Determination of the solution structure of apo calbindin D9k by NMR spectroscopy

J. MOL Biol. (1995) 249, 441-462

.IMB Determination of the Solution Structure of Apo Calbindin Dgk by NMR Spectroscopy Nicholas d. Skelton ~,2, dohan K6rdeF and Walter J. Chazin 2. ~Departnlent of Protein Engineering, Genentech, hlc. South San Francisco CA 94080, USA 2Department of Molecldar Biology, The Scripps Research hlstitllte, La Jolla, CA 92037 USA

*Corresponding author

The three-dimensional structure of apo calbindin D.k has been determined using constraints generated from nuclear magnetic resonance spectroscopy The family of solution structures was calculated using a combination of distance geometr~ restrained molecular dynamics, and hybrid relaxation matrix analysis of the nuclear Overhauser effect (NOE) cross-peak intensities. Errors and inconsistencies in the input constraints were identified using complete relaxation matrix analyses based on the results of preliminary structure calculations. The final input data consisted of 994 NOE distance constraints and 122 dihedral constraints, aided by the stereospecific assignment of the resonances from 21 ]3-methylene groups and seven isopropyl groups of leucine and valine residues. The resulting family of 33 structures contain no violation of the distance constraints greater than 0.17 ,~ or of the dihedral angle constraints greater than 10 °. The structures consist of a well-defined, antiparallel four-helix bundle, with a short antiparallel [3-interaction between the two unoccupied calcium-binding loops. The root-mean-square deviation from the mean structure of the backbone heavy-atoms for the well-defined helical residues is 0.55 ~. The remainder of the ion-binding loops, the linker loop connecting the two sub-domains of the protein, and the N and C termini exhibit considerable disorder between different structures in the ensemble. A comparison with the structure of the (Ca 2.)2 state indicates that the largest changes associated with ion-binding occur in the middle of helix IV and in the packing of helix III onto the remainder of the protein. The change in conformation of these helices is associated with a subtle reorganization of m a n y residues in the hydrophobic core, including some side-chains that are up to 15 ,~ from the ion-binding site. Keywords: calcium binding proteins; three-dimensional protein structure; conformational change; nuclear magnetic resonance

Introduction The ubiquitous calmodulin superfamily of calcium-binding proteins (CaBPs) is involved in numerous Ca-"-mediated intra-cellular and extra-cellular processes, as well as in Ca 2+ uptake, transport and homeostasis. These proteins are characterized by a c o m m o n helix-loop-helix ion-binding motif, termed an EF-hand (Kretsinger, 1972; Kretsinger &

Nockolds, 1973), in which divalent metal ions are bound. N u m e r o u s X-ray crystallographic studies on members of this superfamily (McPhalen et al., 1991) indicate that the basic structural doinains in these proteins are pairs of EF-hands connected by loops of varying length (Seamon & Kretsinger, 1983). However, despite this wealth of information, little is known about the conformational response to binding at atomic resolution, due to the absence of

Present address: J. K6rdel, Department of Structural Biochemistry, Pharmacia Biopharmaceuticals Stockholm, Sweden. Abbreviations used: apo state, calcium-free calbindin D,,k; CaBPs, calcium-binding proteins; (Ca:*)2-state, calbindin D,,; with two calcium ions bound (one to each of sites I and II); (Cd2"),-state, calbindin D,~ with one cadmium ion bound to site II; CORMA, complete relaxation matrix analysis; COSY, correlation spectroscopy; DG, distance geometry; E-COSY, exclusive COSY; FID, free induction decay NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, 2D NOE spectroscopy; P43G, recombinant bovine calbindin D.; mutant with proline 43 substituted by glycine; rEM, restrained energy minimization; rMD, restrained molecular dynamics; S/N, signal-to-noise ratio; TOCSY, total correlation spectroscopy; TPPI, time-proportional phase incrementation; r.m.s.d., root-mean-square deviation(s); p.p.m., parts per million. 0022-2836/95/220441-22 $08.00/0

~ 1995Academic Press Limited

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high-resolution structural comparisons between the apo and Ca2+-bound states of any single calciumbinding domain. This knowledge is important because the conformational response to binding is one of the important factors determining the activity of these proteins. We and others (e.g. Finn et al., 1993; Findlay et al., 1994) have therefore turned to NMR spectroscopy as a tool to obtain comprehensive high-resolution structural analysis of Ca2+-free and Ca2+-loaded states of EF-hand calcium-binding proteins. Calbindin Dgk has been selected as an initial target and our primary model system because it is small (75 residues), extremely stable over a wide range of experimental conditions, and can be efficiently overexpressed in Escherichia coli. This protein is involved in intracellular buffering of Ca 2+ a n d / o r uptake of Ca 2÷ from the intestinal brush border membrane and transport to the basolateral membrane (Christakos et al., 1989; Staun, 1991). Interactions between calbindin Dgk and the calmodulin-binding domain of the plasma membrane calcium pump (Ca2*-ATPase) have been observed in vitro in the presence of Ca 2÷(James et al., 1991), although the in vivo significance of this observation is uncertain. Calbindin Dgkis comprised of two EF-hands with a typical 12-residue Ca2*-binding loop between two helices in the C-terminal site and a 14-residue loop in the N-terminal site that is characteristic of the S-100 subgroup of the calmodulin superfamily (Heizmann & Hunziker, 1991). The protein has been extensively characterized by a wide range of biophysical techniques (Fors6n et al., 1993), including both X-ray crystallography (Szebenyi & Moffat, 1986; Svensson et al., 1992) and NMR (e.g. Akke et al., 1992, 1993; K6rdel et al., 1992, 1993; Skelton et al., 1992a,b).t Here we describe the high-resolution, three-dimensional solution structure of calbindin Dgk in the apo state, determined using NMR-derived distance and dihedral angle constraints in a combined DG and rMD computational approach. The structures have been refined with backcalculation to improve the quality of agreement between the NOEs expected on the basis of the calculated structures and the experimental NOE data. Combined with the structures determined for the (Ca2+)2 state, these results provide the first direct view of the changes induced by Ca 2. binding

-[-Previous NMR studies on calbindin Dgk indicated that wild-type protein is present as two isoforms under equilibrium conditions in solution due to cis-trans isomerization of the G42-P43 peptide bond (Chazin et al., 1989a). The conformationally homogeneous P43G mutant has been introduced to eradicate the problems caused by the presence of two sets of resonances in the NMR spectra and has been shown to cause minimal differences in the structural and dynamical properties of the protein (K6rdel et al., 1990). Subsequent studies have been carried out on the P43G mutant and references herein to calbindin D,~ should be taken to mean this mutant, unless otherwise indicated.

Structure of Apo Calbindin Dgk

in an EF-hand protein and a basis for efforts to correlate conformational response to ion binding and activity for this family of proteins (Skelton et al., 1994). A detailed comparison of the apo and Ca2+-bound structures is presented here, highlighting and quantifying these structural changes.

Results and Discussion The use of CORMA to check input constraints

The final structures described below are the product of many iterations through a protocol of structure calculation and refinement of input constraints. At the end of each round of calculation, the ensemble of structures was analyzed to find instances of: (1) consistent constraint violations, indicative of typographical or other primary errors; (2) NOESY cross-peaks that, although ambiguous in terms of chemical shift, could be unambiguously assigned to a single pair of protons on the basis of the relative spatial proximity of the candidate proton pairs in the structures; (3) prochiral groups that could be stereospecifically assigned on the basis of relative NOE intensities and distance in the structures. Four complete rounds of this protocol were carried out initially. At this stage, in spite of manual checking for typographical or other errors, the ensemble of structures had an average residual restraint violation energy over 20 kcal mo1-1, with several consistent violations in the range 0.1 to 0.2 A and isolated violations of 0.3 A or greater. Although this number is not particularly high among the rMD structures published to date, clearly there are errors in the input restraints. Thus, an additional step was incorporated into the computational protocol to check the consistency of the current structures and the constraints used to generate them. This step involved the use of the CORMA program (Borgias & James, 1989) to calculate the NOE intensities expected for the best eight structures (lowest violation energy) from the current round of calculation and to normalize the experimental and calculated NOE intensities. The mean and standard deviation of every calculated NOE volume (Ic) were then determined and stored in a database, along with the chemical shifts of the two interacting protons. Utilization of a family of structures as opposed to a single structure is preferable, as otherwise the subsequent analysis would be biased towards a single conformation. The complete database was then searched to find entries that matched the chemical shifts of experimental cross-peaks to within 0.03 p.p.m, in ¢01 and 0.015 p.p.m, in co,., and the experimental and calculated cross-peak assignments compared. The possible outcomes of this search are listed in Table 1, along with the appropriate changes made to the experimental constraint list. This procedure is capable of confirming correct constraints (category A), providing assignments for experimental cross-peaks ambiguous on the basis of chemical shift alone (category B), correcting

Structure of Apo Calbindin Dgk

443

Table 1 Summary of the possible outcomes when CORMA calculations Cross-peak Number of used to calculated generate Class constraints~ constraint?~ A Ia Yes

checking the input constraint list against the theoretical constraints derived from Calculated and observed constraints match? ~ Yes

Outcome Assignment verified B 1~ No n.a. Unambiguous assignment may be made: add constraint C Id Yes No Misassignment: change constraint to calculated assignment D >1 No n.a. Confirmation of ambiguous cross-peak E >1 Yes n.a. Multiple ~H - ~H pairs contribute to cross-peak: remove constraint F 0 Yes n.a. Erroneous constraint: suspect error in structures Number of entries in the database of calculated constraints at the 03~and 032chemical shifts of an experimentally observed cross-peak. Was the experimentally observed cross-peak used to generate a constraint? Does the assignment in the database of calculated constraints match the experimental cross-peak assignment? n.a. indicates not applicable. d This category includes instances in which there were several entries in the database of calculated constraints with appropriate chemical shifts but where one of the calculated volumes was greater than three times any of the other calculated volumes; the largest volume was taken as a unique assignment for the cross-peak.

e r r o n e o u s c o n s t r a i n t s ( c a t e g o r y C a n d E), a n d confirming that a cross-peak has contributions from m o r e t h a n o n e p r o t o n p a i r a n d s h o u l d not b e u s e d to g e n e r a t e a c o n s t r a i n t ( c a t e g o r y D). T h u s , the p r o c e d u r e acts as b o t h a c h e m i c a l shift a n d s t r u c t u r a l filter d u r i n g a n a n a l y s i s of the e x p e r i m e n t a l N O E constraints. F o u r m o r e c o m p l e t e r o u n d s of s t r u c t u r e c a l c u lation w e r e c o n d u c t e d , c h e c k i n g t h e r e s u l t s w i t h the b a c k - c a l c u l a t i o n filter each time. D u r i n g the first t w o c y c l e s of this p r o c e s s , e r r o n e o u s c o n s t r a i n t s w e r e c o r r e c t e d ( c a t e g o r y C) or r e m o v e d ( c a t e g o r y E) a n d initially ambiguous NOEs were assigned (category B). A m o r e c o n s e r v a t i v e a p p r o a c h w a s t a k e n for t h e final t w o r o u n d s of c a l c u l a t i o n : c r o s s - p e a k s in category C and E were removed from the exper-

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i m e n t a l list of c o n s t r a i n t s a n d no n e w c o n s t r a i n t w a s a d d e d . C o n v e r g e n c e m a y have o c c u r r e d in f e w e r c a l c u l a t i o n c y c l e s if c o n s t r a i n t s h a d b e e n r e m o v e d a n d c o r r e c t e d o n l y d u r i n g t h e initial r o u n d s . T h e a p p l i c a t i o n of this filter i d e n t i f i e d a s u r p r i s i n g n u m b e r of errors, p a r t i c u l a r l y for N O E s that h a d b e e n p r e v i o u s l y a s s i g n e d as u n a m b i g u o u s ( c a t e g o r y E) b u t that in fact h a d s i g n i f i c a n t c o n t r i b u t i o n s f r o m m o r e t h a n one p r o t o n p a i r ( F i g u r e 1). F o r t h e s e cases, it w a s v e r y l i k e l y that t h e p r o t o n p a i r to w h i c h the N O E w a s a s s i g n e d w a s r e s t r a i n e d too tightl}; l e a d i n g to d i s t a n c e c o n s t r a i n t v i o l a t i o n s a n d e n e r g e t i c s t r a i n in t h e m o l e c u l e . R a t h e r t h a n l o o s e n i n g the constraints, t h e y w e r e s i m p l y not u s e d in s u b s e q u e n t c a l c u l a t i o n s , since it is not p o s s i b l e to a s s e s s the s e p a r a t e c o n t r i b u t i o n s of t h e p r o t o n p a i r s c o n t r i b u t -

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Figure 1. Changes in various structural parameters during iterations of constraint checking with CORMA and re-calculation of structures. A, N u m b e r of errors (thick unbroken line, left ordinate), ambiguous NOEs (broken line, left ordinate) identified in the data (class C and E in Table 1, respectively), and total number of restraints used in the rMD calculations (unbroken line, right ordinate). B, Mean number of violations greater than 0.1 ,~. C, Total energy (thick unbroken line, right ordinate) and violation energy (unbroken line, left ordinate) terms.

444

ing to the cross-peak. Although sacrificing precision, this approach provides an accurate structure. The vast majority of errors (category E) identified in this way were NOEs that had initially been assigned as intra-residue or sequential constraints, hence their removal actually had little effect on tile global fold of the structures. In the structures obtained prior to using the back-calculation filter, there were several examples of poorly defined side-chains for which NOEs were incorrectly assigned; none of the structures was able to satisfy all of the constraints perfectly Correction of the NOE assignment led to substantial improvements in the convergence of these side-chains to a single conformation, thus improving the accuracy and precision as well as decreasing constraint violations, as had been reported previously (Thomas et al., 1991). The data in Figure 1 summarize tile improvement in the structures over four cycles of constraint checking. There is a significant improvement in the average total energy and a fourfold decrease in tile average violation energ)4 indicating that there are both fewer constraint violations and less strain in the structures. In the final structures, there is no consistent violation greater than 0.05 A and the maximum violation is below 0.17 A. A round of constraint checking on the final structures identified no constraint in class C (misassignments) and only 17 in category E (ambiguous NOEs assigned to a single proton pair). Thus, the process is close to convergence at this stage and the final structures are not biased by incorrect constraints. Interestingly, the total n u m b e r of constraints was reduced during this process, yet the quality of the structures improved. The reduction in the total n u m b e r of constraints did not affect the precision of the structures; the r.m.s.d. of backbone heavy-atoms from the mean structure actually decreased by about 0.1 ,~ over the four iterations (data not shown). Similar results with the back-calculation constraint filter have been obtained for (Cd2'h-calbindin D,~ (M. Akke and W.J.C. unpublished results).

Input constraints A total of 1523 unique NOEs were assigned from the various NOESY spectra. For each round of refinement, NOE volumes were converted to interproton distances and constraints were removed if the covalent geometry was more constraining than the assigned u p p e r bound (W6thrich et al., 1983), leaving a total of 994 distance restraints for the final round of rMD calculation (see Table 8). The distribution of these NOEs is shown in Figure 3A. The 994 input restraints included 305 (31%) NOEs between amino acid residues more than four residues apart in the primary sequence (long-range) and 268 (27%) NOEs between amino acid residues two to four residues apart in the primary sequence (medium-range). Calbindin D,,~ contains 60 [3methylene groups; stereospecific assignments were obtained for 22 (45%) of those with non-degenerate

Structure of Apo Calbindin D~k

CI'H2 chemical shifts: 413 (42%) of the rMD restraints involved protons of the stereospecifically assigned methylene and isopropyl groups listed in Table 7. For distance geometry calculations, 32 distance constraints were added for 16 hydrogen bonds but the total n u m b e r of constraints was only 884 due to the pseudoatom corrections introduced for the nonstereospecifically assigned prochiral groups. In addition to the NOE distance restraints, 122 dihedral angle restraints were also included in the rMD calculations (85 in DG). The n u m b e r of distance constraints used for the calculation of the solution structure of (Ca'+)2 calbindin D,,k (1002) was comparable with the n u m b e r in the present s t u d y However, the apo data set contains 124 more long-range and 71 fewer sequential and medium-range NOEs than for the Ca-~*-loaded state. In addition, a greater n u m b e r of dihedral angle constraints and more stereospecific assignments were available for (Ca 2~)2 calbindin D.k. This situation arises because the apo state has more instances of resonance overlap among the backbone amide and C" protons (particularly affecting intraresidue and sequential NOEs), yet the aromatic resonances are much better dispersed, hence it was possible to unambiguously identify a significantly greater n u m b e r of long-range NOEs for certain side-chains within the hydrophobic core.

Choice of final structures for analysis After rMD, the resulting structures were ranked according to increasing restraint violation energy and superimposed for best fit to the structure of lowest restraint energy The average pairwise r.m.s.d. and tile average r.m.s.d, from tile average structure were then calculated for ensembles containing an increasing n u m b e r of structures from the top of this list. The r.m.s.d, values were found to plateau when the n u m b e r of structures included in the comparison exceeded 20 (data not shown), indicating that a minimum of 20 structures should be analyzed to ensure that the ensemble adequately represents the conformational space consistent with the available constraints (Hyberts et al., 1992). In order to facilitate comparisons with the ensemble of 33 NMR solution structures of (Ca2*)2 calbindin D,~ (K6rdel et al., 1993), a family of 33 apo structures was used for further analysis. The 33 structures with lowest restraint violation energy were examined as a series of local overlays. Three structures were noticeably out of family at tile C terminus of helix IV with the deviation starting at Lys71 or Lys72 (Figure 2). These three structures do not contain any significant violation of tile NOE constraints in this region of tile molecule but in all three cases Lys72 has an unusually low ~b value (-81 °, -89 °, -99 °) compared the average ~b angle for the other 30 structures (-60(+4)°). The values of ~],×_,, calculated from the Karplus relationship (Karplus, 1959; Bystrov, 1976; Pardi et al., 1984) for these three structures are 6.8, 7.8, and 8.9 Hz,

Structure of Apo Calbindin Dgk

445

considerably higher than the experimental value of 5.5 H z and the mean calculated value from the other 30 structures of 4.2(+0.5)Hz.i" Due to the discrepancy with the experimentally determined coupling constants, these three structures were replaced in further analysis by the next three structures in the violation energy ranking. The ensemble of 33 final rMD refined structures are shown in Figure 1 of Skelton et al. (1994).

Quality and precision of the ensemble Table 2 summarizes some of the pertinent energy terms for the family of apo calbindin D,k structures, attesting to the agreement with input constraints (low violation energy) and to the well-packed nature of the hydrophobic interior (large negative LennardJones non-bonding term). The 33 apo calbindin D,,~ structures contain very few violations of the input distance constraints, with an average of 1.67 violations greater than 0.1 A per structure, an average constraint violation of 0.028A and an average m a x i m u m constraint violation of 0.13 A. Figure 3B summarizes the sum of violations for each residue; in each case the mean is taken over the n u m b e r of NOE constraints involving a particular residue (K6rdel et al., 1993). As expected, the residues with the largest violations are localized to areas of the structures that have the fewest n u m b e r of constraints and are poorly defined; namely the N-terminal portion of Ca-'+-binding loops I and II, the linker loop between the EF-hands, and the C terminus. Of these four regions, all but loop I have been shown by ~N relaxation measurements to have greater than average internal mobility on the picosecond to nanosecond time-scale in the apo state (Akke et al., 1993), suggesting that the relatively high degree of violations is due to motional processes rendering the volume-distance scaling of NOEs inaccurate, as noted previously (K6rdel et al., 1993). In the N-terminal region of loop I, the relaxation data indicate a significant rate of exchange b e t w e e n distinct conformational substates of the protein. Time-averaging over the conformations sampled is expected to reduce the intensities of some NOEs, possibly below the limits of detectability leading to fewer constraints. Further, the set of NOEs that are i- The three out-of-family structures are not in conflict with the few NOE distance constraints in this part of the molecule, nor in conflict with the Lys72 qb dihedral angle constraint of-155 ° to -45 °. The large range of the latter arises because the local NOE distance constraints used by HABAS can discount only two of the four possible solutions of the Karplus equation and the single constraint range applied in DISGEO and AMBER must include both acceptable solutions. Although not in violation of the input constraints, these structures do have unusually small non-bonded contributions to the molecular energ)¢ indicating that this conformation does not allow optimal packing of helix IV onto the rest of the protein, as is readily visible upon inspection.

Gin75

Gin75

Figure 2. Backbone view of helix IV for the 33 best apo P43G calbindin D,; structures and the three out-of-fami!.y structures (see the text). The overlay was performed using all heavy-atoms between residues 63 and 74; only the N, C" and C atoms of residues 68 to 74 are shown for clarity. The three out-of-family structures are drawn with green lines.

observed may not be consistent with a single static structure, and hence, the corresponding set of constraints are more likely to be violated. The program PROCHECK (Laskowski et al., 1993) was used to assess the stereochemical quality of the ensemble of 33 apo structures. For the well-defined residues (S""~(q~) and S""~(ql) greater than 0.85; see Figure 3D and E), a single structure had Asn21 in a disallowed region of the Ramachandran diagram and one other structure had Glu60 in a generously allowed region of the diagram. Thus, file vast majority of well-defined residues were in acceptable portions of ~, q/space (Figure 4A). When all residues were considered, less than 1% fell into disallowed regions of the Ramachandran diagram, with no residue doing so consistently for all members of the ensemble. The plot of the average (qb, ~]1) angles (Figure 4B) clearly identifies the predominantly helical character of the protein and serves to indicate the high quality of the structure with all residues in allowed regions of ~b, q/space. Residue-specific r.m.s.d, values and S""~(~, ~lJ)have been utilized to assess the precision of the ensemble of apo calbindin D,,k structures. The data in Figure 3(C to E) indicate that the helices and the C-terminal sections of the Ca-"-binding loops are well defined locall)4 with low r.m.s.d, values and high S '''~. The N and C termini of the protein, the linker loop and the N-terminal residues of each Ca~-'-binding loop are the least well defined regions of the protein, with the backbone atoms of Lysl, Lys41, Gly42, Gly43, Ser44, Asn56, Gly57 and Gln75 having r.m.s.d, values greater than 2.0 A and S""~(qb) or S""~(qJ) <0.6. As observed in a n u m b e r of previous

446

Structure of Apo Calbindin D~k

s t u d i e s , the v a r i a t i o n of r.m.s.d, h a s a n i n v e r s e c o r r e l a t i o n to the n u m b e r of c o n s t r a i n t s i n v o l v i n g a p a r t i c u l a r r e s i d u e (see F i g u r e 3 A a n d B). In this case, t h e a v a i l a b i l i t y of ~5N r e l a x a t i o n d a t a ( A k k e et al., 19931 p r o v i d e s d i r e c t e v i d e n c e that t h e s e i l l - d e f i n e d r e g i o n s of the p r o t e i n e i t h e r s a m p l e m u l t i p l e c o n f o r m a t i o n a l s u b s t a t e s or e x h i b i t a b o v e - a v e r a g e , h i g h - f r e q u e n c y i n t e r n a l m o t i o n . T h u s , for a p o calbindin Dgk, the lack of c o n s t r a i n t s a n d s t r u c t u r a l d i s o r d e r is c o n s i s t e n t w i t h i n c r e a s e d m o b i l i t y a n d s a m p l i n g of a s u b s t a n t i a l r e g i o n of c o n f o r m a t i o n a l space. W h e n o v e r l a i d i n d i v i d u a l l y , the e l e m e n t s of r e g u l a r s e c o n d a r y s t r u c t u r e a r e all v e r y w e l l d e f i n e d (e.g. b a c k b o n e r.m.s.d, of helices <0.25 A, Table 3). A l t h o u g h the c o m b i n e d r.m.s.d, for helices I p l u s II is o n l y s l i g h t l y h i g h e r (0.28( -I-0.08) A), the c o m b i n e d r.m.s.d, for helices III p l u s IV i n c r e a s e s s u b s t a n t i a l l y (0.59( -t- 0.15) A). In p a r t i c u l a r , the relative o r i e n t a t i o n of helix III w i t h r e s p e c t to helix IV is not w e l l d e f i n e d ; helix IIl is not p a c k e d onto the rest of the p r o t e i n in a w e l l - d e f i n e d fashion. Interestingly, a h i g h u n c e r t a i n t y for the t h i r d helix of the TR, C f r a g m e n t of t r o p o n i n C h a s also b e e n r e p o r t e d r e c e n t l y ( F i n d l a y et al., 1994). A c o m p a r i s o n of s o l v e n t - a c c e s s i b l e s u r f a c e a r e a s in the a p o c a l b i n d i n D,k s t r u c t u r e s is v e r y revealing: 365( -I-23) ~2 of the s u r f a c e - a c c e s s i b l e s u r f a c e a r e a of helix I is b u r i e d d u e to p a c k i n g onto helices II a n d IV, w h e r e a s the c o r r e s p o n d i n g n u m b e r for helix III is o n l y 1 7 7 ( - I - 3 1 ) A 2. A n e x h a u s t i v e e x a m i n a t i o n of the a v a i l a b l e e x p e r i m e n t a l d a t a on

Table

s t r u c t u r e a n d d y n a m i c s p r o v i d e s no e v i d e n c e for a n i n t r i n s i c p o s i t i o n a l i n s t a b i l i t y for this helix a n d o u r c u r r e n t t h i n k i n g a t t r i b u t e s this u n c e r t a i n t y to i n s u f f i c i e n c i e s in t h e N O E data. T h e m a j o r i t y of h y d r o p h o b i c contacts b e t w e e n helix III a n d t h e rest of the p r o t e i n i n v o l v e p a i r s of l e u c i n e r e s i d u e s whose side-chain resonances are heavily overlapped in h o m o n u c l e a r s p e c t r a . This c a u s e s a n e x t r e m e s c a r c i t y of l o n g - r a n g e c o n s t r a i n t s for helix III, w h i c h a v e r a g e s o n l y 4.0 p e r r e s i d u e as c o m p a r e d w i t h 10.5 in t h e o t h e r h e l i c e s ( F i g u r e 3A). T h e u n c e r t a i n t y in l o o p I a l s o a p p e a r s to b e d u e , in p a r t , to a s c a r c i t y of l o n g - r a n g e c o n s t r a i n t s , as o p p o s e d to l o o p II, w h e r e s i g n i f i c a n t i n t e r n a l m o t i o n s on the p i c o s e c o n d to n a n o s e c o n d t i m e - s c a l e a r e d e t e c t e d ( A k k e et al., 1993). T h e u s e of i s o t o p e - e n r i c h e d s a m p l e s a n d m u l t i n u c l e a r , m u l t i - d i m e n s i o n a l e x p e r i m e n t s for t h e a s s i g n m e n t of N O E s a n d m e a s u r e m e n t of h e t e r o n u clear s c a l a r c o u p l i n g a r e e x p e c t e d to p r o v i d e a c o n s i d e r a b l e i m p r o v e m e n t in the p a c k i n g of helix III a n d the p r e c i s i o n of l o o p I. T h e q u a l i t y a n d p r e c i s i o n of t h e s t r u c t u r a l e n s e m b l e have also b e e n e v a l u a t e d w i t h r e s p e c t to side-chain conformations. The average side-chain Z a n g l e s of all r e s i d u e s e x c e p t Zt of G l u 3 5 c o n s i s t e n t l y c o r r e s p o n d to s t a n d a r d s t a g g e r e d r o t a m e r conform a t i o n s . For Glu35, the c o m b i n a t i o n of 3JH~_HI~a n d 3JN.,-mc o u p l i n g c o n s t a n t s c l e a r l y i n d i c a t e a trans (180 °) X~ r o t a m e r b u t a v a l u e o f - 1 2 1 ( - I - 4 ) ° is f o u n d in the final s t r u c t u r e s . This a p p a r e n t d i s c r e p a n c y s u g g e s t s e i t h e r p i n n i n g (Havel, 1991) of the s i d e - c h a i n or that

2

Summary of residual constraint violations and conformational energies of the 33 apo rMD structures and the 33 DG structures from which they were derived Average number of distance constraint violations Range (A) DG rMD 0.0 < d ~< 0.1 88.7 + 9.5 62.6 _ 5.0 0.1 < d ~< 0.2 26.7 + 5.0 1.7 + 1.7 0.2 < d ~< 0.3 14.4 + 4.6 0.1 + 0.2 0.3 < d ~< 0.5 14.1 + 4.7 0 d > 0.5 4.6 + 3.7 0 Average violation > 0.0 Average maximum violation (A) Range (°) 0.0 < 0 ~< 5 5 < 0 ~< 10 10 < 0 ~< 15 0>15 Average violation >0 ° Average maximum violation (°) E....

0.16 + 0.3 0.024 + 0.003 1.0 + 0.4 0.13 + 0.04 Average number of dihedral angle constraint violations DG rMD 3.8 + 1.3 5.9 + 1.8 0.6 + 0.8 0.9 + 0.7 0.03 +_ 0.17 0.06 + 0.23 0 0 2.7 _+ 1.1 2.5 + 0.7 5.3 + 2.5 6.8 + 2.4 Average AMBER energies (kcal tool ')" E~p

Ea,.,

E,.h,~

DGt' 812 + 376 -168 + 208 885 + 234' rMD -1007 + 13 -422 + 6 3.2 + 0.9 1.1 + 0.4 " E,,,.11= total energy in tile AMBER force field; Eu = Lennard-Jones non-bonded energy term; Ej,,, = NOE distance restraint violation term; Ed.,,,,= dihedral angle restraint violation terrn. b These entries are tile AMBER energies of tile DG structures at the start of the rMD protocol. The high values result from incorporation of tighter 1/r" averaged restraints, and differences between tile preferred covalent geometries within DISGEO and AMBER. ' This value includes contributions from both distance and dihedral angle violations.

Structure of Apo Calbindin Dgk

447

I Ioop l 1 helix I u) LM

100 -

0 Z

80-

0 (1)

,.Q

J,oop. |

I Iinkerl

I helxll

I

helixlll

helix IV

A

6040-

E ¢-

o<~

20-

B

0.010

v

¢-

0.008

oO

0.006

U

0.004

._0

0.002

t"

2.5

o< v

a

2.0 1.5 1.0

rr

-e-

0.5

1.0

v

t'¢0

U~ 0.0 1.0 v

¢0.0 10

20

30

40

50

60

70

residue Figure 3. Plots of NOE distance constraints (A), average sum of NOE constraint violations per constraint for each residue (B), average r.m.s.d, of the backbone atoms of the 33 best MD-refined apo structures calculated from the average structure (C), qbangular order parameter (D), and ~ angular order parameter (E) v e r s u s residue number for apo calbindin D,~. The elements of secondary structure ill apo calbindin D,k are shown by the bars above A. In A all interresidue constraints appear twice, once for each interacting residue. The NOE constraints are grouped into four categories: intraresidue (filled); sequential (shaded); medium-range (hatched); long-range, Jj - il >/5 (open). The values in C were calculated by overlaying the family of structures onto the geometric mean (see Materals and Methods) using backbone N, C ~ and C atoms, and then calculating the r.m.s.d, of these atoms from average structure on a residue-by-residue basis.

Structure of Apo Calbindin Dgk

448

1200

"

iI

m Q.

0.0-

-120.0 -

A o@

•

120.0

•

•

•

#•

II

0.

0.0 °

-120.0

B I

-120.0

I

|

0.0

I

I

120.0

phi Figure 4. Ramachandran (~, ~]1)plots for all 33 of the rMD structures of apo calbindin D,; (A) and the average (~, ~) values for the ensemble (B). In A, data are not included for residues where the ~bor ~ angular order parameter was less than 0.85 (Lysl, Ser2, Ala14 to Asp19, Leu40 to Thr45, Asp54 to Gly59, Ser74 and Gln75).

one or m o r e N O E c o n s t r a i n t s is in error; h o w e v e r , no c o n s i s t e n t violation of G l u 3 5 d i s t a n c e c o n s t r a i n t s can b e found. Besides Glu35, o n l y a few scattered i n s t a n c e s of s i d e - c h a i n c o n f o r m a t i o n s not w i t h i n 30 ° of a s t a g g e r e d Z~ r o t a m e r p o s i t i o n occur in the

e n s e m b l e a n d these are all r e s i d u e s for w h i c h ~JH,-H, a n d 3JN-H[~ either c a n n o t b e m e a s u r e d or i n d i c a t e c o n f o r m a t i o n a l averaging. O t h e r t h a n Glu35, the s i d e - c h a i n s of the 64 n o n - g l y c i n e , p r o l i n e or a l a n i n e r e s i d u e s in apo c a l b i n d i n D,k have b e e n classified into four categories. Classes A a n d B consist of the 39 r e s i d u e s f o u n d to p o p u l a t e a single classical r o t a m e r in all 33 s t r u c t u r e s . T h e m a j o r i t y (23) of these s i d e - c h a i n s are in class A, for w h i c h 3JH~_,, does not i n d i c a t e m o t i o n a l averaging, s t e r e • s p e c i f i c a s s i g n m e n t s w e r e m a d e a n d X~c o n s t r a i n t s w e r e applied. Of note a m o n g these 23, several s i d e - c h a i n s ( L y s l , Ser2, Lys7, G l u l l , Asp19, Asn21, Ser38 a n d Asp47) are p o l a r a n d have a h i g h solvent-accessible surface area, i n d i c a t i n g that s o m e surface r e s i d u e s do have a p r e f e r r e d Z~ c o n f o r m a t i o n . A m o n g the 16 o t h e r s i d e - c h a i n s o c c u p y i n g single r o t a m e r s (class B), ~J,~_,, could not b e m e a s u r e d for six, w h i l s t the r e m a i n i n g ten h a d c o u p l i n g c o n s t a n t s s u g g e s t i v e of s i g n i f i c a n t rotation a b o u t the C~-C" b o n d . T h e m a j o r i t y of the class B s i d e - c h a i n s are s o l v e n t - e x p o s e d a n d the d i s c r e p a n c y b e t w e e n the s t r u c t u r e s a n d scalar c o u p l i n g s m a y arise from the lack of explicit solvent in the r M D c a l c u l a t i o n s ( N o r i n et al., 1994). In the a b s e n c e of solvent, surface s i d e - c h a i n s tend to pack onto the surface of the p r o t e i n , m a x i m i z i n g n o n - b o n d e d contact t e r m s in the force field ( u n p u b l i s h e d results); if solvent w e r e p r e s e n t , the i n t r a m o l e c u l a r interactions that favor one p a r t i c u l a r r o t a m e r p o s i t i o n w o u l d b e replaced b y contacts w i t h solvent a n d m u l t i p l e r o t a m e r s w o u l d m o r e likely b e s a m p l e d . T h e r e m a i n i n g s i d e - c h a i n s all exhibit very low S"'~(K,) a n d o c c u p y two ( n i n e residues, class C) or t h r e e (16 r e s i d u e s , class D) of the classic rotamers. For m o s t of these residues, v a l u e s of 3J.~_,, either i n d i c a t e m o t i o n a l a v e r a g i n g or c a n n o t b e m e a s u r e d . Leu39 is the o n e e x c e p t i o n to this; ~J,,_,., does not i n d i c a t e m o t i o n a l averaging b u t the lack of wellresolved N O E s p r e v e n t s the s i d e - c h a i n o r i e n t a t i o n from being defined more precisely

Table 3 Precision of the apo structures and comparison to the (Ca 2')2 state 33 Apo Structures ~ Apo versus (Ca2`),b Residues" All atomsd Backbone" All atoms J Backbone" Helix I 0.78 4- 0.11 0.25 + 0.07 1.13 + 0.10 0.33 + 0.07 Helix 11 0.79 + 0.07 0.10 4- 0.02 1.48 4- 0.15 0.52 4- 0.05 Helix llI 0.98 4-_0.11 0.19 4- 0.07 1.13 4- 0.12 0.24 4- 0.03 Helix IV 0.78 4- 0.11 0.19 + 0.07 2.05 4- 0.06 1.08 + 0.08 Helices 1.04 4- 0.07 0.55 _+ 0.11 1.96 + 0.06 1.23 _+ 0.07 N-term EF-hand 1.13 4- 0.16 0.60 _+ (/.14 1.53 _+ 0.06 0.88 + 0.10 C-term EF-hand 1.40 4- 0.19 0.96 + 0.17 2.29 + 0.15 1.46 4- 0.11 All well defined ~ 1.00 + 0.06 0.57 + 0.09 1.91 + 0.08 1.20 4- 0.08 All residues 1.59 + 0.15 1.17 + 0.14 2.32 + 0.12 1.72 + 0.14 Average r.m.s.d, from the average structure. b Average r.m.s.d, of mean apo structure from the ensemble of 33 structures for tile (Ca2~)2state (K6rdel et al., 1993). " Residues used for fitting: helix I, 3 to 15; helix 11, 25 to 35; helix III, 46 to 53; helix IV, 63 to 74; N-terminal EF-hand, 3 to 35, C-terminal EF-hand, 46 to 74; all well defined, 3 to 14, 20 to 39, 47 to 53 and 60 to 73, all residues, 1 to 75. a All heavy-atoms. e Backbone N, C~ and C atoms. f Well-defined residues are defined as having all backbone angular order parameters greater than 0.85.

Structure of Apo Calbindin Dgk

449

Interestingly, the side-chains of Leu23, Phe50 and Va161 are located in close proximity to each other in the core of the protein and all occupy single Z~ rotamers in the structures but have ~Jm-,, values indicative of motional averaging. This observation is highly suggestive of the presence of some specific motional process, e.g. the readily observable NOEs involving these three residues are sufficiently similar in the two (or more) conformational substates present in solution that only a single conformation is determined in the present study. During the CORMA constraint checking process, several discrepancies b e t w e e n calculated and observed NOE volumes were observed for constraints involving Phe50, also hinting at some conformational heterogeneity. This hypothesis is s u p p o r t e d by the observation of significant conformational exchange detected by 'SN relaxation for the backbone amide groups of Glu51 and Va161 (Akke et al., 1993). We attribute these observations to some areas of the hydrophobic core of apo calbindin D,,; being somewhat flexible, presumably exhibiting "breathing" motions on a more rapid time-scale than is observed for the (Ca 2.)2 state. Another measure of the quality of the structural ensemble is how well they reproduce the experimental data, for example as reflected in a figure of merit (R-factor) for tile agreement b e t w e e n observed NOE intensities and NOEs back-calculated from the structures. Rather than simply reporting the R-factors of the 33 "final" apo calbindin D,~ structures, the quality of the ensemble has been examined by performing an additional rEM refinement with the force field supplemented by a potential to minimize tile R-factor (Yip & Case, 1989). Tile R-factors before and after the NOE-driven minimization are listed in Table 4. While the minimization does reduce the R-factors substantially, the structures actually change very little; the pairwise r.m.s.d, b e t w e e n structures before and after the additional refinement is 0.037(4-0.003),,~ for backbone heavy-atoms and 0.043( + 0.005) A for all heavy-atoms. This result indicates that the final rMD refined ensemble is of high quality, requiring only minimal conformational changes to obtain substantial improvements in R-factors, as was observed previously for tile (Ca 2' )2 state (K6rdel et al., 1993).

Table 4

R-factors for apo calbindin D.~ structures before and after the NOE volume driven minimization Constraint Constraint+ direct NOE volumes refinement only volumerefinement R(all)~ 0.39 ± 0.02 0.23 4- 0.01 R(intraresidue)~ 0.23 ± 0.01 0.15 ± 0.01 R(interresidue)" 0.65 ± 0.04 0.36 ± 0.02 R' "(All)" 0.088 ± 0.002 0.071 ± 0.002 ~Calculated as Zll,,t.-I,,,hl/Zl,,t., where I,,L,.and l,,,t, are the observed and calculated NOE volumes, respectively. b Calculated as

:£](I,,t.)'/" - (I~.,,J'/"I/Z(I,,L.)

'''.

Description of the solution structure and c o m p a r i s o n with the (Ca2+)2 state

The four-helix bundle global folding topology of apo calbindin D,k is identical with that found for the (Ca'-*)., state (Szebenyi & Moffat, 1986; K6rdel et al., 1989, 1993). Thus, the analysis of differences b e t w e e n the two states requires careful examination at the local level with respect to changes in the elements of secondary structure, as well as the determination of the changes in the side-chain packing and their effects on the tertiary structure. Both local comparisons involving best-fit superpositions of specific structural elements, as well as more global superpositions to elucidate the changes of packing within and between the two EF-hands are necessary. It is also important to consider the precision and accuracy of the structures. For example, it is imperative that comparisons be made only for those side-chains for which there is agreement between the values of the scalar coupling constants and the conformational distribution within the structural ensemble for both states. On the other hand, lower precision within a specific region of one or both structures does not compromise the accuracy of a comparison, as long as the uncertainties are taken into account when determining the magnitude of change that can be considered significant. Secondary structure Figure 5 shows a series of best-fit superpositions of the ensemble of NMR structures for each of the elements of regular secondary structure. The formal assignment of the helical elements is made as described by K6rdel et al. (1993), based on a combination of (i, i - 3) or (i, i - 4) hydrogen-bonding and backbone dihedral angles (cb = - 6 0 ( + 2 0 ) °, ql = - 4 0 ( + 2 0 ) ° ) . The statistics for tile hydrogen bonds present in apo calbindin D,,k are given in Table 5 along with a comparison tile (Ca2*)_,-state. The mean and standard deviation of the backbone qb and q* angles in the apo and (Ca >):-states are compared in Figure 6A and B. In the N-terminal EF-hand, Ser2 is clearly defined as the N-terminal residue of helix I on the basis of hydrogen bonds, although some fraying is evident (Figures 3C and E, 5A): 13 structures (39%) have an N-cap hydrogen bond from Glu5 HN to Ser2 O:, Tile helix extends in regular ~.-helical conformation from Pro3 to Alal5, except for a well-defined kink at Tyrl3, which exhibits a significant deviation of ~ from standard ~-helical conformation (qb = -86( + 7) °) and a mixture of (i, i - 3) and (i, i - 4) hydrogen bonds (Table 5). Helix I terminates at Ala15, as Lysl6 is clearly disordered. By comparison, helix I in the (Ca2+),-state extends from Ser2 to Lysl6 but is otherwise very similar, except that the kink at Tyr13 (~ = - 7 0 ( + 3 ) °) is less pronounced than in tile apo state and only (i, i - 4) hydrogen bonds are observed. Ser24 is both the C-terminal residue of the ]3-interaction and the N-terminal residue of helix II.

Structure of Apo Calbindin Dgk

450

15

15

Glu26

Glu26

Tyr13

Phe36 A

B

62

62

54

54

45

45 C

D 59

59

Figure 5. Local overlays of the elements of regular secondary structure in apo (blue) and (Ca2")2 (red) calbindin D,k: A, helix I; B, helix II; C, helix III; D, helix IV; and E, antiparallel [3-interaction. In each case the geometric mean structure of apo and (Ca2~)2 calbindin D,k (see Materials and Methods) were first superimposed using the atoms in the particular element of structure shown and then each ensemble was superimposed onto its own mean using backbone N, C ~ and C atoms. Specific side-chains discussed in the text are included.

Structure of Apo Calbindin D9k

451

I ,oop,

[h,,,x, ]

I

I ''n"e, I

~

I'oop" I

~

Iho'ix'V]

150 100 50-

•D.

0-

-50

I

-100 -150

150 100 50

"~

o -50 -100 -150

360 300 240

-

180

-

120

-

}

i

,

"°S

}

u m

¢., 0

600

I

10

~ 20

, 30

'I 4O

, 50

I

I

6O

70

residue Figure 6. Summary of the qb, ~ and Z, angles observed in the ensemble of apo (open boxes) and (Ca2")2 (filled boxes) calbindin D,k structures. The mean values of the angles are shown along with the standard deviation calculated from the angular order parameter, as described by Hyberts et al. (1992).

452

Structure of Apo Calbindin D~k

A n N - c a p h y d r o g e n b o n d f r o m G l u 2 7 H N to S e r 2 4 O ~is o b s e r v e d i n 16 s t r u c t u r e s (48%). H e l i x II e x t e n d s to T h r 3 4 i n r e g u l a r ~.-helical c o n f o r m a t i o n . T h e a n g l e o f G l u 3 5 ( - 8 6 ( - I - 4 ) °) is j u s t o u t s i d e t h e h e l i c a l r a n g e b u t all s t r u c t u r e s e x h i b i t a n (i, i - 4) ~ - h e l i c a l hydrogen bond from Glu35 HN. The C terminus of

Table 5 H y d r o g e n b o n d s o b s e r v e d in the e n s e m b l e of apo c a l b i n d i n D,,~ s t r u c t u r e s a n d a c o m p a r i s o n w i t h t h o s e o b s e r v e d for the (Ca 2. )2 state Donor~ Acceptor b Apo' Ca2"(NMR) d Ca2"(X-rayY A. Backbone-backbom' Glu5 HN Ser20 Leu6 HN Ser20 Lys7 HN Pro30 GIv8 HN Glu40 liei-) HN Glu50 Phel0 HN Leu60 Glul 1 HN Lys70 Lysl2 FIN Gly80 Tvrl3 HN lie90 Tyri3 HN Phel00 Alal4 HN Phel00 Alal5 HN Glull O Lysl6 HN Tyr13 O Lvsl6 HN Alal40 (~1n22 HN Aspl90 Leu23 I-IN VaI61O Lys25 HN Gly59 O Leu28 FIN Ser24 O Lys29 FIN Lys25 O Leu30 HN Glu26 O Leu31 HN Glu27 O Leu32 HN Let,28 O Gin33 FIN Lys29 O Thr34 FIN Leu30 O Glu35 HN Leu31 O Phe36 HN Leu32 O Leu39 HN Phe36 O Leu4(} H N Phe36 O Lys41 FIN Ser38 O Lvs41 HN Leu39 O Tiw45 HN Gly43 O Leu49 HN Thr45 O Phe50 FIN Leu46 O GluS1 HN Asp47 O Glu52 ItN Glu48 O Leu53 HN Leu49 O Asp54 HN Phe50 O Lvs55. HN Leu53 O GIv57 FIN Asp54 O Val61 HN Leu23 O Phe63 HN Asn21 O Phe66 HN Ser62 O Gin67 HN Phe63 O Gin67 NH Glu64 O Vai68 HN Glu64 O Va168 HN Glu65 O Leu69 HN Glu65 O keu69 Phe66 Va170 HN Phe66 O Va170 HN Gin67 O Lys71 HN Gin(37 O Lys71 HN Va168O Lys72 HN Va168O Lys72 HN keu(}90 11e73 FIN Leu69 O Ser74 [iN Val70 O Ser74 HN Lvs71 O Gin75 HN Lys71 0 F I N

0

5 32 32 33* 33* 33* 33* 30 14 16 33 32 21 11 31 33* 21 33 33* 33 33 33 33* 33* 33* 21 18 18 3 lb 18 7 33* 32 32 28 33 19 10 32* 19 33 33 {} 33* {} 33*

25 7 31 32 33 33 32 33 33

0

- -

33* {} 33* 0 31" 2 32 33 {} 27

--

33 33 3{} - -

30 32 - -

33 29 4 33 33 33 33 33 32 7 - -

21 - -

-32 33 33 32 33 32 25 22 32 - -

33 - -

10 0 33 -33 --

32 {} 29 {} 29 {} 24 --

y Y Y y Y Y Y Y y y Y N Y N N Y N Y Y Y Y Y Y Y Y y Y N N N N Y Y Y Y Y Y y N Y N Y y N N y N y N y N y N y Y Y N y conl iltued

Table 5 contimted Donor~

Acceptor b

Apo'

Ca2'(NMR) J

Ca:'(X-ray) ~

B. Side-chain to side-chain amt backbone to side-chain hydro,~en bonds

Glu5 HN Ser2 O:' 13 -y Tyrl30"H Thr34 O' 0 18 N Tyr13 O"H Glu35 O" 0 10/4 Y Asn21 HN Aspl9 O"' 11/21 0/32 Y Asn21 H°'N Pro20 O 17 -N Asn21 H"-'N Glu22 O' 0/0 32/0 N Gin22 N'aH Asp19 O '~: 3/4 6/1 Y Gin22 N':H Glu60 O 5/5 19/11 N Ser24 HN Glu27 O'2 10/7 9/0 Y Ser24 O'H Glu27 O" 10/8 17/8 Y Glu27 HN Ser24 O ~ 15 27 Y Gin33 H"N Gin33 O 12 -N Thr34 O~H Leu30 O 33 33 Y Glu48 HN Thr45 O ~ 7 15 Y Lys55 N;FI Leu53 O 1 0 Y Gly57 HN Asp54 O"' 6/6 2/3 Y Gly59 HN Asp54 O *-" 12/5 12/13 Y Glu60 HN Asp58 O "L 2/3 17/7 Y Ser62 HN Glu65 O'2 0/1 0 Y Glu65 HN Ser62 O ~ 3(1 I y Gin67 N':H Phe63 O 0 33 Y Ser74 O~H Val70 O 27 0 Y Underlined amide protons exchange with solvent with a rate constant < 10 '" s ' (Skelton et al., 1992a,b). u In the case of glutamic and aspartic acid residues, the number of structures containing the hydrogen bonds to both carboxylate oxygen atoms are shown as these two atoms are able to exchange position during the rMD. ' Asterisks (*) indicate hydrogen bonds that were enforced during the DG calculations. d Structures described by K6rdel et al. (1993; Protein Data Bank accession number 2BCA); entry indicates how many of the 33 structures in the ensemble contain a particular hydrogen bond. Structure described by Szebenyi & Moffat (1986; Protein Data Bank accession number IlCB); Y, hydrogen bond present; )4 distorted hydrogen bond present; N, hydrogen bond not present.

h e l i x II is P h e 3 6 w i t h 2 / 3 o f t h e s t r u c t u r e s c o n t a i n i n g t h e r e q u i s i t e (i, i - 4 ) hydrogen bond from Phe36 H N . I n ( C a 2 * ) 2 - c a l b i n d i n D,.u h e l i x II a l s o r u n s f r o m S e r 2 4 to P h e 3 6 b u t t h e (i, i - 4) h y d r o g e n b o n d f r o m L e u 3 0 t o G l u 2 6 is p e r t u r b e d b y a k i n k i n v o l v i n g t h e qJ a n g l e of Lys29 (qt[(Ca2~)2]=-66(-I-2)°; q~[apo] = - 5 3 ( + 2)°); t h i s c a u s e s a n o t i c e a b l e d i f f e r e n c e in t h e b a c k b o n e c o n f o r m a t i o n o f t h e t w o s t a t e s ( F i g u r e 5B). In t h e C - t e r m i n a l E F - h a n d , h e l i x III e x t e n d s f r o m L e u 4 6 to A s p 5 4 i n r e g u l a r ~ - h e l i c a l c o n f o r m a t i o n , a l t h o u g h t h e N t e r m i n u s is r a t h e r f r a y e d ; t h e N - c a p h y d r o g e n b o n d f r o m G l u 4 8 H N to T h r 4 5 O : o b s e r v e d i n t h e (Ca2*)~ s t a t e is f o u n d i n o n l y s e v e n o f t h e a p o s t r u c t u r e s (21%). Ill t h e ( C a 2' )2 s t a t e , r e g u l a r ~ - h e l i c a l s t r u c t u r e is o b s e r v e d b e t w e e n T h r 4 5 a n d A s p 5 4 w i t h l e s s f r a y i n g at t h e N t e r m i n u s ( F i g u r e 5C). H e l i x IV e x t e n d s ill a r e g u l a r ~.-helical c o n f o r m a t i o n f r o m S e r 6 2 to S e r 7 4 ( F i g u r e 5D). A n N - c a p (i, i - 3 ) h y d r o g e n b o n d i n v o l v i n g S e r 6 2 O ' is o b s e r v e d i n 30 s t r u c t u r e s (91%). S u r p r i s i n g l y , a n a d d i t i o n a l (i, i - 4) h y d r o g e n b o n d f r o m G l n 7 5 H N to Lys71 O is o b s e r v e d i n 26 s t r u c t u r e s (79%), i n s p i t e o f t h e h i g h o v e r a l l u n c e r t a i n t y i n qb a n d qJ o f G l n 7 5 . T h i s m a y b e d u e to t h e h y d r o g e n - b o n d i n g propensity of the A M B E R f o r c e f i e l d i n s i m u l a t i o n s in vacuo ( M o o r e et al., 1991). T h e r e g u l a r i t y o f h e l i x IV i n a p o

Structure of Apo Calbindin Dgk

calbindin D~k is in marked contrast to the (Ca2")2 state where a mixture of (i, i - 3) and (i, i - 4) hydrogen bonding is present (Szebenyi & Moffat, 1986; K6rdel et al., 1993). Helix IV is distinctly irregular in the (Ca2+)2 state with the values of ql in Phe66, Gln67, Lys72 and Lys73 all being ~ 20 ° higher than in the apo state; a n u m b e r of the backbone dihedral angles values in the Ca2Moaded state are close to or outside the limit for helical conformation (Figure 6). The two EF-hands are packed so as to bring the N-terminal binding loop (loop I) into close proximity to the C-terminal binding loop (loop II), allowing a short anti-parallel ]3-interaction to occur b e t w e e n residues 21 to 24 and residues 59 to 62 (Figure 5E). The evidence for this interaction includes hydrogen bonds from Leu23 H N to Val61 CO and Va161 N H to Leu23 CO, along with backbone q/angles in the range 100 ° to 170 ° for these residues. In approximately 2 / 3 of the apo structures, the anti-parallel [3-interaction is extended in both directions with one additional hydrogen bond (Lys25 H N - G l y 5 9 CO and Phe63 HN-Asn21 CO). Although the average O . . . H distances for these two putative hydrogen bonds are 2.7( + 1.7) A and 2.5( + 0.2) A, respectively, over half of the structures have both of these distances less than 2.1 A. For the (Ca2")2 state, only the two central hydrogen bonds involving Leu23 and Va161 are observed. Backbone dihedral angle differences b e t w e e n the apo and (Ca2")2 state are observed for the qb angle of Asn21, Gln22, Va161 and Ser62, and the qJ angle of Asn21, Gln22 and Va161 (Figure 6). To summarize, in the N-terminal EF-hand, only two significant differences b e t w e e n tile apo and (Ca2")2 state are observed in the backbones of the helices: helix I extends one additional residue into the binding loop and the middle of helix II is kinked in the (Ca2*)_~state. In the C-terminal EF-hand, helix III is less frayed and extends one additional residue at its N terminus in tile (Ca2~)~ state. Helix IV is by far the most severely perturbed, with extensive reorganization of the regular hydrogen-bond pattern relative to that observed in the apo state. Backbone dihedral angles and hydrogen bonding in the Ca2'-binding loops are also altered in response to ion binding, in particular in tile anti-parallel ]3-interaction where two fewer hydrogen-bonds are present in tile (Ca2")2 state and several dihedral angles are significantly different. Side-chain packing In tile N-terminal EF-hand, changes in conformation are very slight for the well-defined sidechains contributing to the hydrophobic core. The side-chain of Glu11 switches from a g* conformation in the apo state to a trans conformation in the (Ca2")2 state; no other side-chain changes X~ rotamer but there is a subtle difference in the Z. value of Phel0 and a significant change in Z'- of lle9 (Figure 5A). In the (Ca 2')2 state, a hydrogen bond is observed from Tyr 13 O:H to Glu35 O' in approximately half of the structures and to Thr34 O' in the other half. Neither of these hydrogen bonds is observed in the apo state,

453

with the side-chain of Tyr13 having a different orientation and Glu35 populating a different Z~ rotamer. Scalar coupling constants clearly indicate that the Glu35 side-chain occupies the trans Xz rotamer in the apo state and the g* rotamer in the (Ca2*)_~ state but the inconsistencies b e t w e e n scalar coupling constants and NOEs in the apo state (vide supra) preclude further analysis. Most of the buried hydrophobic side-chains of helix II occupy very similar positions in the apo and (Ca:*)2 states. Only Leu28 (change in Z2 rotamer), Leu31 (27 ° change in Z2) and Phe36 (38 ° change in Z,) have distinctly different orientations. We note that Leu23 and Leu30 are well defined in both states and are distinctly different; however, for both of these side-chains, 3j,,_,.,, is indicative of conformational averaging in the apo state (vide supra), hence the structures presented may not represent the only conformations occupied by these side-chains. In the C-terminal EF-hand, a description of tile Ca-~+-induced changes in side-chain conformation within helix III are limited by the lack of definition of m a n y side-chains in the apo state (Figure 6). Among the hydrophobic residues, Phe50 and Leu53 have similar orientations in both states (Figure 5C), while Leu46 and Leu49 are disordered in the apo state but well ordered in the (Ca2+)2 state. Figures 5D and 6 show that helix IV is well defined in both states. Most of the mean Xt and X2 angles of interior hydrophobic residues (Phe63, Phe66, Va168, Leu69, Val70 and 11e73) are less then 20 ° different in the apo and (Ca2+)2 states, the exceptions being Phe63 Z2, Leu69 X.~and Ile73 Z~. Despite the similarities of the specific side-chain angles, local best-fit superposition reveals substantial changes in response to Ca 2~ binding (Figure 5D). In this helix, it is the marked differences in backbone conformation that give rise to significant changes in the spatial distribution of side-chains. The full extent of these changes can be fully appreciated only by more global comparisons, as will be discussed in more detail below. The presence of an intricate network of hydrogen bonds in the two binding loops of (Ca 2+)2 calbindin D,~ has been well documented (Szebenyi & Moffat, 1986; Strynadka & James, 1989). Our studies of the apo protein show that the extent of this network is reduced in the absence of the organizing effect of the binding of Ca 2+. For example, a rotation of the side-chain of Asn21 about Z. (from - 7 5 ( + 1 8 ) ° to 176(+19) °) occurs upon Ca 2+ binding and this permits the formation of hydrogen bonds from Asn21 N'~2H to Gln22 O', and in turn from Gln22 N'2H to Gln60 O'. It is important to note that the formation of these hydrogen-bonding networks has an effect on the average structure, and is associated with general rigidification of the binding loops and a significant increase in stability of the protein. The magnitude of the effects on protein dynamics due to Ca2+-binding is exemplified by the >35 ° increase in the thermal denaturation midpoint, substantial reductions in the rates of amide proton exchange with solvent, a reduction in the sampling of conformational substates reported in '+N relaxation

Structure of Apo Calbindin Dgk

454

experiments, and lowered flexibility on the picosecond to nanosecond time-scale (Skelton et al., 1992b; Akke et al., 1993; Fors6n et al., 1993). Global fold

Previous comparisons of EF-hand proteins have made use of interhelical angle measurements to assess differences in global folding of the four-helix bundles in these proteins (Strynadka & James, 1989). Modeling studies of the effect of Ca 2*binding in the N-terminal domain of troponin C predicted large changes within each EF-hand and between the helix pairs I/IV and II/III (Herzberg et al., 1986). Table 6 contains the mean interhelical angles observed in the ensembles of apo and (Ca-'*)-' calbindin D,~ structures, with the corresponding values observed in the N and C-terminal domains of troponin C included for comparison. In no case does the helical angle between apo and (Ca-'*)2calbindin D,,~differ by more than twice the standard deviation in the measurement, suggesting that within the precision of the present structures there is no significant change in interhelical angle as Ca-" is bound. A more detailed discussion of the significance of this finding has been presented elsewhere (Skelton et al., 1994). In contrast to the interhelical angles, there are some significant changes in inter-helical separation, particularly for helix I and IV, and helix III and IV (Table 6). The changes in hydrophobic packing leading to these differences in interhelical angles are discussed below. Global comparisons of the three-dimensional structures of the apo and (Ca>)_, states of calbindin D,,kreveal that the response to Ca 2' binding in the two EF-hands is highly asymmetric (Skelton et al., 1994). The observations of a backbone r.m.s.d, between tile

Table 6

Interhelical angles (distances) in apo and (Ca-")2-calbindin D~ k

Flelices ~ I-ll I-Ill I-IV II-III II-IV Ill-IV

Apo 123 (9.0 -109 118.6 128 (9.6 124 (10.8 -34 112.4 118 (11.0

4- 3 4- 0.3l 4- q _+ 0.5l 4- 4 4- 0.1) 4- 7 + 0.4) 4- 4 + 11.3) 4- 8 4- 0.8)

(Ca>)2 130 + 3 19.1 4- I}.2) -111 4- 6 110.9 4- 0.2) 135 4- 4 (10.1 +_ 11.1) 113 4- 6 (111.0 4- 0.2) -28 4- 4 (12.1 4- 0.3) 110 4- O (8.3 4- 0.3)

TnC-Nb TnC-Cb 136 (9.9) -86 (bL2) 119 110.9) 132 (10.0) -45 111.4) 115 111.7)

11)4 110.4) -126 117.3) 118 110.8) 126 (9.2) -35 (14.4) 108 (9.3)

The interhdical angles, in degrees (and distances in ;\), were calculated according to the protocol of Chothia et al. 11981 ) using a program written by Dr J. Theaker (unpublished). Limits for helices were: 3 to 13, 25 to 35, 46 to 54 and 63 to 72 (calbindin D,;); 19 to 29, 39 to 49, 58 to 66 and 75 to 84 (Tnc-N); and 95 to 105, 115 to 125, 134 to 142 and 115 to 160 (Tnc-C). b TnC-N is the apo N-terminal domain of troponin C molecule, TnC-C is the (Ca>l: C-terminal domain of troponin C. The coordinates for troponin C were obtained from the Brookhavexl Protein Data Bank (accession n u m b e r 5TNC).

two states of only 0.88 A (Table 3) and significant changes in only a small number of side-chains, indicate that the N-terminal EF-hand is essentially preformed. In contrast, the backbone r.m.s.d, for the C-terminal EF-hand is nearly double (1.46 ,~), with repositioning of helix III, significant changes in the hydrogen-bonding pattern and reorganization of the hydrophobic packing of helix IV. As a result of this asymmetry, in much of the following discussion, comparisons between the apo and (Ca2+)2 state will be made after overlaying the structures using the backbone atoms of the N-terminal EF-hand only The essentially preformed nature of loop I in the N-terminal EF-hand of calbindin D,.k is readily apparent from the comparison of the apo and (Ca2+)2 states shown in Figure 7A. The average pairwise displacement of the C" atoms upon Ca 2. binding for Alal5 and Glu27, the first and last Ca > ligatinog residues in loop I, is 0.7(+0.3)A and 0.7(+0.2)A, respectively Helices I and II in apo calbindin D,.kare already positioned with the backbone carbonyl oxygen atom of Ala15 and the side-chain carboxylate group of Glu27 close to the location required for Ca > chelation. Some changes in loop I itself are necessary to bring tile backbone carbonyl groups of Glu17, Aspl9 and Gln22 close to the ion in this loop. We note, however, that residues Lys 16 to Pro20 are somewhat disordered in both the apo and (Ca>): structures, hence a more detailed analysis of this region is not possible at the current stage of refinement. A more pronounced change is observed in the conformation of loop lI in the C-terminal EF-hand (Figure 7B). This occurs because the prospective Ca 2. ligands are not in a suitable position for chelating the ion. Not only is movement of the side-chains of Asp54 and Glu65 necessary, but a shift in the backbone must also be accommodated, with the C" atoms of Asp54 and Glu65 displaced by 2.4( + 0.7) and 1.8(+0.4)A, respectively, upon ion binding (Figure 7B). These changes correspond to a rigid body movement of helix III by approximately 2 A towards loop II as Ca > is bound. The effects of ion binding on helix IV cannot be readily described in a similar manner because there are significant conformational changes within helix IV as Ca > is bound. Figure 7B clearly shows that these changes produce a marked difference in position for many of the interior hydrophobic residues in helices Ill and IV. Given the intimate packing of the helices within calbindin D,k, how are the changes in the C-terminal EF-hand accommodated while maintaining the more modest perturbation of the N-terminal EF-hand? The most significant factors are revealed by considering the hydrophobic contacts between the helices: the subtle changes in side-chain conformations of residues in helices I and II described above are a by-product of the larger movements of helices III and IV. Thus, in helix III, the movement of Phe50 influences the position of Leu23, Leu28 and Va161 whilst the lnovement of Leu49 affects the orientation of the side-chains of Leu28 and Leu32

Structure of Apo Calbindin Dg~

A

Asp19

455

Loop l Gin22

Glu17

Ala15

Glu27

Helix I

Helix II

B Asp54

Helix III

Glu65 Helix IV Leu53 Va168

Leu49

Leu69

Figure 7. Effect of calcium binding on loop I (A) and loop II (B) of calbindin D,,~.The representative structures shown are those that have the lowest backbone r.m.s.d, from the geometric mean structure for the region shown. The apo structure is shown in blue, the (Ca:*)_,structure in red. The structures were overlaid using the N, C ~ and C atoms of residues 3 to 15 and 23 to 36 only (see the text). The calcium ions (yellow spheres) were placed by reference to the X-ray structure of the (Ca2+): state (Szebenyi & Moffat, 1986). The four backbone carbonyl groups and the side-chain carboxylate group that chelate the calcium ion are included in A. The first and last residue involved in ion chelation (Asp54 and Glu65) are included in B, along with some of the hydrophobic side chains that undergo significant rnovement when Ca-'* is bound. (Figure 8A). Leu53 interacts predominantly with helix IV (Phe66, Leu69 and Va161), hence is able to maintain similar contacts in both apo and (Ca2")2 calbindin D,k. Although Leu46 also experiences a large shift in position (data not shown), it interacts mostly with residues in the linker-loop (Leu39 and Leu40). Due to the ill-defined nature of this later segment of the protein, detailed analyses are not possible at the current level of refinement. In helix IV, the change in position of Phe63 and Phe66 influences the positions adopted by Phel0 and

Leu31 in the N-terminal EF-hand (Figure 8B). Note that, although there is a pronounced difference in the backbone in the first turn of helix IV, the Phe66 HN-Ser62 CO hydrogen bond remains intact in both states. Figure 8B reveals that the movement of Val70 influences the positions of the side-chains of Leu6 and Phe36, located in helix I and helix II, respectively. The interactions b e t w e e n the C-termini of helices II and IV are explored in more detail in Figure 9. In the apo state, Val70 packs between helices I and II and makes contacts with Phe36 and Leu6 while I1e73 packs b e t w e e n the linker loop (Leu40) and Phe36. In the (Caa~)2 state, Ile73 has replaced Val70 between helices I and II (directly under Phe36 in Figure 9) and Val70 packs against the edge of helix I (Leu6) away from helix II. This reorganization in the hydrophobic core occurs concomitantly with the shift from ¢~-helical hydrogen-bonding upon Ca-" binding, as i, i - 3 hydrogen bonds are observed to the amide protons of Gln67, Va168 and Ser74 in the (Ca-")2 state. The exact reasons for this marked change in geometry are not clear; however, we presume that the energetic penalty paid by introducing the 3,, hydrogen bonds is recovered by the stabilization associated with Ca 2. binding and the more favorable hydrophobic interactions that Val70 and lle73 can make. The reorganization of these side-chains upon Ca 2. binding is reflected in the large changes in the upfield-shifted methyl resonances of Val70 C ~2 and Ile73 C '~ (0.08 to 0.62 p.p.m, and 0.42 to 0.18 p.p.m., respectively) in consonance with their positioning under the Phe36 aromatic ring (Figure 9). The agreement b e t w e e n the changes observed in the structure and the chemical shift parameters provides a completely independent validation of the results. The descriptions above indicate that substantial rearrangement of helices lII and IV can be accommodated without a substantial movement of the backbone in the N-terminal EF-hand because compensatory changes are made in the packing of the hydrophobic side-chains in helices I and lI. These side-chains act as a buffer zone, shielding the backbone of helices I and II from the perturbations in the C-terminal EF-hand. This leads to subtle changes in the hydrophobic core for side-chains that are up to 15 A from the binding sites, e.g. Phe36 and Leu6. Similar effects have been observed in studies of other proteins, including bacteriophage T4 lysozyme (e.g. Eriksson et al., 1992) and ~. repressor (e.g. Lim & Sauer, 1989), where m a n y hydrophobic residue mutations can be accommodated by slight rearrangements in the surrounding hydrophobic core.

Concluding Remarks The implications of the conformational consequences of Ca 2÷ binding in calbindin D,,; have been discussed previously in the context of correlations between variations in structural response and diversity of function in EF-hand CaBPs (Skelton et al., 1994). Here, we have presented a detailed description of the determination of the structure of the apo

456

protein, an analysis of the quality and precision of the structure, and an in-depth comparison with the (Ca2+)2 state. In contrast to the model for regulatory proteins based on the comparative analysis of the Ca2"-free N-terminal domain and Ca2*-filled C-terminal domain of troponin C (Herzberg et al., 1986), the consequences of ion binding in calbindin D.; are greatly attenuated and distributed in a highly asymmetric manner, being restricted primarily to one EF-hand. As opposed to the substantial opening of the EF-hands and exposure of the hydrophobic core in troponin C, the interhelical angles of calbindin D,~ change very little in response to Ca 2' binding (Table 6) and there is very little change in the exposure of hydrophobic residues (see Figure 4B, Skelton et al., 1994). Nonetheless, there is reorganiza-

Structure of Apo Calbindin Dg.

tion of the packing of the hydrophobic core that affects residues far from the ion-binding sites. The results obtained for calbindin D~k clearly demonstrate that to understand the wide functional diversity of the calmodulin superfamily of CaBPs, it is necessary to consider the affinity, selectivity, and kinetics of Ca "-÷ binding, and to recognize that the response to Ca"-* binding can be tuned over a wide range. EF-hands are nearly always found in pairs, hence the structural changes induced by Ca"-*binding may be viewed as the product of the conformational change within each EF-hand. In the current model for regulatory CaBPs, the shifts within an EF-hand are similar but, since the two EF-hands are packed in a face-to-face orientation, the movements occur in opposite directions relative to the plane defined by

Figure 8. Stereoviews of tile changes ill hydrophobic packing around helix III (A) and helix IV (B) upon Ca 2" binding in calbindin D,,~. Details of color scheme, choice of structure and method of overlay are identical with Figure 7. In both cases, the thick tube represents the backbone of the helix of interest, while side-chain atoms are depicted by thin tubes. Side-chain atoms only are shown for the residues in the N-terminal EF-hand that interact with the helices. Both views are from the core of the protein looking towards the inner (hydrophobic) face of helix III or IV, with the ion-binding site towards the top of the figure.

Structure of Apo Calbindin Dgk

457

protein have been described (Brodin et al., 1986; Chazin et al., 1989b; Skelton et hi., 1992a). Protein was dissolved in

Helix

II

/

Phe36

Ile73 Val70

430 I-d of 95% ~H_,O/5% -'H_,O and the pH adjusted to 5.3 by microliter additions of 1 M NaOH, yielding a final concentration of 4 to 5 mM. Samples in 2H,O solution were prepared by repeated lyopililization from -'H20, with final dissolution in 99.996% -'H20 (MSD Isotopes, Pointe Clare, Qu6bec); after the initial lyopililizations, the pH was adjusted to 5.3 (uncorrected meter reading) by the addition of 0.l M NaO-~H and -'HC1. There is a time-dependent change in the tH NMR spectrum of apo calbindin D,~; after one week in solution, peaks from a second form of the protein are observed at 10 to 15% of the intensity of the original peaks. The evolution of these resonances was carefully monitored during the course of tile analysis of cross-peaks (vMe infra).

NMR spectroscopy Helix IV Figure 9. Comparison of the interaction of hydrophobic side-chains in helix IV with Phe36 in helix II, in the apo and (Ca=')= states of calbindin D,,~. Details of color scheme, choice of structure and method of overlay are identical with Figure 7.

the interface b e t w e e n t h e m ( S t r y n a d k a & James, 1989). This magnifies the effect on the d o m a i n as a whole. With a p r e f o r m e d EF-hand, as in the case of calbindin D,k, structural c h a n g e s occur primarily in only half of the molecule and the synergistic overall effect of m o v e m e n t in o p p o s i t e directions is lost. Thus, the p r e f o r m e d N - t e r m i n a l b i n d i n g site of calbindin D,k a p p e a r s to play a u n i q u e role, leading to a vastly attenuated overall r e s p o n s e to Ca -'+ binding. In light of the p u r p o r t e d r e g u l a t o r y roles for other m e m b e r s of the $100 s u b f a m i l y (Kligman & Hilt, 1988), it will be of great interest to identify w h e t h e r or not their N - t e r m i n a l b i n d i n g loops are p r e f o r m e d and, correspondingl)~ if the conformational r e s p o n s e to ion b i n d i n g is also attenuated in these proteins. We believe that it is the interactions of side-chains b e t w e e n the t w o E F - h a n d s that p r o v i d e the m e c h a n i s m for the vast differences in the conformational r e s p o n s e to Ca -~* b i n d i n g o b s e r v e d across this family of CaBPs. To validate this hypothesis, it will be necessary to identify the specific interactions within the g l o b u l a r d o m a i n that g o v e r n the m a g n i t u d e of the conformational response. To this end, w e are c a r r y i n g out c o m p a r a t i v e analyses of t h r e e - d i m e n s i o n a l structures of Cae+-free and Ca=*-bound states of other EF-hand CaBPs, as well as protein engineering experiments on calbindin D,~ d e s i g n e d to amplify its conformational r e s p o n s e to Ca:* binding.

Materials and Methods Sample preparation The expression of calbindin D,,~ in E. co~i, production of uniformly labeled '~N samples, and purification of the

All experiments were recorded at 300 K on a Bruker AM 600 spectrometer. Scalar correlated experiments (COSY, E-COSY, TOCSY) and NOESY (Macura & Ernst, 1980) spectra in H:O solution were acquired as described (Skelton et al., 1990; Akke et al., 1991; K6rdel et al., 1993). For spectra acquired from : H 2 0 solution, the baselines of the spectra were further improved by sine modulated acquisition in (o~ (Otting et al., 1986). Each NOESY spectrum was acquired with 64 transients for each of 520 t, increments, over spectral widths of 6024 and 12 500 Hz in (0, and (02, respectively The total recycle delay between transients was 2.3 seconds and each 2D spectrum required approximately 20 hours of acquisition time. The mixing times were 20, 40, 60 (x2), 90, 120 and 150 ms in both ~H20 and =H20 solution. The two 60 ms spectra were recorded as the first and last spectrum in each series; comparison of these two spectra permitted the identification of NOE cross-peaks arising from ti~e second form of the protein and provided verification of the absence of effects on relative cross-peak intensities for the signals of the intact protein. A NOESY experiment with a jump-return observe pulse (Plateau & Gu6ron, 1982) was also recorded for the tHe0 sample. The spectrum was acquired with 96 transients for each of 520 t, increments, a mixing time of 160 ms, and the same spectral widths as the other 'H NOESY spectra. A 2D '~N-'H HSQC-NOESY (Bax et al., 1990; Norwood et al., 1990) was recorded for the sample with uniformly ~N-labeled protein with 64 transients for each of 600 t, increments over a '~N spectral width of 1250 Hz. Standard 'H TOCSY and NOESY experiments were also acquired using the '~N-enriched sample to m e a s u r e 3]N-H~ coupling constants (Montelione et al., 1989), as described by K6rdel et al. (1993).

Data processing and analysis The spectra were processed and analyzed using a version of the FTNMR program (Hare Researcl~ Inc., WA) modified by Dr Mark Rance. Spectra were processed with weak Lorentzian-to-Gaussian window functions as described (Akke et al., 1991). The two 60 ms spectra were added to improve the signal-to-noise ratio and all peaks with 6((o2) > 6((0~) were picked manually within FTNMR and the volume measured. The volumes of these peaks were less subject to uncertainty caused by ridges of t~ noise emanating from the sharper resonances of aliphatic protons; this effect was most significant for cross-peaks involving amide protons. In cases of severe peak overlap in (0,, the corresponding cross-peaks with 6((o=)< 6((,)t)

458

were inspected to determine the resonance positions more accurately but the volumes were nonetheless estimated from the ~(c02) > 6(0~,) cross-peaks. Cross-peak assignment was facilitated by the program GENBOUND (Dr Matthew Kalnik, unpublished). Generally, if a peak was unambiguously identified in both the 2H20 and the 'H20 spectra, the peak volume in the ~H20 spectrum was utilized to generate a distance constraint.

Distance constraints Previous studies have suggested that to set the u p p e r bounds on distance constraints by cross-peak integration in a single NOESY experiment, a mixing time of 60 ms represents a suitable compromise between better cross-peak S / N at longer mixing times and minimization of spin diffusion effects at shorter mixing times (K6rdel et al., 1993). This choice was verified by measurement of a series of NOESY spectra and careful examination of the NOE build-up curves. A multiple point calibration scheme (K6rdel et al., 1993) was used to determine NOE volume cut-offs in the 60 ms HaO NOESY spectrum corresponding to u p p e r bound values of 2.8, 3.3, 4.2 and 5.1 A. For overlapped cross-peaks, volumes were estimated, then the constraint distance was increased by one category or set to the maximum distance of 5.1 A, depending on the severity of the overlap. Initial calibration of the 2H20 spectrum was made by normalizing to the volumes of 20 peaks that were well resolved in both 2H20 and 'H20 spectra; all peaks in the -~H~Ospectrum were assigned an u p p e r bound using the normalized cut-offs. Once initial structures were calculated, the scaling factor for the 2H20 data was more accurately determined using CORMA (vide infra). The longer mixing time JR-NOESY and '~N-'H HSQC-NOESY spectra were calibrated independently but with three distance categories corresponding to u p p e r bounds of 3.0, 4.0 and 5.5 A. Cross-peak volumes derived from all spectra involving methyl groups, degenerate methylene resonances and protons of rapidly flipping aromatic side-chains were divided by the number of protons contributing to the resonance before categorization by the above schemes (Yip, 1990). During DG calculations, all lower bounds were set to twice the van der Waals radius of a hydrogen atom, except for those atom pairs that could potentially form hydrogen bonds, for which the lower bound was set to 1.8 A; during rMD the lower bound was set by the force field.

Dihedral angle constraints and stereospecific assignments 3JNf I-II~ scalar coupling constants were measured in COSY spectra acquired and processed with high digital resolution. The coupling constants were evaluated from cross-sections through the cross-peaks analyzed by the method of Kim & Prestegard (1990). 3JH~.,I~ coupling constants were measured in the E-COSY spectrum (Griesinger et al., 1987). 3JN.,, coupling constants were measured in the TOCSY and NOESY spectra recorded using the ~SN labeled sample (Montelione et al., 1989). The homonuclear coupling constants plus intra-residue and sequential NOEs were used as input for the grid search program HABAS (Gfintert et al., 1989) to generate qb, ~ and X. dihedral angle constraints and make stereospecific assignments. The minimum ranges of ~ and ~ used as constraints were 50 ° and 100 °, respectively. All X, ranges were limited to the classical rotamer positions +60 °. Stereospecific assignments and X~ constraints for the CI~H2 groups were used only for residues in which both ~JH~-H,

Structure of Apo Calbindin Dgk

coupling constants were less than 5.0 Hz or where the difference in value of the two coupling constants were greater than 5.0 Hz. The stereospecific assignments of 12 methylene groups were obtained from HABAS calculations and six from the values of 3JH~.Hll and 3JN.ttll coupling constants (Table 7). The methyl groups of Va168 and Val70 were assigned stereospecifically on the basis of 3JH~.H,, intraresidue and sequential NOEs (Zuiderweg et al., 1985). Both C~H-C"H cross-peaks of Pro20 and Pro37 were well resolved allowing stereospecific assignment (Kline et al., 1989). For asparagine and glutamine side-chain amide protons, the proton exhibiting stronger intraresidue NOEs was stereospecifically assigned to Nail 2 and N ' H 2 position, respectively (Kline et al., 1989). Gin67 N~H 2 could not be assigned due to cross-peak overlap. A number of additional stereospecific assignments were made by a statistical analysis of families of structures, as described by K6rdel et al. (1993). The stereospecific assignments used in the final round of structure calculations are listed in Table 3.

Distance geometry calculations The first stage of each round of structure calculations was carried out using the program DISGEO (Havel & Wfithrich, 1984), rewritten in the C computer language by Dr Mike Christiansen for use on either a Convex C-240 or a Cray YMP computer. Pseudoatom corrections and "improper" dihedral angle constraints were applied as described by K6rdel et al. (1993). Initial calculations consisted of 30 to 40 trial embeds, 50 to 75% of which were successful. Following 1000 steps of minimization with the constraints, the structures were refined in four dimensions for a further 1000 steps. For the final round of calculations, the input consisted of 884 NOE distance constraints, 23 qb, 39 ~band 23 XJ dihedral angle constraints (Table 8). Note that dihedral angle constraints spanning regions greater then 180 ° were not enforced in the DG calculations. Constraints enforcing 16 hydrogen bonds, selected according to the criteria discussed by K6rdel et al. (1993), were also used in the calculation: 180 trial embeds were performed, of which 117 were successful.

Restrained molecular dynamics The DG structures were further refined by minimization and annealing within the SANDER module of the AMBER 4.0 suite of programs (Pearlman et al., 1991a,b). Distance restraints were introduced as half-parabolic penalties for proton-proton separations greater than the chosen u p p e r bound (Table 8), with a force constant of 32 kcal mol-' A -~ (K6rdel et al., 1993). Lower bounds were not specifically enforced but effectively imposed by the van der Waals potential of the all-atom force field (Weiner et al., 1986). Non-stereospecifically assigned prochiral groups were treated by 1 / r ~averaging (Clore et al., 1986), which resulted in 994 useful distance restraints, 110 more than in the distance geometry calculations. 45 qb, 52 ~ and 25 X, dihedral angle restraints were included. Hydrogen-bond restraints were not enforced in the rMD calculations. The calculations were performed in vacuo with a distance-dependent dielectric and net charges of glutamate, aspartate and lysine side-chains reduced from +1 to +0.2 to compensate for the absence of explicit water molecules. Peptide bonds were restrained in the trans conformation within 10 ° of planarity by use of specific restraints with a force constant of 50 k c a l m o l ~ r a d - L Absolute stereochemistries of all chiral groups were enforced by explicit

Structure of Ape Calbindin Dgk

459

Table 7 Stereospecific a s s i g n m e n t s of p r o c h i r a l g r o u p s of a p o c a l b i n d i n D,k Residue Lysl Ser2 Pro3 Lys7 Phel0 Glul I Tyr13 Asp19 Asn21 Ser24 Leu28 Leu31 Glu35 Phe36 Pro37 C'~H2 Ser38 Asp47 Leu53 Glu60 Ser62 Phe63 Phe66 Asn21 Gin22 Gln33 Asn56 Gln75

Chemical shift (p.p.m.)" Coupling constants (Hz) ~ H ~2 H r~ 3/H,.,2 ~J,,~,l~3 ~JNH~2 ~JMI,3 1.74 1.85 11.3 2.8 4.45 4.09 -4.5 -1.5 2.07 2.50 1.54 1.81 6.7 10.9 2.96 3.34 5.7 11.4 -0.7 -1.5 2.19 2.07 10.7 4.6 2.52 2.74 11.8 2.1 -2.3 -6.1 2.57 2.83 4.8 10.9 -0.8 -2.2 2.81 2.98 10.6 4.9 -1.5 -4.6 4.38 4.10 4.0 4.0 1.43 2.27 5.6 11.3 -0.8 -0.8 1.07 1.65 -0.8 -1.5 1.62 1.54 3.7 12.1 -1.5 -0.7 2.97 3.29 11.0 4.3 -0.8 -5.3 3.25 3.54 3.94 4.04 3.8 4.8 -1.1 -4.5 2.62 2.67 11.0 4.4 -1.5 -4.5 1.68 1.46 10.4 4.7 1.82 2.00 10.3 4.0 4.51 4.09 4.0 4.0 2.32 2.57 6.0 12.4 -1.5 -1.5 3.23 3.36 6.0 11.5 -1.5 H6., i Ho.,2 7.05 7.98 6.59 7.35 6.81 7.23 6.96 7.72 6.80 7.49 CY.,~H~ C,.,~2H~ 9.9 -1.5 0.86 0.99 0.44 0.08 9.0 0.92 1.05 0.48 0.43 0.97 1.03 0.50 0.91 0.70 0.79 referenced to the H20 signal at 4.75 p.p.m, and are generally accurate to

Method' H J M H H S J H J H H H/J J H M H J S H H H H M M M M M

Val68 M Val70 M Leu6 S Leu23 S Leu30 S Leu31 S Leu53 S Chemical shifts are +0.01 p.p.m. The experimental uncertainty in these values is +0.5 Hz. ' H, derived with the program HABAS; M, based on intra-residue NOEs to Pro C~H, Asn C"H2, and Gln C'H2,or the method of Zuiderweg et al. (1985) for Val methyl groups; J, from the values of 3],~2, ~]~,a, ~]N,2, and %1~; S, by reference to rMD structures.

r e s t r a i n t s to p r e v e n t inversion d u r i n g the a n n e a l i n g cycle. A 10 ps a n n e a l i n g protocol identical w i t h that u s e d b y K6rdel et al. (1993) w a s u s e d that i n v o l v e d initial equilibration, rapid h e a t i n g to 1200 K, a n d slow cooling to OK. For the initial r o u n d s of calculations, the 10-20 s t r u c t u r e s w i t h l o w e s t DISGEO p e n a l t y f u n c t i o n s w e r e subjected to this a n n e a l i n g protocol a n d a n a l y z e d . For the final calculation, 69 of the 117 DISGEO s t r u c t u r e s w e r e refined in this m a n n e r .

Constraint checking with CORMA calculations C O R M A calculations (Keepers & James, 1984; Borgias & James, 1990) w e r e p e r f o r m e d on a SPARC-2 workstation. Eight s e p a r a t e calculations w e r e p e r f o r m e d at each stage of the refinement, w i t h p r o t o n p o s i t i o n s d e r i v e d f r o m each of the eight r M D s t r u c t u r e s w i t h lowest violation e n e r g y T h e m e a n theoretical N O E v o l u m e for each p r o t o n - p r o t o n pair (I~), was t h e n calculated. P r o g r a m s w e r e w r i t t e n in the AWK l a n g u a g e ( A h o et al., 1988) to s e m i - a u t o m a t e the

Table 8 N u m b e r s of c o n s t r a i n t s u s e d in the v a r i o u s stages of the final r o u n d of calculation Upper bound and NOE volume constraints" Dihedral angle (deg.) 60 ms 'H20 60 ms 2H20 160 ms JR 150 ms '~N Total qb ~ 21 DG 481 261 47 63 884 b 23 39 23 rMD 567 305 68 54 994 45 52 25 Backcalc. 254 (421) 237 (193) 68 54 613 (614) 45 52 25 "The direct NOE volume constraints are given in parentheses. b Includes 32 constraints from 16 hydrogen bonds.

Structure of Apo Calbindin Dgk

460

comparison between tile theoretically observable NOEbased constraints and the constraints actually used to generate tile structures. To facilitate comparison, experimental NOE volumes (1,.) were converted to an absolute scale (the intensity of a single proton at 0 ms mixing time) by multiplying by the average ratio of I,/1, for all well-resolved cross-peaks involving only achiral or stereospecifically assigned protons. Separate CORMA calculations were performed and separate scaling factors determined for tile 'HaO and 2H.,O data sets. Occupancies were set to 0.0 for all NH and OH protons in the :H20 CORMA calculation and for hydroxyl and amine protons in tile 'H_,O calculation. Calculated NOE volumes less than 0.0003 were not considered in the analysis; for reference, this is approximately 2c~. of the cross-peak volume of the intense sequential HN-H 'x NOE observed in the helices.

Angular order parameters (S ''"~) were calculated for ~, qJ, X,, dihedral angles (Hyberts et al., 1992); an order parameter of unity indicates that the angle is the same in all structures, whereas an order parameter of zero indicates that there is no preferred orientation about a particular torsion angle. The ensemble of apo calbindin D,k structures were compared with the ensemble of structures determined in solution for the (Ca2")2 state (K6rdel et al., 1993; accession number 2BCB). A mean (Ca-")2 structure was determined in a similar fashion to that described above for the apo state. A variety of methods were used to overlay the apo and (Ca -~)2 ensembles and these are described in more detail below. The coordinates of the 33 final rMD structures and the input constraints have been deposited in the Brookhaven Protein Data Bank under accession number 1CLB

NOE-driven refinement The final structures were further refined by 250 additional steps of energy minimization with the force field supplemented by a NOE-driven energy term using tlle Remarc routine of the SANDER module (Yip & Case, 1989; Pearlman et al., 1991b). In all, 613 NOE volume restraints were used from cross-peaks that were well enough resolved and sufficiently free of spectral artifacts to allow accurate integration and for which corresponding protons were stereospecifically assigned. This left 614 standard distance restraints and all 122 dihedral angle restraints (Table 8). At every step of the minimization, tile complete relaxation matrix was calculated for the structure and NOE volume restraints determined from a parabolic energy term of the form k x f x (I,.-/,,)2 where l, is the NOE volume calculated on the basis of tlle current structure, 1, is tile observed NOE cross-peak volume, k = 2 kcal tool-' ~2, and.f is a scale factor. To prevent intense intra-residue NOEs from dominating the refinement, f was set to 1/I,, and limited to a maximum value of 40 and a minimum value of 5. Analysis of the solution structures A geometric mean structure was generated after r.m.s.d. best fitting the N, C" and C atoms of helical residues onto the structure with lowest violation energy. The r.m.s.d. values for variot, s parts of the structure were then calculated with respect to this mean structure, as described below. Prior to superpositioning, the atom names of symmetry-related pairs of atoms (Tyr and Phe C " / C '*-"and C'~/C'2; glutamate O'~/O'"; aspartate O " ' / O "2) were adjusted to follow IUPAC-IUB nomenclature using the computer program ROTFIX (Garry Gippert, unpublished). The overlaid structures were visually inspected using MMS (Dr Stephen Dempsey; unpublished), INSIGHT-ll (Biosym Inc., San Diego) or MIDAS (Ferrin et al., 1988) running on Silicon Graphics workstations. Hydrogen bonds were identified using the ANALYSIS module of AMBER 4.0 (Pearlman et al., 1991a). A particular hydrogen bond was deemed to be present if the X . . . H distance was less than 2.5 A and the X . . . H-X angle was larger than 135 ° . Solvent-accessible surface areas were calculated using the program DMS (Computer Graphics Laboratory, University of California, San Francisco). A probe radius of 1.4 A was employed to calculate tile total accessible surface of each of tile 33 final apo structures as well as the mean and standard deviation of the contribution from each residue. Corresponding calculations were also made for tile 33 final solution structures of the (Ca 2")., state (K6rdel et al., 1993).

Acknowledgements We gratefully acknowledge Professor Sture Fors6n for his long-standing collaboration and encouragement, Dr Mark Rance for continued assistance with experimental techniques, Eva Thulin for protein expression and purification, Dr Matthew Kalnik for providing the program GENBOUND, Dr Mikael Akke, Dr David Case, Dr Tom James and Garry Gippert for help and inumerable discussions on various aspects of the structure calculations, and a referee for helpful comments. This work was supported by the National Institutes of Health (GM 40120) and in part by a fellowship to W.J.C from the American Cancer Society (JFRA-294). W.J.C. thanks Genentech, lnc. for their cooperation during the final stages of this research and preparation of the manuscript.

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Structure of Apo Calbindin Dgk

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Structure of Apo Calbindin Dgk

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Edited by B. Honig (Received 9 November 1994; accepted 13 February 1995)