the linker of calmodulin lacking glu84 is elongated in solution, although it is bent in the crystal

PROTEINS Structure, Function, and Genetics 25335-341 (1996)

The Linker of Calmodulin Lacking Glu84 Is Elongated in Solution, Although It Is Bent in the Crystal Mikio Kataoka,' Anthony Persechini? Fumio Tokunaga,' and Robert H. Kretsinger3 'Department of Earth and Space Science, Faculty of Science, Osaka University, Toyonaka 560, Japan; 'Department of Physiology, Rochester University School of Medicine, Rochester, New York 14642; 3Department of Biology, University of Virginia, Charlottesville, Virginia 22903

ABSTRACT The solution structure of a mutant calmodulin (des84) lacking Glu84 in the central helix linking the two calmodulin lobes is substantially different from its crystal structure. As determined by small-angle X-ray scattering, the radius of gyration and the maximum linear dimension of des84 in the presence of 0.1 mM calcium are 20.8 A and 62.5 A, respectively. These respective dimensions are larger than those expected from the crystal structure of des84,18.5 A and 55.0 A, and smaller than those expected from the crystal structure of wild type, 22.8 A and 67.5 A. The distance distribution function of des84 indicates that it assumes an elongated, dumbbell shape in solution. The solution scattering profile of des84 is indistinguishable from that of wild-type calmodulin. The calcium-dependent binding of melittin to des84 causes a change in its shape from elongated to spherical, as seen with other calmodulins. In comparison with calcium-saturated des84, calcium-free des84 is slightly elongated; a slight compaction is observed with native calmodulin. The observed differences between the averaged solution structure and the crystal structure of des84 suggests that an ensemble of structures is available to calmodulin in solution and that its target need not induce a change in its conformation. These results support the the- ory that the linker region of the central helix of calmodulin functions as a flexible tether. 0 1996 Wiley-Lisa, Inc.

Key words: solution X-ray scattering, calcium- dependent conformational change, calmodulin-melittin complex, deviation from crystal structure

INTRODUCTION Calmodulin, a ubiquitous calcium-modulated pro-

tein in eukaryotic cells, is the most extensively studied member of the family of proteins that contains multiple EF-hand domains.lT2 The ~ r y s t a l ~ - ~ and so- lution6 structures of native calmodulin are unusual in that eight residues of the central a-helix that con- nects the N- and C-terminal lobes of the dumbbell- shaped molecule are exposed to solvent on all sides.

0 1996 WILEY-LISS. INC.

Seaton et al.7 used small-angle X-ray scattering (SAXS) to show that calmodulin retains its elongated structure in solution, although deviation of the solution structure from the crystal structure of calmodulin was indicated by the SAXS studies of Heidorn and Trewhella,' and Barbato et a1.6 concluded from nuclear magnetic resonance (NMR) studies that the Asp78Ser81 region of the linker is disordered, even though it retains its helical character in solution.

Based on their mutagenesis studies Persechini and Kretsinger', lo proposed that the linker region of the central helix functions as a flexible tether, thereby enabling the two calmodulin lobes to enfold the calmodulin-binding domain. The predicted large, calcium-dependent change in the conformation of calmodulin was first revealed by SAXS studies of calmodulin complexed with a model target pep-

The results of these SAXS experiments were consistent with the model of Persechini and Kretsinger," although differences in overall dimensions and the distance between peptide and each lobe were noted.''^^^ The details of these structural changes were elucidated to atomic resolution both by NMR spectroscopy18 and X-ray crystallography. l9

We14 studied calmodulins lacking Glu83-Glu84 and Ser81-Glu84 (des83-84 and des81-84) by SAXS and determined that these mutants are elongated and have an overall dumbbell shape. The observed changes in radius of gyration (Rg) and d,,, can be explained reasonably by the two- or four-residue deletion from an ideal a-helix. Based on the crystal structure of native calmodulin, we inferred that the linker regions of des83-84 and of des81-84 are elongated and probably helical in solution.

Abbreuiations: SAXS, small-angle X-ray scattering; dmax, maximum linear dimension of molecule; Rg, radius of gyration; des84, or des83-84 or des81-84, bacterially expressed calmodulin in which Glu84 or Glu83 and Glu84 or Ser81, Glu82, Glu83, and Glu84 have been deleted.

Received October 12, 1995; revision accepted January 16, 1996.

Address reprint requests to Dr. Mikio Kataoka, Department of Earth and Space Science, Faculty of Science, Osaka Univer- sity, Toyonaka 560, Japan.

336 M. KATAOKA ET AL.

The crystal structure of calmodulin lacking Glu84 (des84) with four Ca' + ions bound" shows interesting and significant deviations from the structure of native calmodulin. The linker region of the central helix in des84 is bent - 30" at Lys75 and - 90" at Ile85. Although there is no target present, the shape of des84 approaches that of the calmodulin-target complex. Des84, like des81-84, des83-84, and native calmodulin, can fully activate bovine brain cal- cineurin, plant NAD kinase, and skeletal muscle myosin light chain kinase activities.'l Furthermore, the solution structures of des83-84 and of des81-84 are rather close to the elongated dumbbell shape seen for ca1m0dulin.l~ Hence, it is important to de- termine the solution structure of des84 to see whether it resembles these other solution structures or its crystal structure. We have found that the solution structure of des84 is similar to that of native calmodulin and different from the more compact crystal structure. Des84 undergoes a large calcium- dependent conformational change upon binding of melittin similar to what is observed with native calmodulin.

MATERIALS AND METHODS Preparation of Proteins

Des84 was expressed in Escherichia coli and pu- rified as described by Persechini et al.'l Bovine brain calmodulin was prepared by the method of Masure et a1.22 Melittin was purchased from Sigma and repurified as described by Kataoka et al." Freeze-dried calmodulin and melittin were dissolved in water as stock solutions. Protein concentrations were determined by quantitative amino acid analysis. Samples for SAXS measurements were prepared by dialysis against the appropriate buffer, as described by Kataoka et al." Final dialyzed samples contained 100 mM KC1, 50 mM MOPS (pH 7.21, 0.02% sodium azide, and either 0.1 mM CaC1, or 1.0 mM EGTA. For samples containing melittin, the dialysis solution included 1.0 pM melittin.

Solution Small-Angle X-Ray Scattering Experiments

SAXS experiments were performed with the SAXES diffractometerz3 at the Photon Factory, Tsukuba, Japan, using synchrotron radiation, A = 1.488 A monochromated by a doubly flat crystal monochromator and focused by a bent cylindrical glass mirror. The design of the beam line and the details of the experiments were described previ-

The small-angle X-ray scattering intensity distri- ously.15.23

bution from a protein in solution is expressed by:

I(Q) = I(0) exp(-Rg2Qz/3)

where Q = 4min0/A and 20 and A are the scattering angle and wavelength of X-rays.z4 The Rg value

is obtained from the slope of the Guinier plot, In I(Q) vs. Q".

Scattering curves at infinite dilution were obtained by linear extrapolations of a series of scattering curves obtained at five or more protein concentrations in the range of 1-20 mg/ml. Extrapolations were made directly and by Zimm plots.z5 Both methods gave the same final scattering curves. Rg values were obtained mainly by Guinier analysis for the extrapolated scattering curve.z4

The distance distribution function, P(r), was calculated by the indirect Fourier transform method developed by Moore.26 The P(r) function was also evaluated using the GNOM program kindly pro- vided by Svergun et al.27 The Rg and P(r) functions for the crystal structure" were calculated based on Monte-Carlo samplingz8. The P(r) function was computed as a histogram of distances between two sam- ple points binned every 2.5 A. The Rg was calculated as:

Rg' = J' rzP(r)dr/2 J' P(r)dr

The d,,, of the molecule is derived from the point a t which the P(r) function approaches 0.

RESULTS Comparison of the Solution and Crystal Structures of des84 With Those of Wild-Type Calmodulin

The crystal structure of des84 with four calcium ions bound is significantly different from that of wild-type calmodulin.20 In the crystal structure of wild-type calmodulin, there is a long continuous a-helix consisting of helix F2 of the N-terminal lobe, the central helix linker, and helix E3 of the C-terminal In contrast, the linker region of the central helix in des84 in the crystal is bent - 30" at Lys75 and - 90" at Ile&j2' (see Fig. 4 in Raghu- nathan et al."). The solution structure of des84 was examined by SAXS and compared with the solution structure of bovine brain calmodulin and also with the crystal structures of des84 and native protein. Despite substantial differences in the two crystal structures, the solution scattering profile from calcium-saturated des84 is indistinguishable from that of calcium-saturated bovine brain calmodulin (Fig. 1). The maximum dimension and the general P(r) profile of des84 are similar to those observed for bovine brain calmodulin. This indicates that the solution structures of des84 and bovine brain calmodulin are very similar. The SAXS profile of bovine brain calmodulin is explained reasonably as the elongated dumbbell-shaped s t ru~ tu re .~ ,~ , " Hence, des84 is also likely to be elongated in solution.

A P(r) function for des84 was calculated from its crystal structure and compared with the observed solution P(r) and with the P(r) calculated from the crystal structure of wild-type calmodulin (Fig. 2).

desGlu84-CALMODULIN IS ELONGATED IN SOLUTION 337

Fig. 1. Comparison of the solution X-ray scattering profile of Ca2'-des84 (0) with that of bovine brain Caz+-calmodulin (0). The scattering profiles extrapolated to zero protein concentration are presented as a Guinier plot.

The three are quite distinct. The observed P(r) for des84 has a peak at 18 A and a shoulder around 40 A. Such a bimodal P(r) is consistent with a dumbbell-shaped s t r u c t ~ r e . ~ ~ ~ * ~ ~ The longest chord for des84 is 62.5 A, identical to what has been observed with bovine brain c a l m o d ~ l i n . ~ ~ ~ , ~ ~ The P(r) for the crystal structure of des84 consists of a peak at 20 A and a shoulder around 35 A, indicating that the two lobes are closer together in the crystal structure than they are in solution. The LX value for des84 is 55 A in the crystal structure, which is much shorter than the d,, value, 62.5 A, observed for des84 in solution. The Rg value for des84 in solution is 20.8 A; the value calculated from the crystal is 18.7 A. These observations indicate that the solution structure of des84 is significantly different from its structure in the crystal. The similarity of the scattering profiles for des84 and bovine brain calmodulin suggests that the structure of des84 in solution is an elongated dumbbell, as suggested for native calmodulin.

The differences between the observed P(r) for native calmodulin and that calculated from crystal structure of this protein are evident, as previously reported by Heidorn and Trewhella.8 The calculated P(r) for native calmodulin contains two peaks around 18 A and 40 A, with an intervening trough, and suggests that two lobes are slightly more sepa- rated in the crystal than in the solution structure. The La, and Rg values calculated from the crystal structure of the native protein are 22.8 A and 67.5 A, respectively, larger than the values observed for either des84 or native calmodulin in solution.

It is also clear that the observed P(r) for des84 cannot be explained by a simple linear combination of the P(r)s calculated from the crystal structures for native calmodulin and des84, although the observed P(r) is largely intermediate in shape. Determina-

Y -

O 1 0 2 0 3 0 4 0 5 0 " 6-0

r (A)

Fig. 2. Comparison of the distance distribution functions. 0, the observed P(r) for Ca2+-des84; 0 the calculated P(r) for the crystal structure of Ca2+-des84; 0, the calculated P(r) for the crystal structure of Ca2+-native calmodulin.

tions of the solution structure of apo-calmodulin by NMR29,30 show how the average of the ensemble of solution structures is shorter than the single crystal structure. Since SAXS gives information only for the time- and ensemble-averaged structure in solution, our results likely reflect des84 conformations ranging between two extremes, one being the elongated dumbbell-shaped structure seen in the native calmodulin crystal structure and the other the bent structure seen in the des84 crystal.

Effect of Bound Calcium and Melittin on the Solution Structure of des84

Scattering profiles for calcium-saturated des84, apo-des84, and for calcium-saturated des84-melittin are shown in Figure 3. Rg values were obtained from the slopes of regression lines of the Guinier plots, using data within the region Q" < 0.1 A-", where the criterion RgQ 5 1.3 was satisfied.31 More rigor- ous analyses were performed with the indirect Fou- rier transform method of Moore.26 The Rg value obtained by Moore analysis is identical to the value obtained by Guinier analysis, indicating that the quasi-point focus optics produced essentially no slit smearing. The Rg values are listed in Table I.

As is seen in Figure 3, the Guinier plot for des84- melittin is linear over the entire range; however, the Guinier plots for calcium-saturated des84 and apo- des84 both show inflections around Q" = 0.01; the data from Q" = 0.01 to 0.02 lie on straight lines. Such a Guinier plot with two different linear regions is characteristic of a dumbbell Thus, we conclude that des84 is dumbbell-shaped in solution in the presence or absence of bound calcium.

Rg values measured for des84 are plotted against protein concentration in Figure 4. The measured Rg value for des84 shows a minimal concentration dependence, although Seaton et al.7 have observed a distinct concentration dependence for bovine brain calmodulin. In the case of native calmodulin ex-


TABLE I. Structure Parameters Obtained by SAXS Measurements

Rg (A) (A) des84 + Ca2 + 20.9 ? 0.2 62.5 +- 2.5 -Ca2+ 21.5 * 0.2 64.5 * 2.0 +Ca2+, + melittin 19.4 * 0.3 50.0 * 2.5

Bovine brain calmodulin f C 2 + * 18.7 55

+Ca2++ 20.9 ? 0.2 62.5 t 2.5 +Ca2+* 22.8 67.5 des83,84 +Ca2+§ 19.5 * 0.4 52.5 * 2.5

*Calculated value for the crystal structure.20 'The value obtained by the present measurements. *Calculated value for the crystal str~cture.~ $Taken from Kataoka et al.14

I I I I I 0.000 0.005 0.010 0 . 0 1 5 0.020

Qz (A 2,

Fig. 3. Scattering profiles of Ca2+-des84 (0), of apo-des84 (a), and of the Ca2+-des84-melittin complex (0) in the form of Guinier plot. Each scattering profile was obtained by extrapolation of at least five different scattering curves with different protein concentrations. For clarity, each plot is shifted along the In I axis. Rg was obtained using Guinier region, where QRg 5 1.3 obtains.

pressed in bacteria, a similar minimal dependence of the Rg value on the protein concentration was observed.12 Extrapolating to zero protein concentration gives Rg values of 20.7 * 1.6 A for the calcium- saturated des84 and 21.2 * 1.8 A for apo-des84. The extrapolation was performed using a plot of Rg2 vs. protein concentration rather than Figure 2.8,33 The Rg values obtained are essentially identical to those from Guinier plots extrapolated to zero protein concentration (Table I). As is the case for bovine brain calmodulin, the Rg value for the complex of des84 with melittin does not show any concentration dependence, and the extrapolated Rg value is 19.3 %

0.6 A (data not shown). Previous SAXS studies have shown that calcium-

saturated native calmodulin undergoes a large conformational change on binding the bee venom peptide melittin.11,14 Other synthetic peptides capable

2 2

1 6

0 5 1 0 1 5 2 0 2 5

Protein Concentration (rnglrnl)

Fig. 4. Typical protein concentration dependence of the radii of gyration of Ca2+-des84 (0) and of apo-des84 (0). The solid line represents the least-square linear fit of the plot for Ca2+- des84 and the broken line the plot for apo-des84. The Rg values were derived from Guinier analysis.

of binding to calmodulin also cause similar conformational changes in calmodulin12*13~15~16,34 Perse- chini et al.'l showed that three deletion mutants, des84, des83-84, and des81-84, retain the ability to bind melittin and a peptide, M-13, representing the calmodulin-binding-domain of myosin light chain kinase. SAXS measurements of the complexes between melittin and des83-84 or des81-84 show peptide-dependent conformational changes similar to those observed for the native calmodulin-peptide complex.'* The Guinier plot for des84-melittin is linear up to Q2 = 0.02, suggesting that the conformation of des84 is similarly altered by peptide binding.

This conclusion is also consistent with the distance distribution function, P(r), presented in Fig- ure 5. As expected, in the absence of calcium a peptide-dependent conformational change is not observed for des84 (data not shown). Similarly, in the absence of calcium no interaction with melittin was inferred for either native calmodulin, des83-84, or d e ~ 8 1 - 8 4 . ~ ~ ~ ~ ~ The distance distribution functions for calcium-saturated des84 in the presence and absence of melittin were obtained using the indirect Fourier transform method of Moorez6 (Fig. 5). The P(r) function was also evaluated using a different indirect Fourier transform method with a com- pletely different alg~rithm.'~ Both methods gave essentially identical results. As explained above, the P(r) for calcium-saturated des84 in the absence of melittin is consistent with an elongated dumbbell- shaped structure.

In contrast, the P(r) for des84-melittin contains a single peak at about 20 A. This is characteristic of a spherically shaped molecule. The longest chord of the complex, as deduced from the P(r) function, is 50 A, which is slightly longer than that observed for

desGlu8CCALMODULIN IS ELONGATED IN SOLUTION 339

1 0 0 , I I I I I I

Fig. 5. Comparison of the distance distribution function, P(r), for Caz+-des84 (0) with that of the Ca2+-des84-melittin complex (0). Error bars are indicated or contained within the area of the symbol.

bovine brain calmodulin complexed with melittin." Therefore, the P(r) functions also confirm a dumbbell to spherical shape change for des84 upon com- plexation with melittin.

DISCUSSION Our SAXS study indicates that des84 assumes an

elongated, dumbbell shape in solution as do native calmodulin, des83-84, and des81-84. Furthermore, on binding a target analog, like melittin, all four of these proteins become nearly spherical in shape.

The crystal structure4 and the NMR solution structure6 of native calmodulin are both dumbbell shaped. In marked contrast, the crystal structure of des84" reveals that the linker region of the central helix is bent - 30" at Lys75 and - 90" at Ile85. The observed P(r)s for native calmodulin and des84 are essentially identical (data not shown). However, the computed P(r)s for their respective crystal structures are significantly different from each other (Fig. 2). Interestingly, the observed P(r) for des84 differs substantially from the P(r) calculated from its crystal structure (Fig. 2).

It is evident that the central helix linker region of calmodulin, and of its deletion mutants, is highly flexible in solution and can assume an ensemble of conformations. The present results indicate that with des84, in contrast to native calmodulin, an ap- parently minor bent conformation from this ensemble is selected upon crystallization.

SAXS studies of calmodulin combined with those of troponin C suggest some additional, more subtle phenomena evidenced in the effects of calcium binding, as well as comparisons of the P(r) functions for des84, des83-84, and des81-84 in the presence and absence of bound peptide. Troponin C contains four EF-hand domains and is the calcium-binding component of the heterotrimer troponin, which imparts calcium sensitivity to skeletal and cardiac muscle.

Its SAXS P(r) curve' is consistent with the dumbbell shape in the ~ r y s t a l . ~ ~ , ~ ~

In the absence of bound calcium ion, troponin C, native calmodulin, and all three central helix deletion proteins examined are dumbbell shaped in solution. However, it is interesting that both native calmodulin and des81-84 show a slight decrease in their Rg values upon addition of EGTA, while des83-84 shows almost no effect,14 des84 shows a slight increase, and troponin C shows a distinct increase in Rg following loss of calcium.37 The crystal structure of troponin C has calcium ions bound only to the EF hands in the C-terminal lobe. Based on the difference in structure between the N- and C-terminal lobes in this structure, Herzberg et al.38 proposed a model for the change in conformation associated with the binding of calcium by troponin C and calmodulin. However, such conformational changes do not fully account for the observed calcium-dependent changes in Rg values for the five proteins, and additional subtle conformational changes associated with calcium binding remain to be elucidated.

The change in Rg values with deletion of one, two, or four residues from the central helix of calmodulin would not be expected to result in a smooth, mono- tonic decrease in length as assessed by SAXS. In native calmodulin the centers of mass of the two lobes are displaced from the axis of the central helix and are trans to one another. The deletion of two residues not only brings the two lobes 3.0 A closer, as projected on the helix axis, but also places the two lobes cis to one another. The deletion of two more residues in des81-84 shortens the helix 3.0 A more but puts the two lobes trans to one another, thereby increasing their ~eparat i0n. l~ However, the finding that the linker of des84 is bent in the crystal indicates that we might not be able to extrapolate the structures of the deletion proteins reliably in solution from the structure of native calmodulin. Heidorn and Trewhella' have noted that the Rg and d,, values for native calmodulin are slightly smaller than expected based on the crystal structure, and proposed a model for the structure of calmodulin in solution containing a bend at residue 81 in the central helix. Alternatively, the observed P(r) for native calmodulin was described as reflecting a linear combination of the calculated P(r) for the crystal structure and the P(r) for a compact structure derived by molecular dynamics ~imulation.~' The observed P(r) for des84, however, is not a simple linear combination of the calculated P(r) for the native and des84 calmodulin crystal structures (Fig. 2). We propose that des84, as well as native calmodulin, assumes a range of conformations in solution rather than just the two seen in the crystal and the fully extended form. The observed P(r) reflects the ensemble-averaged structure.

Our final point is that native calmodulin, as well as des84, des83-84, and des81-84 all assume a


nearly spherical shape upon binding a target helix such as melittin. The similarity of the P(r) functions describing these different calmodulin-peptide complexes implies that in each the relationships of the N- and C-terminal lobes to each other (and to melittin) are similar. However, since one, two, or four residues have been removed from the central helix linkers in the deletion proteins, these regions must be conformationally distinct in the different calmodulin-peptide complexes. Furthermore, the distribution of calmodulin-binding residues varies among various calmodulin-binding peptides. The exact ori- entation of the lobes of calmodulin would be ad- justed in each complex utilizing the flexibility of the linker.

CONCLUSIONS The solution structure of des84 calmodulin is an

elongated dumbbell, very similar to that of native calmodulin, while in the crystal structure of des84 the central helix is bent -30” at Lys75 and -90” at Ile85. We suggest that des84, as well as native calmodulin and other deletion mutants, occupies an ensemble of conformations in solution, most of which are elongated; only one component is selected for crystallization. The overall shape of the complex of des84 with melittin is spherical, as is the complex of calmodulin and other central helix deletion mutants with melittin and with other target peptides.

ACKNOWLEDGMENTS This work was supported in part by grants from

the Ministry of Education, Science, and Culture of Japan to M. K. and from the Council for Tobacco Research to R. H. K. and a PHS grant (DK44322) to A. P. We thank K. Mihara and H. Kamikubo for their help with solution scattering experiments a t the Photon Factory, Tsukuba, Japan. The SAXS measurements a t the Photon Factory were performed under the approval of the Photon Factory Program Advisory Committee (proposals 92-066 and 94-G072).

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the linker of calmodulin lacking glu84 is elongated in solution, although it is bent in the crystal

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