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1 Department of Biological Chemistry and Molecular Pharmacology and 2 Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, USA
3 Department of NMR-Based Structural Biology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany
Reprint requests to: James J. Chou, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; e-mail: james_chou{at}hms.harvard.edu; fax: (617) 432-2921.
(RECEIVED April 19, 2005; FINAL REVISION June 7, 2005; ACCEPTED June 21, 2005)
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Abstract |
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, a protein of previously unknown structure that is critical for physiological relaxation of vascular smooth muscle. This approach can be extended to solve coiled-coil structures with higher order assemblies. Keywords: coiled coil; residual dipolar coupling; NMR; cGMP-dependent protein kinase I
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051528905.
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Introduction |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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helices. They are ubiquitous in nature, as they are found in >5% of open reading frames (ORFs) in eukaryotic genomes (Newman et al. 2000). The length of coiled coils vary from protein to protein, with the longest being several hundred residues and the shortest being around 30 residues (Rose and Meier 2004). Very long coiled coils such as those found in intermediate filaments and myosin domains usually have structural and mechanical functions, whereas shorter coiled-coil domains often facilitate specific protein–protein oligomerizations in signaling and regulatory pathways (Lupas et al. 1991). The primary sequences of coiled coils consist of a characteristic repeating heptad of amino acids, denoted (abcdefg)n, in which the a and d positions are predominantly hydrophobic, and the e and g positions are typically charged or polar. Assembly into oligomers results from hydrophobic residues packing together such that knobs pack into holes (Crick 1953; Harbury et al. 1993). Thus, some structural aspects of coiled coils are conserved and can be predicted from protein primary sequences (Lupas et al. 1991; Berger et al. 1995). The molecular weight of typical coiled coils studied by solutionNMRspectroscopy is small (<15 kDa) because the highly extended helical conformation results in a large rotational diffusion anisotropy (Tirado and de la Torre 1980). Since the amide N-HN bond vectors all roughly point toward the long axis of the molecule, 15N relaxation rates are unusually large and are in some cases comparable to globular proteins of ~45 kDa (Mackay et al. 1996). As a result of the extended helical conformation that is rich in methyl-bearing residues such as leucine, valine, and isoleucine, the methyl resonances are typically poorly resolved. Moreover, since NOEs are qualitative distance restraints, the subtle deviation from the backbone of ideal straight helices can only be detected by a large number of local and intermolecular NOEs quantified with different NOESY mixing times. At the same time, without long-range information, small local inaccuracies in the structure determination can be propagated into large deviations over the length of the coiled coil.
Residual dipolar couplings (RDCs) are exquisitely sensitive to bond vector orientation (Tolman et al. 1995; Tjandra and Bax 1997), and are therefore uniquely suited for characterizing the bending of -helices (Chou et al. 2002) that occurs in coiled-coil structures. It is not possible to pack the constituent monomers into a dimer using only RDCs, since dipolar couplings do not provide translational information. However, assembly can be accomplished using the conserved properties of coiled-coil packing because without exceptions, the a and d positions of the heptad repeat make up the core of the helix–helix interface, and the e and g positions occupy the region between the core and the solvent. This permits the development of an RDC-based approach for rapid and accurate structure determination of coiled-coil dimers that does not involve the time-consuming process of assigning NOE-derived distance restraints. It involves (1) measuring RDCs for proteins marginally aligned in the filamentous phage (Pf1) liquid crystalline medium, (2) identifying the coiled-coil motif by RDC-based molecular replacement analysis, (3) defining the subtle curvature and supercoiling of the constituent helices by RDC refinement, (4) assembling parallel and anti-parallel models of the coiled-coil dimer using knowledge-based intermolecular distance restraints, and (5) deriving the correct monomer–monomer orientation by comparing experimental RDCs with those predicted from the three-dimensional charge distribution and shape of the alternative structural models.
The approach is demonstrated here for the coiled-coil domain of the cGMP-dependent protein kinase I (cGK1), which functions in the nitric oxide (NO)-mediated relaxation of vascular smooth muscle (Lincoln 1994). The state of contraction or relaxation of vascular smooth muscle cells is closely coupled to phosphorylation and dephosphorylation of the regulatory myosin light chain, which is in part regulated by the binding of cGK1 to the myosin-binding subunit (MBS) of the myosin phosphatase (Surks et al. 1999). It has been recently demonstrated that this binding is mediated by the interaction between a coiled-coil-containing region (residues 1–59) of cGK1 and the leucine/isoleucine zipper of MBS (Surks and Mendelsohn 2003). A number of mutagenesis studies have suggested that cGK11–59 forms a coiled-coil dimer of which the zipper region may be responsible for interaction with MBS (Atkinson et al. 1991; Surks and Mendelsohn 2003). Hence, the high-resolution structure of the cGK1 coiled-coil dimer, which is not yet known, is the first step toward the understanding of cGK1-MBS recognition.
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Results and Discussion |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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1–591–59 was made using the programs COILS (Lupas et al. 1991) and PAIRCOIL (Berger et al. 1995). Both programs identified the region to be approximately 38 amino acids in length, commencing at residue 9 and continuing through residue 46 (Fig. 1A
). The structured helical region of cGK11–59 was independently examined by 15N R1 and R2 relaxation rates (Fig. 1B), which indicated that for residues 9–44, the T1/T2 ratio is quite uniform but decreases sharply as the sequence is extended beyond 9–44 at both N- and C-terminal ends. The results from sequence prediction and relaxation measurements indicate that it is valid to treat the backbone of residues 9–44 as a rigid molecule for dipolar coupling refinement.
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1–59 was weakly aligned under a strong magnetic field by adding 15 mg/mL of the filamentous phage Pf1. The observed amide chemical shift resonances of cGK11–59 in Pf1 liquid crystal were essentially identical to that of the isotropic sample without Pf1, indicating that perturbation of the protein by the alignment medium is minimal. Three types of backbone dipolar couplings—1DNH, 1DC'C, and 1DC'N—were measurement for structural analysis.
A preliminary model of the monomeric helix in the cGK1 coiled coil was found by fitting experimental RDCs to segments of coiled-coil -helices in the database, analogous to the molecular fragment replacement method described previously (Delaglio et al. 2000). Here we used the 2.7 Å crystal structure of the 14-heptad repeat coiled-coil domain of cortexillin I from Dictyostelium discoideum (Burkhard et al. 2000). This parallel coiled-coil dimer is 101 residues long and contains a full superhelical turn. The backbone RDCs of residues 9–44 were systematically fit to all 36-residue segments of the crystal structure in a sliding window manner. While the segments are qualitatively similar, their agreements with experimental RDCs varied significantly with the Pearson correlation coefficient (r) ranging from 0.84 to 0.95 and the quality factor (Q), from 0.31 to 0.53 (Fig. 2A), further emphasizing the strength of dipolar couplings in describing the less obvious features of -helices. Remarkably, when sliding position a of the first heptad of cGK11–59 against the crystal structure, both r and Q showed a pronounced wave-like pattern having the exact heptad periodicity that is characteristic of coiled coils. This is because in a supercoiled helix, the helical turns at the packing interface (residues a, d, e, and g) are slightly compressed while those facing outside (residues b, c, and f) are stretched, analogous to the bending of a spring. This leads to periodic disagreement when RDCs are fit to a segment of coiled coil that is out of register. Q is maximum when RDCs for the coiled-coil interior positions a and d are in register with the exterior positions f and b, respectively, of the structural model. Alternatively, Q is minimum when the heptad repeat is in register, or nearly so. Surprisingly, the quality of fit when RDCs for positions a and d of cGK19–44 corresponded to positions g and c of the structural model was as good as or better than when the heptads were fully in register. The source of this degeneracy is unknown, but may be attributable to the specific details of how cGK19–44 assembles, the resolution of the structural model, or the accuracy of the RDC measurements. When performing similar fittings to a regular -helix, no patterns could be recognized (Fig. 2B). The wave-like patterns of dipolar couplings have been used to characterize helical structures (Mesleh et al. 2002). Here we show that the correlation between dipolar couplings and known coiled-coil structures also acquires a wave-like pattern when there is systematic bending of a helix, and the heptad periodicity can be used to directly conclude whether or not the protein under study is a coiled-coil even in the absence of sequence-based coiled-coil predictions.
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) was subjected to dipolar coupling refinement to yield the monomer structure. Refinement was performed using the program XPLOR-NIH (Schwieters 2002) according to the low-temperature simulated annealing (SA) protocol described previously (Chou et al. 2000b, 2001) (see Materials and Methods for details). Values of the magnitude (Da) and rhombicity (R) of the alignment tensor required by the XPLOR protocol were determined from the extended histogram method (EHM) (Bryce and Bax 2004) to be –19.9 Hz (normalized to 1DNH) and 0.13, respectively. An ensemble of 20 structures were calculated with experimental RDCs, consisting of three backbone couplings per residue, as well as side chain 
1 angles derived from 3JNC
and 3JC'C scalar couplings. To examine whether RDC refinement was meaningful, a series of cross-validations were performed. This was achieved by randomly excluding 15% (16 out of 108) of the backbone couplings that are uniformly distributed along the peptide during refinement and using them to validate the refined structure by an SVD fit. The process was repeated 100 times to allow for meaningful statistics. Before refinement, the average Q was 0.21 with standard deviation of 0.06. After refinement, the average Q is improved to 0.15 with standard deviation of 0.06.
Building alternative dimer models
Having an accurate structure of the constituent helices, which exhibits a subtle bending expected for a coiled-coil structure, a preliminary model of the dimer was built by satisfying the characteristic side-chain packing of the residues in the heptad repeats. A representative set of leucine–leucine parallel coiled-coil interactions, which had been obtained at high-resolution using X-ray crystallography, were extracted from the Protein Data Bank (PDB) in order to implement knowledge-based restraints on the intermolecular packing. Table 1 presents the intermolecular distances between the -carbons of leucines and isoleucines at the a and d positions for parallel and anti-parallel dimers. To investigate whether it is possible to use RDCs to distinguish parallel packing from anti-parallel packing in the absence of biochemical data, both parallel and anti-parallel dimers were assembled. The models can be built either manually or using a structure calculation protocol. Here the dimers were built in XPLOR by using the rigid-body dynamics option (Schwieters et al. 2002). In this case, the backbones of individual monomers were treated as rigid body while the side chains were allowed to move. The calculations were done in the presence of experimental 1 restraints and knowledge-based intermolecular distance restraints. For the parallel dimer, the distance between intermolecular C atoms for leucines in heptad position a (L12, L26, and L40) and position d (L22 and L36) were set according to the values listed in Table 1. For the antiparallel dimer, the distance between intermolecular leucine C and isoleucine C1/2 atoms were set to 5.0 Å . At the end of calculations, no distances larger than 0.25 Å were violated and in both cases the supercoiling is left-handed, consistent with all known heptad-based coiledcoil structures. Therefore, without additional considerations, RDC refinement alone cannot be used to distinguish between parallel and anti-parallel packing.
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and 
derived from the model, and side-chain 1 angles derived from three-bond J couplings—are assigned to both monomers. To keep the dimer intact, the same knowledge-based distance restraints used above were enforced. The 20 parallel structures, which were subsequently proven to have the correct packing mode (see below), are shown in Figure 3A. The refinement statistics are given in Table 2.
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, were used. To account for structural uncertainty and dynamics, the alignment tensor was averaged over an ensemble of 20 structures with lowest dipolar energy calculated for parallel and anti-parallel dimers. When using only the shape-based prediction component of the program, we found that the alignment tensors were both axially symmetric, with magnitude of –11.4 ± 0.2 Hz and –11.3 ± 0.2 Hz for the parallel and the anti-parallel structures, respectively. However, by activating both steric and electrostatic prediction components in PALES (Zweckstetter et al. 2004), the experimental RDCs agreed very well with RDCs predicted from the parallel dimers (average correlation coefficient r=0.94) but very poorly with those derived from the anti-parallel structures (r=0.39); this indicated that the parallel dimer is the correct structure. The structure of the parallel dimer is shown in Figure 3, B and C.
The C2 symmetry
In an ideal case of rigid molecule, AXX is parallel to the C2 axis (Tjandra et al. 1996; Prestegard et al. 2000). To examine the degree of correlation between molecular symmetry and the principal axes of the alignment tensor, the 20 structures were fitted to all RDCs of residues 9–44 to determine the Euler angles. Upon rotating the PDB coordinates to the principal axis frame, the molecular symmetry axis is almost, but not exactly, parallel to the AXX of the tensor, with an average angle of 2.7 ± 1.8° between the two axes (Fig. 3C). The small deviation from the ideal scenario could be attributed to errors in the structure and/or RDC measurement.
Conclusions
We have demonstrated that NMR dipolar couplings can be used to identify the coiled-coil heptad repeats, and by employing knowledge-based constraints, determine the high-resolution structure. Since measurement of RDCs requires only sequence-specific assignment of protein backbone chemical shifts, which is relatively straightforward using standard triple-resonance experiments (Kay et al. 1990), this approach for rapid structure determination can be easily automated. The structure of the coiled-coil region of cGK1 determined here is new, and will contribute to the understanding of molecular recognition between cGK1 and MBS. Although the approach is demonstrated for a coiled-coil dimer, it is expected to be applicable for higher order assemblies such as coiled-coil trimers and tetramers. Since the 15N R2 relaxation rate is largely governed by the -helical length, it will not significantly increase upon the addition of helices into higher order oligomers. The ability to quickly and accurately determine the structure of coiled coils will facilitate our understanding of the multitude of protein–protein interactions mediated by this motif.
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Materials and methods |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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1–59 gene encompassing the coiled-coil region was cloned into the pGEX-2T vector and expressed in Escherichia coli with GST fused at the N terminus. The amino acid sequence after thrombin cleavage was GSPGIPGSTT-1SELEEDFAK10ILMLKEERIK20ELEKRLSEKE30EEIQELKRKL40HKCQSVLPVP50STHIGPRTT. The natural sequence of cGK1 starts from Thr 1. The transformed cells were grown in either LB-rich media (for unlabeled protein) or M9-minimal media (for isotope-labeled protein). For isotope labeling, the M9 media were substituted with 15N-labeled ammonium chloride. For triple-resonance experiments, uniformly 15N-, 13C-, 2H-labeled protein was prepared by growing the cells in 90% D2O with 15N-ammonium chloride and 13C-glucose. A total of two NMR samples were used for the present study, each prepared in 260 µL of 95% H2O/5% D2O (pH 7.0), using 280-µL Shigemi microcells. The isotropic sample contained 1 mM protein, 20 mM potassium phosphate, 10 mM NaCl, 1 mM EDTA, 5 mM DTT, and 1 mM azide. The aligned sample used for structure determination contained 15 mg/mL of the filamentous phage Pf1 (Asla Labs), 1 mM protein, 20 mM potassium phosphate, 10 mM NaCl, 1 mM EDTA, 5 mM DTT, and 1 mM Azide.
NMR measurement
All NMR experiments were conducted on Bruker spectrometers equipped with cryogenic probes at 30°C. Sequence-specific backbone assignments of residues 1–55 were accomplished using a standard suite of triple-resonance experiments including the TROSY versions of HNCA, HNCACB, HNCACO, and HNCO (Kay et al. 1990; Salzmann et al. 1999).
Three types of backbone dipolar couplings were measured: 1DNH, 1DC'C, and 1DC'N. The 1H-15N couplings were measured at 600 MHz (1H frequency) by interleaving the 3D HNCO experiments (Ikura et al. 1990), with and without 1H CPD decoupling (Kontaxis et al. 2000), both acquired with 50 msec of mixed-CT 15N evolution. The 13C'-13C couplings were obtained at 500 MHz from the standard 3D HNCO recorded with 100 msec of 13C-coupled 13C' evolution. The one-bond 13C'-N couplings were measured at 600 MHz using the 3D TROSY-HNCO in a quantitative-J manner (Chou et al. 2000a). On the basis of the length of the time domain data and the signal to noise (Kontaxis et al. 2000), the estimated average errors for 1DNH and 1DC'C are ±0.2 Hz and ±0.1 Hz, respectively. For 1DNC' derived from the quantitative J experiment, the average error is estimated to be ±0.2 Hz based on the error analysis previously described (Chou et al. 2000a).
Measurements of 3JC'C and 3JNC coupling constants for determining side-chain 1 rotamers were carried out at 500 MHz for couplings involving 13C' and at 600 MHz for couplings involving 15N, using standard two-dimensional quantitative J methods (Hu and Bax 1997; Hu et al. 1997a,b). Longitudinal (T1) and transverse (T2) 15N relaxation data were recorded at a 1H spectrometer frequency of 500 MHz. The pulse sequences used closely followed that of Farrow et al. (1994) with sensitivity enhancement and coherence selection via pulsed-field gradients.
Data processing and spectra analyses were done in NMRPipe (Delaglio et al. 1995). RDCs were extracted by subtracting isotropic couplings from the aligned couplings. Fitting of the dipolar couplings to structures was done by singular value decomposition (Losonczi et al. 1999), using the program PALES (Zweckstetter and Bax 2000). The goodness of fit was assessed by both Pearson correlation coefficient (r) and the quality factor (Q) (Cornilescu et al. 1998).
Structure calculation protocol
Dipolar coupling refinement of structural models were performed using the program XPLOR-NIH (Schwieters et al. 2002) according to the low-temperature simulated annealing (SA) protocol described previously (Chou et al. 2000b, 2001). Structural restraints were applied only for residues 9–44, the rigid -helical region as indicated by 15N relaxation data, C/C
chemical shifts and RDC values.
In the case of monomer refinement, the starting model was a segment from the crystal structure of cortexillin that best matches the experimental RDCs. Refinement used the three sets of measured RDCs (1DNH, 1DC'C, and 1DC'N) and side-chain 1 angles derived from 3JNC and 3JC'C scalar couplings. Backbone torsion angle restraints were derived from the starting model, all having a flat-well (±10°) harmonic potential with force constant fixed at 200 kcal mol–1 rad–2. Hydrogen bond distance restraints of 2 Å and 3 Å (O—HN and O—N, respectively) were enforced for the rigid helical region, with flat-well (±0.2 Å) harmonic potentials, and a force constant fixed at 20 kcal mol–1 Å–2. For dimer refinement in which knowledge-based intermolecular distance restraints for leucine residues in the a or d heptad position are used, these artificial NOE restraints are also enforced by flat-well (±0.2 Å) harmonic potentials, with the force constant fixed at 30 kcal mol–1 Å–2. For side chains that are more or less "locked" into nearly ideal 1 staggered rotamers on the basis of comparing of 3JC'C and 3JNC couplings, flat-well (±30°) harmonic 1 potentials are applied with force constant fixed at 20 kcal mol–1 rad–2. In addition to the experimental 1 restraints, a weak database-derived "Rama" potential function in XPLOR (Kuszewski et al. 1997) is ramped from 0.02 to 0.2 (dimensionless force constant) for the general treatment of side-chain rotamers. Finally, RDC restraint force constant is ramped from 0.0002 to 0.1 kcal mol–1 Hz–2 (normalized for the 1DNH couplings). Other force constants, commonly used in NMR structure calculation, are k(vdw)=0.002 
4.0 kcal mol–1 Å–2; k(impr)=0.1 1.0 kcal mol–1 degree–2; and k(bond angle)=0.4 1.0 kcal mol–1 degree–2. During the annealing run, the bath is cooled from 300 to 20 K with a temperature step of 10 K, and 6.7 psec of Verlet dynamics at each temperature step, using a time step of 3 fsec.
The coordinates of cGK19–44 have been deposited to the PDB with ID 1ZXA.
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Acknowledgments |
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construct and helpful discussions throughout. This study was supported by an Atorvastatin Research Award from Pfizer to A.C.R., a DFG Emmy Noether-Grant (ZW 71/1-4) to M.Z., and the Smith Family Award for Young Investigators and the PEW Scholarship to J.J.C. |
References |
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 S. Rumpel, R. Lakshmi, S. Becker, and M. Zweckstetter Assignment-free solution NMR method reveals CesT as an unswapped homodimer Protein Sci., November 1, 2008; 17(11): 2015 - 2019. [Abstract] [Full Text] [PDF]
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 J. G. Wise and P. D. Vogel Subunit b-Dimer of the Escherichia coli ATP Synthase Can Form Left-Handed Coiled-Coils Biophys. J., June 15, 2008; 94(12): 5040 - 5052. [Abstract] [Full Text] [PDF]
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 J. R. Burgoyne, M. Madhani, F. Cuello, R. L. Charles, J. P. Brennan, E. Schroder, D. D. Browning, and P. Eaton Cysteine Redox Sensor in PKGIa Enables Oxidant-Induced Activation Science, September 7, 2007; 317(5843): 1393 - 1397. [Abstract] [Full Text] [PDF]
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