A better grip on calmodulin

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  • JAMES F. HEAD PROTEIN CONFORMATION

    A better grip on calmodulin Two atomic resolution structures show just how

    calmodulin binds to a peptide that represents the natural binding site of a calmodulin-activated enzyme.

    A vast literature on calmodulin has accumulated over the two and a half decades since the proteins discovery. Any- one who has performed a literature search of a database using calmodulin as a keyword, is painfully well aware of this fact. Most of these studies are part of the gradual incremental progress toward our present understanding of calmodulin and its numerous interactions. However, some papers represenlt clear landmarks in our progress. Such a landmark has recently been posted with the pub- lication of two papers, each giving atomic resolution de- tails of a complex fo.rmed between calmodulin and a binding peptide obtained from a target enzyme, myosin light chain kinase. These two papers are of particular interest not only to the study of Ca2+ regulation but also because they present complementary NMR [l] and X-ray crystallographic [ 21 studies of similar protein structures. The former represents a new frontier in the determina- tion of solution protein structures and demonstrates the power of current multidimensional NMR procedures, in this case giving atomic resolution details for a complex with a molecular weight of nearly 20 kD.

    Calmodulins principal function is to serve as a Ca2+- sensitive switch in the cytosol, regulating the activity of a wide variety of enzymes. The central importance of calmodulin in cellular regulation is emphasized by its ubiquitous presence in the cells of eukaryotes and the re- markable conservation of its amino-acid sequence across phyla. Most, though not ail, of the actions of calmodulin conform to a generalized outline. In the resting, or un- stimulated, cell large amounts of calmodulin are free in the cytoplasm (although in some cell types, significant quantities apparently remain associated with membrane structures through alternative binding mechanisms). Fol- lowing cell stimulation that leads to Ca2+ influx or intra- cellular Ca2+ release, calmodulin binds Ca2+ and under- goes a conformational change. In this activated state, the protein is able to bind to calmodulin-binding domains on its various target enzymes to form a complex with en- hanced enzymatic activity. As free Ca2+ is pumped from the cell or back into intracellular stores, calmodulin gives up its complement of lCa2 + ions and the calmodulin-en- zyme complex dissociates, reducing the enzymic activity to resting levels.

    From this outline it is apparent that there are three states of calmodulin in the (cell that are of particular physio- logical interest: the ofl? state, when free Ca2+ levels are below about lo-7 M; the activated state, with free Ca2+ at about lo-5 M; and the on state, when calmodulin is bound to its target in the presence of Ca2 f . It is also clear that detailed structural studies of calmodulin in its various

    Volume 2 Number I1 1992

    states are crucial to the understanding of the mechanism of action of the protein.

    Before 1985, no atomic resolution information was avail- able about any of these states, although there were some preliminary analogy-based models. Structural data were available only from low resolution spectroscopic tech- niques and chemical modification/proteolysis studies. Then, in 1985, Babu et al. 133 published the X-ray crys- tallographic structure of the activated form of calmod- ulin, that is bound to Ca2+ but not to a target protein. This dumbbell shaped structure, which is similar to the just previously published structure of the homologue, troponin-C [4], has since given rise to much interest and some controversy.

    The physiological significance of the long and exposed central c1 helix separating the two bells in calmodulin has been the subject of considerable speculation and experimentation. Several lines of evidence led Persechini and Kretsinger [5] to suggest that this region of the molecule might serve as a flexible tether permitting the two bells or lobes of the molecule to swing together, each binding on either side of a peptide domain on the target enzyme, enfolding the peptide and activating the enzyme.

    The calmodulin-binding domain of many target enzymes has been found to consist of a discrete portion of c1 helix with basic amphipathic properties, having basic hydrophilic residues along one side of the helix and hydrophobic residues along the other. Such peptides re- tain their ability to bind calmodulin with high affinity even when cleaved from the remainder of the enzyme. These peptides and homologues have thus provided valuable simple models for investigating the calmodulin-enzyme interaction. It has also been known for some time that the binding of calmodulin to a target depends on the Caz+ induced exposure of hydrophobic surfaces on calmod- ulin. A hydrophobic surface domain is indeed apparent on each of the lobes of the crystal structure of activated calmodulin. Based on these and other facts, Persechini and Kretsinger [6] proposed a model for the complex formed between calmodulin and the calrnodulin-bind- ing peptide of myosin light chain kinase. This model included the formation of a kink in the middle of the central helix of calmodulin, bringing the lobes to either side of the peptide. The hydrophobic surfaces of each lobe would then be in appbsition with the hydrophobic side of the target peptide, and the peptide helix chelated in a b&dentate complex. Results from small angle X- ray scattering studies supported this model: in solution,

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  • the binding of calmodulin to a target peptide changes the dumbbell-shaped calmodulin molecule into a more compact structure [ 7-91,

    With the recent atomic resolution studies of Ikura et al. [ 1 ] and Meador et al. [ 21, we are linally able to see details of the calmodulinpeptide interactions. Ikura et al. de- scribe the NMR structure of calmodulin complexed with a 26residue peptide corresponding to the calmodulin- binding domain of myosin light chain kinase from verte- brate striated muscle. The x-ray crystallographic structure reported by Meador et al. is of calmodulin complexed with a 20.residue peptide corresponding to the same do- main but from the smooth muscle form of the enzyme. These two binding peptides share a 19-residue stretch of homology. Although only seven of these residues are identical, most of the rest correspond to conserved sub- stitutions. The NMR structure is not yet fully relined, omit- ting a number of assignments. It is described by the au- thors as a lowresolution structure equivalent to . . . . . . a second generation NMR structure. The crystal structure is refined to 2.4A resolution.

    Both structures show essentially the same features: the two lobes of calmodulin swing around to enfold the CI- helical target peptide as predicted previously (Fig. l), but with an even more dramatic change in the central linker region of the calmodulin than had generally been previ- ously believed. A large portion at the center of the linker becomes uncoiled, allowing the two lobes to extend over greater distances to engulf the peptide. The lobes extend so far that they are able to surround the peptide and, according to the crystal structure, form interactions with one another on the far side. Such a configuration would help to lock the peptide in place. The two lobes them- selves remain largely unaltered from the conformation observed in the unc80mplexed activated calmodulin struc- ture of Babu et al. [ 31. The complex formed is ellipsoidal, with the target peptide running in a tunnel through the center of the calmodulin at an angle of about 45 to the long axis of the ellipsoid.

    Both studies describe extensive interactions between peptide and calmodulin - 185 contacts ( < 4A) consist- ing of 15% hydrogen bonds, 80% van der Waals cdntacts and seven salt bridges are reported in the crystal structure [2], while in the NIWZ structure more than 80 percent of the surface of tlhe peptide in contact with calmod- ulin is buried [ 11. In both cases, the orientation of the peptide is such that the N-terminal part of the peptide interacts principally with the C-terminal lobe of calmod- ulin and the C-terminal portion of the peptide interacts with the N-terminal lobe of calmodulin. The hydropho- bic face of each lobe of calmodulin interacts with the hydrophobic residues of the peptide, as previously pre- dicted, but me orientation of the two lobes is such that these two faces together are able to form a hydrophobic arc [which] interfaces with the hydrophobic side of the helical peptide [2].

    Another important feature noted in both studies is that the methionine residues of calmodulin interact exten- sively with the peptide. The conformational flexibility of methionine side chains, together with the flexibility of

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    ! Fig. 1. A schematic illustration of calmodulin (red) before and after binding to a target peptide. N and C indicate the amino- and carboxyl-terminii of both molecules.

    the unwound linker region of calmodulin, allows for accomodation of a wide variety of binding peptides with diEerent topological features. This adaptability is clearly of value to a protein that must bind so many targets and is consistent with the promiscuous binding of calmod- ulin to a great array of peptides and proteins, some of physiological importance, some not.

    While the similarities reported in me two studies are pre- dominant, it is evident that there are also some differ- ences. As noted by Meador et al. [ 21, there are some dissimilarities in orientation and distance of separation at the interface of the two lobes of cahnodulin, where they interact on the far side of the peptide. There are also dif- ferences in the extent of the uncoiling of the linker region of calmodulin. In the NMR structure, residues 74 to 82 are reported to be disordered, whereas in the crystal struc- ture, residues 73 to 77 are in extended StfUChlre, with the residues before and after in well-defined helices. Whether these and any other distinctions that might emerge result from differences in the target peptides themselves or in the experimental conditions remains to be determined.

    Several earlier studies have suggested that calmodulin can bind to some peptides in conformations that must dif- fer from that of the myosin light chain kinase peptide. One variation on the theme could result from the re- markable 2-fold symmetry of the present complex, per- pendicular to the axis of the peptide. It is possible, as noted previously in the modelling studies of Persechini and Kretsinger [6], that for some peptides the orienta- tion of the helix could reverse in the calmodulin tun- nel while retaining extensive interactions. It is evident, however, from solution scattering studies of calmodulin complexed with other binding peptides that still other modes of interaction must exist, giving rise to significantly different overall conformations.

    Another, as yet unresolved, issue concerns me struc- tural consequence of calmodulin binding on the target

    @ 1992 Current Biology

  • enzyme. Does calmodulin enhance enzymic activity sim- ply by pulling the binding peptide out of a sterically hindering conformation, as suggested by many studies? &r-e there other, longer range consequences on the en- ,iyme structure? Given the diversity of enzymes activated by calmodulin, it is likely that no single mechanism will apply in all cases. Ultimately, high resolution structures of entire calmodulin-enzyme complexes should give some of the definitive answers. Until such times, the structures of these two calmodulin-peptide complexes give us a far better understanding of the significance of past re- sults and a valuable basis on which to build future ideas, models and experiments

    References WJJRA M, CLORE GM, GRONENBORN AM, ZHU G, KIEE CB, BAX A: Solution structure of ,a calmodulin-target peptide complex by multidimensional NMR Science 1992, 256:632-638.

    MEAIXR WE, MEANS AR, @JIoCHO FA: Target enzyme recogni- tion by calmodulin: 2.4.A structure of a cahnodulin-peptide complex. Science 1992, 2571251-1255.

    BABU YS, SACK JS, GREEM~OUGH TC, BUGG CE, MEANS AR, COOK WJZ Three dimensional structure of calmodulin. Nature 1985, 315:37-40.

    4. HEmERG 0, JAMES MNG: Structure of the calcium regulatory muscle protein troponin-C at 2.8A resolution. Nature 1985, 313653659.

    5. PERSECHINI A, KREISINGER RH: The central helix of calmod- ulin functions as a flexible tether. J Biol Gem 1988, 263:12175-12178.

    6. PERSECHLNI A, KRETSINGER RH: Toward a model of the calmoduhn-myosin light chain ldnase complex: implications for calmodulin function. J Curdiovusc Pharm 1988, 12(suppl 511-12.

    7. KATAOKA M, HEAD JF, SEATON BA, ENGELMAN DM: Melittin bind- ing causes a large calcium-dependent conformational change in calmodulin. Proc Nat1 Acad Sci USA 1989, 86:6944-6948.

    8. YOSHINO H, MINARI 0, MATSUSHIMA N, UEK3 T, MIYAKE Y, MATSUO T, IZIJMI Y: Calcium-induced shape change of calmodulin with mastoparan studied by solution x-ray scattering. J Biol Chem 1989, 264:1970619709.

    9. HEIDORN DB, SEEGER PA, ROKOP SE, BLUMENTHAL DK, MEANS AR, C~SPI H, TREWH!ZLL~ J: Changes in the structure of calmod- t&n induced by a peptide based on the cahnodulin-bind- hg domain of myosin light chain kinase. Biocbemist?-y 1989, 28~6757-6764.

    James F. Head, Department of Physiology, Boston Uni- versity School Of Medicine, Boston, Massachusetts 02118, USA.

    Volume 2 Number 11 1992 611

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