measured automatically in Sparky. R ₁ and R ₂ values were extracted for each peak by fitting signal intensities to a two-parameter single-exponential decay, in Origin, version 7.5 (OriginLab). The NOE values represent the ratio of the signal intensities in the presence and absence of proton saturation. These relaxation data were analyzed with Tensor2 (Dosset et al. 2000).

The overall rotational correlation time (τ _c) was estimated from the subset of amide vectors having R ₂/R ₁ values within 1 SD of the mean value (Tjandra et al. 1995). The isotropic rotational diffusion model yielded a τ _c value of 6.49 ± 0.03 nsec, corresponding to a rotational diffusion coefficient of 2.56 × 10⁷ sec⁻¹. Axially symmetric and fully anisotropic models did not afford significant improvements in χ².

Internal mobilities were examined with the Lipari-Szabo model-free formalism (Lipari and Szabo 1982a,b). Tensor2 employs the five models suggested by Clore et al. (1990a^,b) and the model selection strategy described by Mandel et al. (1995).

Coordinate and data deposition

The atomic coordinates and structural restraints have been deposited in the Protein Data Bank (PDB 2JWW). ¹H, ¹⁵N, and ¹³C assignments have been deposited in BioMagResBank (BMRB 15,517).