J. Mol. Biol. (1992) 228, 1177-1192

Calmodulin Rajagopal

Structure Refined at l-7 A Resolution

Chattopadhyayal, William E. Meadorl, and Florante A. Quiocho1*3



R. Means2T

‘Howard Hughes Medical Institute 2Department of Cell Biology 3Department of Biochemistry College of Medicine, Houston, TX 77030, U.S.A.

(Received 25 June 1992; accepted 11 August


We have determined and refined the crystal structure of a recombinant calmodulin at 1.7 w resolution. The structure was determined by molecular replacement, using the 2.2 a published native bovine brain structure as the starting model. The final crystallographic R-factor, using 14,469 reflections in the 10.0 to 1.7 a range with structure factors exceeding 0.50, is 0.216. Bond lengths and bond angle distances have root-mean-square deviations from ideal values of 0.009 a and 0.032 8, respectively. The final model consists of 1279 nonthree Asp118 hydrogen atoms, including four calcium ions, 1130 protein atoms, including side-chain atoms in double conformation, 139 water molecules and one ethanol molecule. The electron densities for residues 1 to 4 and 148 of calmodulin are poorly defined, and not included in our model, except for main-chain atoms of residue 4. The calmodulin structure from our crystals is very similar to the earlier 2.2 a structure described by Babu and coworkers with a root-mean-square deviation of 0.36 A. Calmodulin remains a dumb-bell-shaped molecule, with similar lobes and connected by a central a-helix. Each lobe contains three a-helices and two Ca ‘+ binding EF hand loops, with a short antiparallel P-sheet between adjacent EF hand loops and one non-EF hand loop. There are some differences in the structure of the central helix. The crystal packing is extensively studied, and facile crystal growth along the z-axis of the triclinic crystals is explained. Herein, we describe hydrogen bonding in the various secondary structure elements and hydration of calmodulin.

Keywords: 1.7 a calmodulin

inclusion of calcium (Babu et al., 1988; this study; Taylor et al., 1992), the structure of the N-terminal domain of a related protein, troponin C, containing no calcium, has been determined (Herzberg & James, 1985a; Satyshur et al., 1988). The calmodulin structure in t#his study is the highest resolution study among calmodulin structures available, and will be described here, emphastructure of calmodulin not aspects sizing elaborated upon by Babu et al. (1988). The structure of a peptide-calmodulin complex has been solved in our laboratory recently at 2.4 13 (1 a = 0.1 nm) resolution (Meador et al., 1992) using a 20 residue peptide and different crystal growth conditions compared with this study. The solution structure of another calmodulin-target peptide complex has been determined by multidimensional n.m.r.1 (Ikura et al., 1992).

1. Introduction Calmodulin performs a large number of functions in living cells. It belongs to a family of calcium binding proteins that contain EF hands or helixloop-helix motifs (Kretsinger & Nockolds, 1973; Herzberg & James, 19853; Satyshur et al., 1988; Szebenyi & Moffat, 1986; Babu et aZ., 1988). It modulates the activities of several other proteins, including protein kinases, NAD kinase, phosphodiesterase, calcium pumps, as well as motility proteins (Manalan & Klee, 1984; Means, 1988; O’Neil & 1990). A highly conserved protein DeGrado, throughout evolution, it contains only 148 amino acid residues. Binding of calcium gives calmodulin its functional configuration which, in turn, recognizes a variety of protein targets. Although the calmodulin has not been crystallized without the t Present address: Department Duke University U.S.A.






$ Abbreviations

of Pharmacology, Durham, EC 27710,

resonance; MPD, mean-square.


used: n.m.r., nuclear 2-methyl-2,4-pentanediol;

magnetic r.m.s.,


0 1992 Academic Press Limited

R. Chattopadhyaya


2. Materials

and Methods

(a) Calmodulin pu$;fication, crystallizakion and X-ray data collection Calmodulin was expressed in a bacterial system et al., 1987) using the common vertebrate (Putkey sequence. As expected in a recombinant protein, Lysl15 was not trimethylated as found in higher organisms. The synthetic 23 amino acid residue peptide contained the calmodulin-binding domain of smMLCK (Olson et al., 1990). Crystals were grown by the hanging-drop method using small nat,ive calmodulin crystals as seeds. The mother liquor contained a molar ratio of 1.25 : 1 of peptide to calmodulin, a protein concentration of 12 mg/ml, in 50 mM-NaOAc (pH 50), 50 mml-MgCl z , 5 mM-CaCl,, 17.5 y’ (v/v) 2-methyl-2,4-pentanediol (MPD), 7.5 y0 (v/v) ethanol. The reservoir contained the same salt concentrations, but 35% MPD and 15% ethanol. Long rod-like crystals were grown from the smaller seeds in about 1 month. A crystal of approximate size 91 mm x 0.1 mm x 95 mm was broken off from a larger piece and chosen for data collection. The triclinic crystal of cell dimensions t=25.14A, a=3@17A: 1,=53.60& a = 9362", p = 97.30”, y = 8917” was used to collect’ all the data. The cell dimensions are close to those described by Babu et al. (1988), but not identica,l. The cell dimensions of our crystals are slightly larger (1.5%) in the a-direction and in c-direction (0.6%) and slightly smaller (63%) in the b-direction. The space group is Pl for both crysta.ls. Using a San Diego Multiwire two-dete&or system mounted on a Rigaku RU-200 (CuKa, 40 kV, 110 mA), 46,091 reflections were measured over 4 days and processed to yield 15,417 unique refiections. Decay and absorption corrections were applied.

(b) AVoEec&ar replacement Though the cell dimensions of our crystal are very close to those previously reported, when the unrotated 2.2 A of model of Babu et al. (1988) was tested, an R-factor 0.613 resulted using 15 to 2.5 A data. Using co-ordinates for Babu’s model (entry PDBSCLN.ENT> Brookhaven Protein Data Bank) and Crowther’s rotation function (Crowther, 1972) for 8.0 to 40 A data, and 5” steps for the 3 Eulerian angles, the rotation function peak was determined using the program MERLOT (Fitzgerald, 1988; Chattopadhyaya & Chakrabarti, 1988). There were no other peaks having greater than 60% of the rotation function value of the correct peak. A 2nd narrow rotation function search using 1” step size for p gave a more accurate set of Euler angles. As the crystal is triclinic; the origin is arbitrary, and there is only 1 calmodulin molecule per unit cell. Therefore, the translation problem does not exist. The center of the molecule was placed at (65; 65; 65) and rigid body refinement initiat,ed. Several rounds of RMINIM (Ward et al., 1975) within MERLOT, successively using 15 to 5 A. 15 to 4 8, 15 t.o 3 A and 15 to 2.5 A data reduced the R-factor to a final value of 6329 using an overall temperature factor of 25 AZ and all data to 2.5 A. An overall temperature factor was found to be 24.8 A2 from a Wilson plot in the PROTEIN package for the dataset. The net rotation for converting the 2.2 A model to ours is about 5”.

(c) Rejhement PROLSQ (Hendrickson 1985) refinement was started with 1126 protein atoms and 4 calcium ions after applying

et al.

the final rotation found from the final round of RMISLM. The R-factor fell from 0.328 to 0280 using 10.0 to 24 ?I data in 16 cycles using positional refinement only and an overall temperature factor of 25 X2. Using 190 to 2.0 a data, the R-factor reduced from O-308 to 0298 using positional refinement, and then to 0254 when t’hermal refinement was allowed. Roughly 30 water molecules were introduced in each round of refinement by inspection of FO- F, and 2F,--F, maps, and moved to new locations if the B-factors exceeded 60 8’. Finally, after adding a total of 125 water molecules in 5 stages, the R-factor reduced to 0225 using 190 to 1.7 x data. The protein structure remained close to ideality during the entire refinement. Water occupancies were set at unity for all water molecules and not, refined. At, this stage, examination of regions of the unit cell not occupied by the calmodulin molecule revealed that (I) there was a small crescent-shaped electron density in the hydrophobic pocket in the N-terminal lobe of eaimodulin occupying the pocket, a.nd (2) there was a stronger density inside a similar pocket in the C-terminal lobe of calmodulin. This density, however; seemed to extend outside the pocket. in 2 directions, being continuous over a large distance, although demented in sections. It was concluded that the density in the N-terminal pocket could be explained adequa,tely by an ethanol molecule, arising from the crystallization mother liquor. The 2nd density, in the C-terminal lobe, however, was quit’e long when contoured at O-60. Initially, it was surmised that this was due t,o the peptide analog that binds with an association constant in the nanomolar range. Finally, the existence of the peptide was rejected as the R-factor did not improve significantly upon its inclusion and the 2F,-F, map remained discontinuous at !a level. Water molecules best fitted the densities. After the refinement was eomplet’ed: 13 residues from t.he N-terminal end of calmodulin were delet,ed. and an




of the L)istances (A) Bond Angle Dihedral Pianes (A) (:hiral volumes (&k3) contacts (A) Single Multiple Hydrogen bonded Torsion angles (deg.) Planar Staggered Orthonormal Thermal (delta B, AZ) &fain bonded Main angle Side honded Side angle Structure factor weights? A-term B-term

in the ,final rou,z&

refinement Target sigma

Final value

omo 0.040 0060 @020 @I.50

0009 0032 0040 0.007


0.500 0500 0500

0199 0304 0281

30 150 2@0

I.6 214 26%

40 5.0 5.0 &O

2.0 25 3.0 42

5% -306

65 - 38~0

t The final values are fitted values of ?‘, - F,



at 1.7 A

Figure 1. A backbone model for the ealmodulin molecule is shown in stereo. Calcium ions are shown as triple concentric circles. The calmodulin N terminus is at the top right and the C terminus at the top right section of the bottom lobe. Side-chains and water molecules are excluded from this Fig. Residue numbers are shown for every 10th residue starting with residue 5. In this view, the EF hand loops for the N domain are at, top center and those for the C domain at the bottom left. The non-EF hand loops are also clearly seen at the left side of the N domain and at the right side of the C domain. The 7 cc-helices comprising residues 5 to 19 (I), 29 to 37 (II), 45 to 55 (III), 65 to 92 (IV), 102 to 111 (V), 118 to 128 (VI) and 138 to 146 (VII) can also be clearly seen from this backbone model.

OMIT map calculated. The section came back in the OMIT map including density for side-chains. The X-ray data and final co-ordinates have been deposited at the Brookhaven Protein Data Bank (accession number PDBlCLL ENT).

3. Results (a) Crystallographic


Each unique reflection was measured roughly three times on average. Cumulative completeness is 89.9% up to 1.70 A, and 78% of the theoretical reflections have intensities greater than two sigma. Of the 15,417 unique reflections measured, 14,469 reflections were used in the refinement; this represents roughly 2.2 times as many reflections used in the refinement compared with that of Babu et al.

(1988). The final R-factor is 0216 for 14:469 reflections in the 10.0 to 1.7 A range having structure factors greater than O&r, and 0225 including all 15:417 reflections. The Luzzati root-mean-square error gives a,n upper limit of 0.12 A in the 1.7 A structure. The deviations from ideality of the model from

standard geometry as well as target sigmas used in the PROLSQ refinement are listed in Table 1. Ideality of the model is extremely good. There were 50 distances (bond, angle and dihedral) in the structure that deviated by more than twice the target sigmas in Table 1. There is only one short hydrogen bond (2.1 A) within the calmodulin molecule between Glu82 and Arg86 side-chains, and this was noted in the 2.2 A structure described by Babu et al. (1988). Other hydrogen bonds less than 2.5 A generally involve water molecules as acceptors. The short 2.0 A intermolecular contact between the carbonyl oxygen of Va191 and NH1 of Arg126 from a neighbor observed earlier by Babu et al. (1988) was absent in our structure. We used less stringent, thermal refinement parameters compared with Babu et al. (1988) as they improved the R-factor and removed positive or negative difference densities at the atoms. The final values of the thermal parameters are only slightly higher than those described by Babu et al. (1988). (b) Calmodulin


and hydration

A global view of our structure is given in Figure 1. The molecular structure is shown in more detail in


R. Chattopadhyaya

et ai.

Figure 2. The K-terminal domain is shown including all side-chains and most of the water molecules (circle) in the region in stereo. Polar side-chains interacting with water molecules are labeled. (Ball and stick.) Part of the cent~ral a-helix can be seen close to the viewer, going left and down towards the c-domain, which is not shown in this Fig. Residues 8 to 72 are displayed in this Fig. The view is close to that in Fig. 1, but not identical. Calcium ions 1 and 2 are shown as triple concentric circles.

Figures 2 to 4, including side-chains and water molecules. Figure 2 shows the N-terminal lobe, Figure 3 the central cc-helix, and Figure 4 the C-terminal lobe. The overall conformation of the calmodulin molecule is very close to the dumb-bellshaped model described by Babu et al. (1988) with seven a-helices comprising residues 5 to 19 (I), 29 to 37 (II), 45 to 5.5 (III), 65 to 92 (IV), 102 to 111 (V), 118 to 128 (VI) and 138 to 146 (VII). By running the program DSSP of Kabsch & Sander (1983), Babu et al. (1988) considered residue 38 as part of helix II and residue 147 as part of helix VII. n’evertheless, even long hydrogen bonds along with shorter ones are described in Table 2 (helix IV) and Table 4 (all others). Two thirds of all residues in calmodulin are in a-helical conformation. As Figure 2 shows~ some of the intervening residues, 20 to 31, provide their side-chains as ligands for the calcium ion, and similarly some other residues in the range 56 t,o 67 also provide ligands for the other calcium ion in the N-terminal lobe of calmodulin. Six ligands for the calcium ions in the N-terminal lobe come from these three side-chains (Asp, Asn, Glu) and one from the main-chain carbonyl group of a Thr residue (Thr26 or Thr62). A seventh ligand is always provided by a water molecule in all four calcium co-ordination sites examined. Calcium co-ordination in the EF hand loops has been adequately described in the 2.2 A study (Babu et al., 1988) and is briefly described here in Table 5. The remaining intervening region between two a-helical regions is in residues 38 to 45, where a hairpin turn can be seen at the left back portion in Figure 2. The turn is made possible by Gly40. This turn was not specifically mentioned, although displayed in the earlier 2.2 A study (Babu et al., 1988). Water molecules are seen to envelop the surface lobe. Polar side-chains interacting of the N-terminal

with water molecules jGin8, Glull, Lysl3, Lys62, Lys30, Arg37, Asn42. Glu46, Gln49, AspSO, Asn53, 611~54) have been labeled in Figure 2. Some neutral side-chains (e.g. Thr29, Ser38) are also hydrated, but these are not identified. Some side-chains (like Thr26, Thr28, Asp56, Asp58, Asn60, Thr62) that provide ligands for the calcium ions are also hydrated. Other water molecules are close to mainchain carbonyl groups. There is a cluster of water molecules between Lys13 and Asp24 (see top right side of Fig. 2). Some of these are not contacting protein atoms directly. There is a string of seven water molecules on top of the N-terminal lobe running between Glu54 and Lys30. A small fra,ction of water molecules lie close to ma’in-chain nitrogen atoms (e.g. Asn42). Only two water molecules hang close to the hydrophobic pocket. One of these hydrogen bonds to the main-chain earbonyl group of Met51 and the other is a second shell water near it. The ethanol molecule found in the hydrophobic pocket has been omitted in Figure 2, but displayed in Figure 5. The central helix (IV), comprising residues 65 to 92, is shown in Figure 3. Ramachandran angles for the central helix are listed in Table 2, and these values may be compared with those from the earlier 2.2 A structure given in parentheses. The helix as seen in Figure 3 is quite straight. The main-chain hydrogen bonding pattern of an a-helix is maint,ained throughout. Of the two ends of the central helix, some residues form part of the two hydrophobic pockets; these have been marked by residue numbers in Figure 3 (Phe68, Met71, Met72 at the top; Phe92 at the bottom). Polar residues are also marked in Figure 2, and it is seen that some of the wa,ter molecules are close to main-chain carbonyl groups (Glu67, Ala73, Glu83), while other water 0 (Arg’i4, molecules are close to polar side-chains Lys75, Lys77, Asp78, Asp80, Glu82, Arg86, Glu87).


Structure at I.7 A

Table 2 Ramachandran angles and H-bond lengths for central a-helix Residue Asp64 Phe65 Pro66 Glu67 Phe68 Leu69 Thr70 Met71 Met72 Ala73 Arg74 Lys75 Met76 Lys77 Asp78 Thr79 Asp80 Ser81 Glu82 Glu83 Glu84 Ile85 Arg86 Glu87 Ala88 Phe89 Arg90 Va191 Phe92 Mean Std. Dev.

Phi (“)

Psi (“)

-57( -55) -55(-53) -66(-68) -61(-55j -57(-52) -57(-74) - 72( - 64) -57(-55) -Bl(-54) -5O(-39) -66(-67) -56(-78) -4O(-67)

--52(-55) -41(-40) -44(-50) -45( -55) -56( -45) - 39( - 34) -41(-52) -44( -47) -63(-73) -37(-54) -41(-18) -61(-26) -46( -47) -37(-15) -27(-11) -35( -09) -6O( -63) -47(-27) -22( -33) -46(-46) -38(-35) -34(-21) -53(-61) -24(-44) - 39( - 46) -35(-34) -36(-39) -43( - 48)

-68(-68j -67(-99) -75(-95) -62(-94)

-47( -51) -71(-81) -74(-68) -66(-66) -66(-69) - 64( - 74) -61(-48) -72(-52) -63(-68) -69(-77) -72(-63) -626 (-66.2) 8.2 (132)

- 42.0 (-403) 10.1 (152)

H-bond length 310 2.85 3.13 319 2.80 2.91 326 2.83 290 3.05 3.34.t 298 303t 3.217 3.19 340t 329t 313 2.917 304 308 300 323 324 3.35 2.87 4371 6.83 686 308 617

These are details of bonds for helix IV. Angles within brackets are those from the 2.2A calmodulin studied by Babu et al. (1988). Hydrogen bond distances between main-chain carbonyl oxygen atoms of residue rz with main-chain nitrogen atoms of residue n + 4 in A. Calculation of mean and standard deviation in H-bond length calculation included 26 entries in the Table, leaving out the last 3. t These distances indicate an additional H-bond donor/ acceptor within 4 A of the carbonyl oxygen. en of Arg90 is closer to the nitrogen of Asp93,

The hydroxyl groups of Thr70 and Ser81 are also hydrated. There appear to be two water molecules close to the sulfur atoms of Met71 and Met72 sidechains, and one of them has a second shell water. The side-chains of Glu82 and Arg86 clearly form salt-linking and hydrogen bonding interactions, as noted earlier (Babu et al., 1988). This is a particularly strong salt-link, oppositely charged atoms being only 2.1 A from each other. The only other salt-link in the central helix, albeit a weak one (4.3 A), is between Lys75 and Asp78 (Fig. 3). There are no additional intermolecular salt-bridges for the central helix in our crystals. As Figure 4 shows, residues 93 to 104 and 129 to


140 provide the ligands for the two calcium ions in the C-terminal domain by virtue of their negatively charged side-chains or main-chain carbonyl atoms. Negatively charged side-chains are provided by Asp, Glu and Asn, while Tyr99 and Gln135 provide main-chain carbonyl groups as ligands. Again, like the EF hand loops in the N-terminal lobe, the seventh ligand to the calcium is always a water molecule. The other intervening residues, 112 to 117, between the two last a-helices in the molecule also make a turn, facilitated by Gly113. A total of 14 hydrophobic residues defining the hydroplhobic pocket are all numbered (same as those in Table 5 of Babu et al. (1988)). The polar residues are highlighted in Figure 4. It is seen that some of the residues that form ligands to calcium ions also have water molecules close to them (Asp93, Asp95 Asn97, Glu104, Aslp129, Asn137) on another side. Other polar residues on the protein surface (Lys94, Arg106, His107, Asnlll, Glu114, Lysll5, Aspl18, Glu120, Asp122, Glu139) have water molecules in the vicinity of their sidechains. Some Thr residues on the surface also form hydrogen bonds to water molecules (ThrllO, Thrl17, Thr146). Secondary water molecule:s are also seen, although they are few in number. There are several water molecules close to main-(chain carbonyl groups and few near main-chain NH groups. Both lobes of calmodulin seem to contain about equal numbers of bound water molecules. Solvent occupancies were not refined and the final criterion for determining high-temperature factor waters was the appearance of positive F,, - F, at 2a level or 2F,- F, at la level, when the water wabsnot included in the structure factor calculation. Therefore, some waters with B-values as high as 70 A2 were retained. This is justified as many protein side-chains in this crystal have B-values in the fifties and sixties. We have used twice as Imany (139/69 = 2.01) water molecules compared with the structure of Babu et al. (1988), but the higher number is justified on two grounds; first,, our crystals diffract to 1.7 A and, secondly, calmodulin is a dumb-bell-shaped protein with a lot of exlposed surface area relative to globular proteins, so a#high amount of hydration per unit mass is to be expected. Of the 139 water molecules, 88 are within 3.5 A of possible hydrogen bond donors or a,cceptors; 15 more water molecules are within 401 A of possible donors or acceptor atoms; 25 .water molecules are not within 40 A of hydrogen bond donors or acceptors, but have other water molecules within the same range; finally, 11 are more than 40 A away from possible hydrogen bond donors or acceptors from protein molecules and other water molecules. See also section (f), below, for water molecules bridging calmodulin-calmodulin interactions in the crystals. Hydrogen bonding in calmodulin has been reported in detail in Table 4 and classified according to the secondary structure. All the residues in the protein were checked for hydrogen bonds made by

R. Chattopadhyaya


et al.

Figure 3. The central cl-helix is shown in stereo, and its polar residues labeled. (Ball and stick.) The view is diRerent from Figs 2 and 4. The view is chosen so that the helix is seen vertical in this Fig. Some of the main-chain-side-chain and side-chain-side-chain bonds can also be seen for residues belonging to this helix (described in Table 4, sections D and E).

the main-chain amide groups. It turns out that, except for eight residues for which the hydrogen bonds were donated to water molecules and one residue (Glu45) that possesses no partner closer than 3.5 8, all other residues had their main-chain amide participating in hydrogen bonds to acceptors within the protein molecule. Other kinds of hydrogen bonds in the protein have also been tabulated.

compared with a density corresponding to that of a water molecule. The ethanol molecule is within van der Waals’ distance to two Phe side-chains (19, 68), as well as with Met71, Leu32 and Va155 side-chains. The 2F,- E‘, map for this section is displayed in Figure 5.

(d) Hydrophobic (c) Hydrophobic

pocket in the N-terminal


There appears to be a crescent-shaped density inside the amino-terminal hydrophobic pocket that is easily explained by a molecule of ethanol, as ethanol was used for crystallization. This density is too close to some hydrophobic side-chains to be explained as a water molecule, and too large

pocket in the C-terminal


We are surprised to find quite a few ordered water molecules in this hydrophobic pocket, instead of the ethanol molecule just described for the N-terminal one. Initially, this led to hopes of seeing the peptide used in the crystallization, but the idea was discarded later as the inclusion of a peptide did not lower the R-factor significant,ly and the density remained quite weak and discontinuous in t(he



at 1.7 d


Figure 4. The C-terminal domain,is shown including all side-chains and most of the water molecules in the region in stereo. The view is the same as in Fig. 2. Just as in Fig. 2, the polar side-chains interacting with water molecules are labeled. Part of the central helix is seen at the back of this Figure, away from the viewer, at the right side of the drawing. Calcium ions 3 and 4 are shown as concentric circles. A total of 7 water molecules, which are found in the C-terminal hydrophobic pocket, are shown in black. These are not near any polar groups on the surface. Some more water molecules like these exist in the model, but are not shown here. They are close to Glu84 and Ala88 (not marked) in this Figure.

ETigure 5. levt 31. There wit h Met71, the crescent 3-o A apart

The hydrophobic pocket in the N-terminal lobe is shown in detail with superposed 2F,-F, density at lo seems to be a molecule of ethanol in this pocket, interacting with two Phe side-chains (19 and 68), as well as Leu32 and Va155 side-chains. A corresponding ethanol molecule in the C-terminal pocket is not inferred as shape of the density was not seen there; instead, it is explained as 2 water molecules (see Fig. 4) more th ,an from each other. The program CHAIN (Sack, 1988) was used for density fitting and molecular modeling :.

R. Chattopadhyaya


et ai.

Ezigure 6. Superposition of the central a-helices in the present (1.7 8) study and in the previous 2.2 ?I study. The ator ns fro1n the present study are color-coded according to atom type, and the 2.2 A structure is entirely red. Differences in ma in-chain atom positions are as great as @75 a near the middle of the helix, in the region 72 to 82. The differences neaax the ends of the central helix are minimal.

2F,-F, map contoured at la. Therefore, the existence of the peptide could not be proven well and we have modeled the strongest portions of it as 11 ordered water molecules.

of the 1.7 A calmodulin structure with the previous 2.2 A structure

(e) Comparison

The refined structure obtained from our study is very close to the earlier structure, wit,h the rootmean-square deviation between the two sets of 1130 atoms, including residues 5 to 147, and the four calcium ions being O-36 d; the root-mean-square deviation between main-chain atoms only is O-25 8, and between side-chain atoms only, it is @44 8; that for four calcium ions is @13 a. An a-carbon superposition of the two structures will not be very informative due to their near-identity. Plots showing real space fit for main-chain atoms and side-chain atoms as a function of the residue number were made (not shown). For the mainchain, the r.m.s. differences are generally less than @25 8, except for 29 residues. Considering the mainchain atoms only, the largest differences are mainly in the central a-helix, Asp131 and Ala147. For the

side-chain comparison, several residues had r.in.s. differences more than @5 .t% (Thr5, Lysl3, Thr29, Xet36, Gln41, Asn42, Ile52, Thr70, Lys75, Met)76, Lys77, Thr79, Glu83, Va191, Arg106, Lysl15, Va1136, Glul39, Met145 and Ala147). The r.m.s. differences were first calculated for all atoms common to the two structures; for main-chain as well as side-chain atoms, then simple averages calculated within each residue. The side-chain of Lysl15, the trimethyla,ted residue from mammalian sources, did show a 0.81 w r.m.s. difference between the two structures, and the main-chain atoms of Lysl15 showed an 1p.m.s. difference of @22 8. Average group temperature factors for both main-chain and side-chain atoms in the two strueLures are compared in Figure 7. Side-chain entries are missing at glycine residues. Elements of the secondary structure at the residues are also shown. Entries for our structure are joined by a zig-zag line, whereas those for the earlier study are not connected and shown as circles. For main-chain atoms, it is seen that (1) EF hand loop regions possess i;he lowest temperature factors, but the fourth EF hand loop between residues 129 to 137 has higher thermal motion compared with the other three in both structures; (2) for t,he two terminal sr-helices (I and VII), temperature factors generally








,Ct,EF,CL turn


60 40 20 0


I I I I I I I I I I I I I I I 20








Residue number Figure 7. Temperature factors compared for side-chain and main-chain atoms between the 2 studies; circles represent the 2.2 a study and points connected by straight lines represent the 1.7 A study. Secondary structure is indicated at the top and a temperature factor comparison of the 2 structures subdivided into these 13 components given in the text. Main-chain averages are at the top and side-chain averages at the bottom. Breaks in the bottom graph indicate glycine residues.

increase towards the termini; (3) among the remaining five a-helices, the central a-helix shows the highest temperature factors; (4) the two turns between a-helices (1 in each lobe) possess temperature factors higher than the EF hand loops and the four low-value B-factor a-helices, although less compared to the central and terminal helices. As to differences in the values between the two structures, (1) the first a-helix (I) in our study has about the same average value, but more variation about the average; (2) the first EF hand loop has a significantly lower temperature factor (approx. 5 A2) in our study compared with the earlier one; (3) for the second a-helix (II) and the first non-EF turn, values are more or less the same in both structures; (4) for the third helix (III), our structure has a slightly lower temperature factor on average; (5) for the second EF hand loop, the average value is more or less the same in both structures; (6) for the central a-helix (IV), our structure has a significantly lower temperature factor compared with the earlier study, although the values are still high; (7) for the second non-EF turn, values are more or less the same in both structures; (8) for the fifth helix (V), we find consistently lower temperature factors, as well as for the adjoining EF hand loop; (9) the same behavior is noticed for the sixth helix (VI) and the fourth EF hand loop, in fact, for all residues in the

at l-7 A


range 98 to 144, our structure has consistenly lower B-values; (10) for the C-terminal helix (VII), our structure has lower temperature factors, especially for inner residues. The lower panel of Figure 7 shows a comparison of the side-chain average temperature factors, and no general comments could be made as for the mainchain. In general, temperature factors are quite close in both structures. The only general comment that could be made is that in the central a-helix, side-chains for residues 75 to 86 have consistently lower temperature factors in our structure compared with the earlier study. Also, among .the 14 hydrophobic side-chains defining the pocket in the carboxy-terminal lobe of calmodulin (listed in Table 5 of Babu et al. (1988)), 12 have lower temperature factors in our study, the two exceptions being Va1121 and Met145. In contrast to small differences in the two lobes, larger differences emerged in the central helix between the two structures. This is seen from Figure 6. Differences in main-chain atom positions are as much as 0.75 A near the middle of the central helix, in the region 72 to 82. The two ends of the central helix can be juxtaposed like other patrts of the calmodulin molecule. Some of the polar sidechains like Lys75 and Lys77 have new positions. Thr79 side-chain conformation has also changed. A perusal of Table 2, where Ramachandran angles for the central helix are listed, shows that mean values of Ramachandran angles for t’he 28 residues (65 to 92) constituting the central o:-helix are close to the mean values of phi, psi of (- 63, -40) found by Baker & Hubbard (1984) from a survey of a-helices in known protein structures. It is interesting to note that for the angle phi, this study has an average of -62.6, extremely close ho the mean value of -63, while for the angle psi, the earlier structure of Babu and coworkers has a mean value of -403, close to the value of -40 of Baker & Hubbard (1984). However, standard deviations from the mean angles are smaller for our structure for both angles. Values of corresponding angles for the 2.2 A study are taken from Table 3 of Babu et al. (1988), and given in brackets in Table 2 here. Thus, the smaller spread of Ramachandran angles; from the mean values in our structure shows a better a-helix in our structure. Also, the mean hydrogen bond length from the 26 entries gives a mean hydrogen bond distance of 3.08 A, and Baker & Hubbard (1984) report a mean value of 2.99 A for this distance in a-helices in their survey. A rather large hydrogen bond length of 3.8 A was reported for Thr79 in the earlier study (Babu et al., 1988), but it is considerably shortened here. Some of the longer hydrogen bond lengths are marked by a dagger in Table 2, indicating the presence of additional hydrogen bond donors/acceptors within 4 A of the carbonyl oxygen atom, normally provided by sidechains. For many residues having long hydrogen bonds at the center of the helix, the Ramachandran angles have moved closer to the mean values (-63, -40) compared with the 2.2 A study, with. large


R. Chattopadhyaya et al

(b) Figure 8. (a) Calmoduln-calmodulin interactions in the crystal, between (0, 0,O) and (0, 1, I); these are between non-EF hand loops from 2 molecules with several intermolecular hydrogen bonds. The structure of these non-EF hand loops in the crystal may have been determined by these bonds and hence alternative conformations may be present in solutions for these loops. (b) Calmodulin-calmodulin interactions in the crystal, between (0, 0,O) and (0, 0, 1). Mole&e (0, 0,O) is shown on the left and (0, 0, 1) to the right. The C-terminal lobe of (0, 0,O) is partially shown, and similarly the N-terminal lobe of (0, 0, 1) is partially shown, including only those that interact with the neighbor. Water molecules bridging the 2 are included.


Structure at 1.7 d

Table 3 Summary of translation-related intermolecular


contacts in crystal

Number of Translation in crystal for neighbor

Protein-protein contacts

Protein-water contacts

Water-water contacts




















010 001

0 9

0 4

1 0


changes in residues 79 to 81. A more ideal central a-helix is consistent with reduced temperature factors in that region of the molecule as noted earlier.

(f) Calmodulin-calmodulin

interactions in

the crystal There are several different types of calmodulincalmodulin interactions in these crystals, and Figure 8(a) and (b) is used to illustrate those with the largest number of protein-protein contacts. As seen from Table 3, there are some proteinwater contacts between molecules (1, 0,O) and (0, 0, 0), forming a cluster between the N termini of adjacent molecules in the x-direction. Some of these water molecules hydrogen bond to carbonyl and NH groups in the main-chain. Others are near Asn42 side-chain from the upper molecule (0, 0,O) or Asp64 side-chain of the lower molecule (1, 0, 0). Atoms from the C-terminal lobe of calmodulin (0, 0,O) are close to the two N-terminal EF hand loops as shown in this diagram. The C-terminal EF hand loops of molecule (0, 0,O) are seen to interact with the N terminus of molecule (1, 0, - 1). Some more interactions in this region are likely, but they could not be discerned as the first three N-terminal residues are disordered. Tyr99 from molecule (0, 0,O) is seen to interact with Glu7 side-chain from molecule (1, 0, - 1); Glu139 side-chain from (0, 0, 0) contacts the main-chain NH of Glu6 from (1, 0, - 1). Molecule (0, 0,O) is linked with (1, - 1, - 1) via a cluster of water molecules. There is only one protein-protein contact, that between an Asp118

Comments Protein-water contacts between adjacent N-terminal lobes along an axis 3rd and 4th EF hands of molecule (0, 0,O) with N terminus of (l,O, -1) Some contacts through water molecules; H107 CEl of (0, 0,O) near N 60 0 of (1, - 1,O) contacts, only 1 proteimExtensive protein-water protein contact; residues 102 to 126 of (0, 0,O) near 1st and 2nd EF hands of (1, -1, -1) Contacts between turn in residues 37 to 42 in (0, 0,O) and turn in residues 114 to 120 in (0, 1, 1) (Fig. 8(a)) Only 1 water-water contact Largest number of calmodulin-calmodulin contacts; N terminus and C terminus of (0, 0,O) contact pil terminus and C terminus of (0, 0, 1) through many salt-bridges, hydrogen bonds and water-mediated contacts (Fig. 8(b))

NH from (0, 0,O) and an Asp24 ODl from (1, - 1, - l), which is also liganded to one ‘of the calcium ions. There is also a salt-link between Asp22 side-chain from (1, - 1, - 1) with an Arg106 sidechain from (0, 0,O). Some of the water molecules are bonding to main-chain atoms, others near polar side-chains, and the rest secondary water molecules near other water molecules. In Figure 8(a), residues 37 to 42 of (0, 0,O) and 114 to 120 of (0, 1, 1) are seen to contact each other. It will be remembered that region 37 to 42 includes the end of the second a-helix and the beginning of the first non-EF turn in the N-terminal lobe, while region 114 to 120 constitutes the second nlon-EF turn and the start of the sixth a-helix in the C-terminal lobe. These turns were defined as sections of the molecule between the m-helices that do not participate in the EF hand loops. It was noted previously that these regions have higher temperature factors compared with the domain ol-helices and the EF hand loops. There appear to be several hydrogen bonds in this region, namely, (1) between Arg37 from (0, 0,O) and Glu114: from (0, 1, 1); (2) between Asn42 from (O,O, 0) and Glu120 from (0, 1, 1); (3) between Asn42 from (0, 0,O) and Lysl15 main-chain carbonyl from (0, 1, 1). There are some protein-water contacts (not shown in Fig. 8(a)). calmodulin-calm’odulin The most extensive contacts are in the z-direction involving molecules (0: 0, 0) and (0, 0, 1) (Fig. 8(b)). In these contacts the first a-helix of molecule (O,O, 0) seems to interact with residues in the third a-helix of molecule (O,O, l), as well as the Arg74 side-chain from the central helix of molecule (0, 0, 1). In the region shown in the bottom part of Figure 8(b), the


R. Chattopadhyaya

et al.

Figure 9. Environment around Asp129 in the 4th EF hand loop of calmodulin. Asp129 has been emphasized by drawing thicker bonds for it compared with other residues shown. The calcium ion represented by concentric circles is at the center, with the 7 ligands described in Table 5 for calcium ion number 4 surrounding it. Residues 129 to 135 and 140 are displayed together with the calcium ion and a water molecule. Asp129 062 atom makes a hydrogen bond with the main-chain nitrogen atom of Gly134 as shown.

C-terminal part of the central a-helix, and sidechains of Lys94 and HislO?, all from molecule (0, 0, 0), interact with side-chains from the sixth a-helix of molecule (O,O, 1). Some water molecules are found between the seventh a-helix of (0, 0, 1) and the central a-helix of (0, 0, 0), contributing to stabilize the interaction between the two molecules. A water molecule also bridges Lys94 of (0, 0,O) and Glu119 main-chain carbonyl of (0, 0, 1). There is a salt-link between His107 from (0, 0,O) and Glu119 side-chain from (0, 0, 1). There are two other hydrogen bonds between Arg90 and Va191 main-chain carbonyl oxygen atoms from (0, 0,O) and Arg126 side-chain from (O,O, 1). Arg90 side-chain from (O,O, 0) forms a long hydrogen bond with Glu127 main-chain carbonyl oxygen from (0, 0, 1). In the upper portion of the Figure, Ala10 CB and Glu14 side-chain of (0, 0, 0) both point towards Arg74 sidechain from (0, 0, 1). Ser17 side-chain forms a hydrogen bond with a water molecule in this region, but this is now shown. In addition, Lys21 side-chain and its main-chain carbonyl oxygen from (0, 0,O) interact with Asp50 and Asn53 side-chains from (0, 0, l), respectively. Thus, in summary, there appear to be multiple hydrogen bonds and salt-links that hold together neighboring molecules in the z-direction.

(g) Some new structural


Residue 129, which appears at the beginning of the fourth EF hand loop in calmodulin, was judged to be Asn in the earlier study (Babu et al., 1988). We have modeled it as aspartic acid. The 129 side-chain provides a ligand for the calcium in the fourth EF hand loop; however, the otker oxygen atom in the side-chain is found to be 2.85 d away from a mainchain nitrogen from Gly134. In addition, tempera.ture factors of Asp129 QEl and OE2 atoms are nearly equal, but if the residue is assumed to be Asn the nitrogen had a lower temperature factor compared with the oxygen atom in its side-chain. Revised sequencing is consistent with Asp, not Asn (Marshak et al., 1984). The fourth EF hand loop is shown in Figure 9, emphasizing the residue 129. Thus, all four residues at the beginning of the four EF hand loops in calmodulin are now Asp. The previous study (Babu et al., 1988) did not deal with a double conformation for a surface polar side-chain, Aspll8. The two conformations were found to be equally important in our study. Assignment of 50% occupancy to each eonformaLion yielded nearly equal temperature factors for the two alternate conformations. Both conformations make hydrbgen bonds to the side-chains of


Structure at I.7 d


Table 4

Table 4 (continued)

Hydrogen bonding in calmodulin Donor atom

Acceptor atom

Bond length

Donor atom rj

Acceptor atom

B. Short antiparallel

Helix II Leu32 N Gly33 N Thr34 N Va135 N Met36 N Arg37 N Sm.38 N Helix III Glu47 N Leu48 N Gln49 N Asp50 N Met51 N 111~52N Asn53 N Glu54 N Va155 N

Leu4 0 Thr5 0 Thr5 0 Glu6 0 Glu7 0 Gln8 0 Gln8 0 Ile9 0 Ala10 0 Glull 0 Phel2 0 Lys13 0 Glu14 0 Ala15 0 Phel6 0 Thr28 Thr29 Lys30 Glu31 Leu32 Gly33 Thr34

289 258 2.54 322 331 3.06 322 2.81 306 321 3.02 2.92 356 317 289

0 0 0 0 0 0 0

301 2.91 303 2.99 307 3.03 319

-66 -66 -58 -52 -68 -68 -74 -66 -63 -62 -57 -56 -62 -62 -84

-56 -58 -66 -63 - 5.8 -60 -76




A. Alpha he&es Helix I Gln8 N Gln8 N He9 N Ala10 N Glull N Glull N Phel2 N Lys13 N Glu14 N Ala15 N Phel6 N Serl7 N Leul8 N LeulS N Phel9 N

Bond length

-52 -52 -39 -44 -32 -32 -35 -38 -43 -44 -47 -29 -34 -34 -38 -40 -48 -34 -48 -46 -29 -13

beta sheets

N-domain Ile63 N Ile27 N Wat237 Phe65 N Watl81 Thr29 N

Ile27 0 Ile63 0 Gly25 0 Wat237 Gly61 0 Watl81

2.94 2.63 2.75 270 302 2.76

- 109 -110

C-domain Va1136 N IlelOO N Wat227 Tyr138 N Wat224 Ala102 N

IlelOO 0 Va1136 0 Gly98 0 Wat227 Gly134 0 Wat244

2.97 284 287 305 2.92 3.04

-113 -90

C. Two non-EF hand turns Gly40 N Met36 0 Gly40 N Arg37 0 Gln41 N Met36 0 Gly113 N Met109 0 Gly113 N ThrllO 0 Glul14 N Met109 0

341 326 327 319 2.98 2.86

Donor atom

Acceptor atom

132 125




-38 124 117





88 88 -120 93 93 -112

16 16 124 10 10 109

Bond llength

D. Four EF hand loops Thr44 Thr44 Glu45 Ala46 Glu47 Leu48 Gln49 Asp50 Met51

0 0 0 0 0 0 0 0 0

3.11 2.90 2.82 3.16 3.13 323 2.84 308 373

-69 -62 -63 -62 -61 -68 -51 -58 - 100

-37 -40 -41 -39 -46 -41 -46 -31 -20

Helix IV? Helix V LeulO6 N ArglOG N His107 N Va1108 N Met109 N ThrllO N Asnlll N Leull2 N

SerlOl Ala102 Ala103 Glu104 Leu105 ArglO6 His107 Va1108

0 0 0 0 0 0 0 0

294 281 3.08 3.10 3.04 3.16 323 299

-58 -62 -69 -61 -63 -67 -63 -89

-42 -38 -49 -33 -37 -39 -22 -5

Helix VI Glu120 N Va1121 N Asp122 N Glu123.N Met124 N Ile125 N Arg126 N Glu127 N Ala128 N

Thrl17 Thrl17 Asp118 Glu119 Glu120 Va1121 Asp122 Glu123 Met124

0 0 0 0 0 0 0 0 0

3.24 310 285 3.13 326 2.98 2.93 3.18 3.71

-65 -68 -60 -66 -66 -65 -51 -57 -104

-40 -40 -39 -37 -43 -50 -44 -31 -10

Helix VII Phe141 N Va1142 N Gln143 N Met144 N Met145 N Thrl46 N Ala147 N

Am137 Tyr138 Glu139 Glu140 Phe141 Va1142 Gln143

0 0 0 0 0 0 0

295 2.79 309 308 2.79 2.79 3.07

-67 -65 -56 -71 -66 -60

-44 -38 -44 -34 -68 -58

Asp20 N Lys21 N Asp22 N Gly23 N Asp24 N Gly25 N Thr26 N Thr28 N Lys30 N Glu31 N Asp56 N Ala57 N Asp58 N Gly59 N Asn60 N Gly61 N Asp64 N Glu67 N Asp93 N Lys94 N Asp95 N Gly96 N Am97 N Gly98 N SerlOl N Ala102 N Glu104 N Asp129 N Ile130 N Asp131 N Gly132 N Asp133 N 01~134 N Am137 N Glu140 N

Phel6 0 Glu31 0” Glu31 0” Asp22 0 Asp22 0” Asp20 Od’ Asp24 0” Glu31 0” Thr29 Oy’ Thr28 0 Ile52 0 Glu67 Oe2 Asp58 0” Asp56 06’ Asn60 06i Asp56 Odl Glu67 0” Asp64 0” Phe89 0 Glu104 OeZ Asp93 0 Asp93 0 Asp93 OS’ Asp93 Odl Glu104 0”’ SerlOl Oy SerlOl 0 Ile125 0 Glul40 0”’ Asp131 Odz Asp129 061 Asp129 06’ Asp129 Odz Glul40 0” Am137 061

392 330 332 302 336 2911 324 331 292 317 276 34:3 298 350 298 280 3.213 304 287 343 2.99 290 3.09 288 3:!3 347 336 249 295 391 3.118 3lL9 205 273 2.70

E. Main-chain-side-chain Ser17 Oy Lys21 Ns Gly25 N Lys30 N

bonds Lys13 0 Leul8 0 Asp20 Od’ Thr29 Oy’

2.77 2’73 2!11 2.!12

R. Chattopadhyaya et al. -


Table 4 (continued) Bond length

Donor atom

Acceptor atom

Ser38 Oy Thr44 N Gly61 N Met71 N Gly98 N Gly134 N Glu140 N Ala147 N

Va135 0 Glu47 OE’ Asp56 06’ Thr70 Oy’ Asp93 0” Asp129 062 Asn137 Ndz Thrl46 Oy’

276 2.90 280 281 2.88 2.86 271 2.69

I?. Side-chain-side-chain

bonds Asp24 Odl Thr29 Ori Glu86 oel Glu82 Oe’

276 2.49 214 2.35

Thr26 Lys30 Arg86 Tyr138


Distances in Angstroms and Ramachandran angles in degrees for donor residue. t Helix IV or the central helix has been described in Table 3 and illustrated in Fig. 3.

Arg106 directly for one conformation and via a water molecule for the second conformation. Main-chain hydrogen bonding distances have been summarized in Table 4. In the first section of this Table, the a-helices have been described, except for the central one, which has been described in Table 2. It was found that the two short antiparallel

Table 5 Calcium ligands in calmodulin Calcium

Ligand atom


Asp20 OD2 Asp22 ODl Asp24 OD2 Thr26 0 61~31 OEl Glu31 OE2 Water 0 Asp56 OD2 Asp58 OD2 Am60 ODl Thr60 0 Glu67 OEl 61~67 OE2 Water 0 Asp93 OD2 Asp95 OD2 Asn97 ODl Tyr99 0 Glu104 OEl GlulO4 OE2 Water 0 AspI ODI Asp131 OD2 Asp133 OD2 Gln135 0 Glu140 OEl 61~140 OE2 Water 0




Distance 2.42 250 2.40 2.35 2.37 2.42 2.62 213 2.30 237 2.29 255 2.40 231 2.23 2.53 235 2.20 2.44 259 2.60 2.31 245 2.30 230 2.57 2.55 2.63

(2.34) (242) (261) (2.46) (2.28) (2.38) (2.42) (221) (2.48) (246) (2.17) (2.49) (2.31) (2.37) (2.14) (2.22) (2.39) (206) (2.32) (2.76) (2.01) (2.17) (256) (2.07) (2.38) (2.57) (2.32) (2.63)

The bracked numbers are from the 2.2 A study of Babu et al. (1988).

beta sheets were held together by only two hydrogen bonds each, unless water bridges were also included. These have been included as they were found to exist at both ends of the two short beta stretches. For example, Water 237 is a hydrogen bond acceptor from Phe65 amide and is a donor to Gly25 earboxyl oxygen, thus bridging the two strands. A total of four such water molecules, two for each domain, were found. There are two stretches of turns in calmodulin that are not EF band turns: these are also stabilized by three hydrogen bonds each in the course of their turns. These non-EF turns have not been described before and consist of residues 39 to 44 (intervening ol-helices II and III) and residues 112 to 117 (intervening a-helices V and VI). Both of these turns have a Gly residue, which participates in two of the three hydrogen bonds through its main-chain amide group. There are some additional hydrogen bonds that have not been listed in Table 4 and they are basieally of three categories: (I) hydrogen bonds of protein atoms with water molecules; (2) hydrogen bonds at the ends of the various ol-helices, often participating in 310 helices; (3) intermolecular hydrogen bonds in the crystal. The calcium co-ordination is no doubt the important feature of the EF hand loops. But there are hydrogen bonds within the protein in the EF hand loops in addition to the bonds to the calcium ion. These are pointed out in Table 4. It was found that all main-chain amide groups donated a hydrogen bond except for Glu45. The calcium ligands are described in Table 5 and the small differences bet,ween our model and the earlier model of Babu et al. (1988) are noted. Our model seems to be free from the three shortest calcium ligand distances as reported in the earlier model. Also, for each calcium, the Glu OEl and OE2 atoms have become more equidistant from the calcium in our model compared with the earlier model.

. Discussion In view of tight association predicted for this and similar peptides with calmodulin (O’hieil & DeGrado, 1990), it is frustrating not to find a sufficiently ordered peptide in our crystal. Most of these peptides have dissociation constants in the nanomolar range. However, the conditions of crystallization might disfavor the formation of such a tight as partly hydrophobic solvents like complex, ethanol. and MPD may associate with calmodulin, competing with the peptide. Indeed, a molecule of ethanol is predicted from our study to lie in the N-terminal hydrophobic pocket. Another factor that might have disfavored crystallization of the complex is that the conditions we have used for crystallization are those established for calmodulin alone, a condition that allowed peptide-free cai-



modulin molecules to selectively form the crystal over peptide-bound calmodulin molecules. Calmodulin hydration has been described in detail above. Ordered water molecules were found in the vicinity of potential donors and acceptors from both main-chain and side-chains. Polar side-chains are hydrated as found in other protein structures. We also noticed the recurrence of the side-chains forming ligands to calcium having a water molecule on the other side. Finally, we had to position water molecules in some unconventional locations; namely, near non-polar side-chain atoms. Such water molecules are, however, close to other water clusters. One such big cluster has been used to replace the putative peptide density in the C-terminal hydrophobic pocket. Similarities and differences in the two structures (i.e. 1.7 a and 22 8) as well as their temperature factors have been discussed. The larger differences between the two structures occur in the region of the central a-helix, with the helix in the present study being more ideal and having shorter hydrogen bonds in some of the residues in the center of the helix. It is possible that due to the differences in crystal packing forces, the central a-helix, which is known to be deformable (Trewhella et al., 1990), took a nonideal structure in the 2.2 ,& study. In the absence of such packing forces, it relaxed to a more ideal geometry in our study. The calmodulin-calmodulin

crystal have been extensively




studied. The crystals

appeared as rods along the z-direction, and this is reasonably explained due to the existence of

multiple hydrogen bonds, salt-links and bridging water molecules between successive molecules in that direction. The water-bridged short beta sheets and the non-EF hand turns have also been characterized. Hydrogen bonds in the EF hand loops in addition to the well-known calcium co-ordination have been enumerated. We thank Dr Nand K. Vyas for helpful discussions and Dr Mark F. A. van Berkum for supervising the calmodulin preparations. This study was suported in part by a Howard Hughes Medical Institute grant to F.A.Q., Glaxo Drug Discovery grant to F.A.Q. and A.R.M., and NIH grant GM33976 to A.R.M.

References Babu, Y. S., Bugg, C. E. & Cook, W. J. (1988). Structure of calmodulin refined at 2.2 A resolution. J. Mol. Biol. 204, 191-204. Baker, E. N. & Hubbard, R. E. (1984). Hydrogen bonding in globular proteins. Progr. Biophys. Mol. Biol. 44, 97-179. Chattopadhyaya, R. & Chakrabarti, P. (1988). Solving DNA structures by MERLOT. Acta Crystallogr. sect. B, 44: 651-657. Replacement Crowther, R. A. (1972). The Molecular Method (Rossman, M. G., ed.), pp. 174-177, Gordon and Breach, New York.

at 1.7 A


Fitzgerald, P. M. (1988). MERLOT: an integrated package of computer programs for the determination of -crystal structures by molecular replacement. J. Appl. Crystallogr. 21, 273-278. Hendrickson, W. A. (1985). Stereochemically rest)rained refinement of macromolecular structures. Methods Enzymol. 115, 252-270. Herzberg, 0. & James, M. N. G. (198%). Structure of the calcium regulatory muscle protein troponin-C at 28 A resolution. Nature (London), 313, 653-659. Herzberg, 0. & James, M. N. G. (1985b). Common structural framework of the two Ca2+/Mg2+ binding loops binding prloteins. of troponin C and other Ca” Biochemistry,

24, 5298-5302.

Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, C. B. & Bax, A. (1992). Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science, 256, 632-638. Kabsch, W. & Sander, C. (1983). Secondary structure and solvent exposure of proteins from atomic coordinates as given by the Brookhaven Protein Data Bank. Biopolymers,

22, 2577-2637.

Kretsinger, R. H. & Nockolds, C. E. (1973). Carp muscle calcium-binding protein. J. Biol. Chem. 248, 33133326. Manalan, A. S. & Klee, C. B. (1984). Calmodulin. .4dvan. Cyclic




Res. 18, 227-278.

Marshak, D. R., Clarke, M., Roberts, D. M. C Watterson, D. M. (1984). Structural and functional properties of calmodulin from the eukaryotie microorganism Dictyostelium disccoideum. Biochemistry, 23, 28912899. Meador, W. R., Means, A. R. & Quiocho, F. A. (1992). Target recognition by calmodulin - 24 A structure of a calmodulin-peptide complex. Science, 257, 12511255. Means, A. R. (1988). Molecular mechanisms of action of calmodulin. Recent Progr. Horm. Res. 44, 223-286. Olson, N. J., Pearson, R. B., Needleman, D. S., Hurwitz, M. Y., Kemp, B. E. & Means, A. R. (1990). Regulatory and structural motifs of chicken gizzard myosin light chain kinase. Proc. Nat. Acad!. Sci., U.S.A.

87, 2284-2288.

O’Neil, K. T. & DeGrado, W. F. (1990). How calmodulin binds its targets: sequence independent recognition of amphiphilic a-helices. Trends Biochem. Sci, 15, 59-64. Putkey, J. A., Donnelly, P. V. & Means, A. R. (1987). Bacterial expression vectors for calmodulin. Methods Enzymol. 139, 303-317. Sack, J. (1988). CHAIN - a crystallographic modelling program. J. Mol. Graph. 6, 224-225. 0 Baylor College of Medicine. Satyshur, K., Rao, S. T., Pyzaalska, D., Drendel, W., Greaser, M. & Sundaralingam, M. (1988). Refined structure of chicken skeletal muscle troponin C in the two-calcium state at 2 A resolution. J. Biol. Chem. 263, 1628-1647. Szebenyi, D. M. E. & Moffat, K. (1986). The refined structure of vitamin D-dependent calcium-binding protein from bovine intestine. J. Biol. Chem. 261, 8761-8777. Taylor, D. A., Sack, J. S., Maune, J. F., Beckingh,sm, K. & Quiocho, F. A. (1992). Structure of a recombinant calmodulin of Drosophila melanogaster refined at 2.2 A resolution. J. Biol. Chem. 266, 21375521380. Trewhalla, J., Blumenthal, D. K., Rokop, S. E. & Seeger, P. A. (1990). Small-angle scattering studies show distinct conformations of calmodulin in its complexes


R. Chattopadhyaya

with two peptides based on the regulatory domain of the catalytic subunit of phosphoryl kinase. Biochemistry, 29, 9316-9324. Ward, K. B., Wishner, B. C., Lattman, E. E. & Love,


et al. W. E. (1975). Structure of deoxyhemoglobin A crystals grown from poioyethylene glycol solutions. J. Mol. Biol. 98, 161-177.

by B. W. Matthews

Calmodulin structure refined at 1.7 A resolution.

We have determined and refined the crystal structure of a recombinant calmodulin at 1.7 A resolution. The structure was determined by molecular replac...
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