Biochemistry 1992, 31, 5269-5278 5269 Backbone Dynamics of Calmodulin Studied by 15N Relaxation Using Inverse Detected Two-Dimensional NMR Spectroscopy: The Central Helix Is Flexible? Gaetano Barbato,*9$ Mitsuhiko Ikura,* Lewis E. Kay,* Richard W. Pastor,ll and Ad Bax*.* Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, and Biophysics Laboratory, Center for Biologics Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, Maryland 20892 Received December 19, 1991; Revised Manuscript Received March 31, 1992 ABSTRACT: The backbone dynamics of Ca2+-saturated recombinant Drosophila calmodulin has been studied by lSN longitudinal and transverse relaxation experiments, combined with lSN( ‘HJ NOE measurements. Results indicate a high degree of mobility near the middle of the central helix of calmodulin, from residue K77 through S81, with order parameters (S2) in the 0.54.6 range. The anisotropy observed in the motion of the two globular calmodulin domains is much smaller than expected on the basis of hydrodynamic calculations for a rigid dumbbell type structure. This indicates that, for the purposes of 15N relaxation, the tumbling of the N-terminal (L4-K77) and C-terminal (E82-S147) lobes of calmodulin is effectively independent. A slightly shorter motional correlation time (7c = 6.3 ns) is obtained for the C-terminal domain compared to the N-terminal domain (7, - 7.1 ns), in agreement with the smaller size of the C-terminal domain. A high degree of mobility, with order parameters of -0.5, is also observed in the loop that connects the first with the second EF-hand type calcium binding domain and in the loop connecting the third and fourth calcium binding domain. Calmodulin (CaM) is a ubiquitous intracellular protein of 148 residues (M, 16.7K) that plays a key role in coupling Ca2+ transients caused by a stimulus at the cell surface to events in the cytosol (Cohen & Klee, 1988). It performs this role by calcium-dependent binding to a host of intracellular en- zymes. Its crystal structure (Babu et al., 1988) shows that the protein consists of two globular domains, each containing two calcium binding sites of the “EF-hand” type, connected by a continuous 26-residue a-helix, often referred to as the “central helix”. Consequently, in the crystalline state CaM adopts a dumbbell type structure. Small-angle X-ray scat- tering studies of CaM in solution (Seaton et al., 1985; Heidom & Trewhella, 1988; Matsushima et al., 1989) agree on the presence of the two globular lobes in calmodulin, but disagree on the question of whether these lobes are separated by a large fmed distance as in the crystal structure, or by either a “flexible tether” type central helix or a bent one. Very recently, the crystal structure for recombinant CaM from Drosophila melanogaster was reported (Taylor et al., 1991). Although there were some differences with the structure of bovine brain calmodulin (Babu et al., 1988), the overall shape of the two molecules is very similar. At the time the present study of the CaM dynamics was conducted, the coordinates of the recombinant protein were not yet available, and in our discussions we refer to the structure of the bovine CaM structure, although our studies are actually carried out using recombinant CaM from Drosophila melanogaster. Using recently developed triple-resonance NMR techniques, we previously made complete assignments of the ‘H, 13C, and 15N NMR spectra of this protein and determined its secondary structure in aqueous solution (Ikura et al., 1991a,b). NOE data indicated that the so-called “central helix” is disrupted This work was supported by the Intramural AIDS Anti-viral Pro- gram of the Office of the Director of the National Institutes of Health. *National Institutes of Health. ‘On leave from Universita di Napoli, Federico 11, Dipartimento di I( Food and Drug Administration. Chimica, Via Mezzocannone 4, 80134 Napoli, Italy. from residue Asp78 through Ser-8 1. Spera et al. (199 1) found that the hydrogen exchange rates of residues located near the middle of the central helix are fast, suggesting that hydrogen bonding for these amides is either weak or absent. The NO& observed for these residues are relatively weak and do not point to a helical structure. The secondary shifts (Le., the deviations from random coil chemical shifts) of the Ca carbons and C@ carbons for these residues are also relatively small in thii region of the protein (Ikura et al, 1991a,b). These NMR data suggest that there may be a significant degree of mobility in the protein near the middle of the central helix. The present study measures the NMR relaxation properties of backbone ISN nuclei in engineered calmodulin by using highly sensitive indirect detection techniques (Nirmala & Wagner, 1988; Kay et al., 1989). The relaxation data both provide unambiguous measures for the degree of internal motion of individual backbone amides and report on the an- isotropy of the molecular reorientation. Our analysis largely followed similar studies conducted previously for staphylo- coccal nuclease (Kay et al., 1989) and interleukin-l@ (Clore et al., 1990a). However, recently improved techniques for measurement of I5N relaxation rates, which suppress the effect of cross correlation between dipolar and chemical shift an- isotropy (Boyd et al., 1990; Kay et al., 1992; Palmer et al., 1992), are used in the present study. In addition, these pulse schemes have been modified to remove the need for H20 presaturation. This permits the study of amides for which the proton exchanges rapidly with solvent, as occurs for the most mobile residues of calmodulin. Relaxation data are analyzed using the model-free approach of Lipari and Szabo (1982), and apparent rotational corre- lation times are derived for the individual amides. The de- pendence of the measured rotational correlation time on the orientation of the N-H bond vector in a molecular axis system is used to provide information on the degree of anisotropy of the molecular tumbling. This degree of anisotropy in mo- lecular reorientation is compared with the anisotropy evaluated from hydrodynamic calculations on a variety of slightly dif- This article not subject to U.S. Copyright. Published 1992 by the American Chemical