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An open and shut case
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Mikael Akke and Walter J. Chazin A powerful new NMR technique applied to the ubiquitous Ca2+ sensor, calmodulin, reveals significant conformational flexibility within each globular domain, which contributes to its ability to bind a wide range of targets. These measurements of residual dipolar couplings between nuclear spins demonstrate a fast and accurate method for pinpointing structural features that cannot be delineated reliably by traditional NOE analysis.
The pace toward understanding the dynamic nature of protein structure has been accelerating over the past decade. One protein under intense investigation is the ubiquitous Ca2+ regulatory agent calmodulin (CaM). On page 932 of this issue of Nature Structural Biology, Chou et al.1 provide convincing new evidence of the multifaceted and flexible structure of CaM by applying a relatively new solution NMR technique. As shown elegantly by Bax, Prestegard and their coworkers2−4, residual dipolar couplings (RDCs) between nuclear spins provide powerful constraints that enable a fast and accurate method for protein structure determination, which in a variety of situations — such as in the present case — outperforms more traditional approaches. The great value and even greater potential of using RDCs in NMR analysis has been amply reviewed elsewhere3,4, and so here we focus on the implications of the new results with respect to how CaM functions. The present report reveals a significant difference between the X-ray crystal structure of Ca2+-saturated calmodulin and the time- and ensemble-averaged conformation in aqueous solution. Beyond the significance with respect to interpretation of CaM’s functional structure, these studies also provide a striking example of the importance of complementary applications of X-ray crystallography and NMR spectroscopy. It is such multidirectional investigation of three-dimensional structure and dynamics that is required to understand in depth how proteins function. Calcium signaling is effected by a transient increase in the intracellular Ca2+ concentration and is transduced by Ca2+binding proteins that can carry out Ca2+dependent actions in the nucleus or cytoplasm. The Ca2+-binding unit in many of these proteins is the EF-hand motif, which has been identified in all living organisms5 and is one of the five most common protein motifs in animal cells6. 910
Fig. 1 Schematics of the transduction of Ca2+ signals into activation of a biochemical pathway by calmodulin. Ca2+ signals cause a ∼100-fold increase in the concentration of Ca2+ ions in the cell. The affinity for Ca2+ in each of CaM’s globular domains is tuned such that Ca2+ binds only after a Ca2+ signal is given. The binding of Ca2+ causes a large change in the conformation of each domain, resulting in exposure of a considerable hydrophobic surface, which serves as a binding site for target proteins. The effect on the N-terminal domain of CaM is shown with the hydrophobic accessible surfaces colored yellow. Activation of a biochemical pathway is achieved through cooperative binding by the two Ca2+-activated CaM domains to a helical segment from the target protein, in this case shown for the CaM binding region of the myosin light chain kinase12.
The EF-hand consists of two helices and an intervening Ca2+-binding loop. Two or more EF-hands pack together to form a globular domain that serves as the basic functional unit. It was recognized early on that the interhelix angle of the EF-hand can change dramatically upon Ca2+ binding7, causing the domain to switch from a compact ‘closed’ structure to an ‘open’ structure. The opening of the protein upon binding Ca2+ results in exposure of a hydrophobic surface where target proteins bind (Fig. 1). The magnitude and type of structural rearrangements induced by Ca2+ binding provide the individual EF-hand proteins with their distinct functions. The evolution in conceptualizing the conformational changes in CaM and related proteins stands out as a strikingly illustrative example of the power of structural biology to improve our understanding of the molecular basis for biological function. It
is also a very good example of the complementarity of NMR spectroscopy and X-ray crystallography in structural biology research. The first crystal structures of Ca2+− CaM revealed a dumbell-shaped structure8. However, data from small-angle X-ray scattering suggested that CaM in solution should have a significantly less extended shape than observed in the crystal9. This controversy was settled definitively by analysis of key structural and motional parameters determined in solution NMR experiments, ranging from J-coupling constants to 15N relaxation measurements of the global diffusion tensor and the local backbone order10,11. Indeed, the central helix linking the two domains is unstructured and kinked near its midpoint in aqueous solution. The paper by Chou et al.1 contributes relevant data showing that the N- and C-terminal domains align with the magnetic field to
nature structural biology • volume 8 number 11 • november 2001
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© 2001 Nature Publishing Group http://structbio.nature.com
news and views different degrees, which indicates that the relative orientation of the two domains is modulated dynamically in solution. The functional relevance of the flexibility in the central helix is supported by previous structures of CaM–target peptide complexes, determined by both NMR12 and X-ray crystallography13. These structures showed that the N- and C-terminal domains wrap around and clasp onto the peptide like two hands on a stick (Fig. 1), and the middle segment of the central helix is completely unwound and disordered. As for the individual domains, the data available prior to the current report by Chou et al.1 suggested that there were few differences between the structure of CaM in solution and in crystals10,14. Now, the RDCs measured by Chou et al.1 convincingly show that there are in fact significant structural differences. The solution structure calculated with RDC-derived input data reveals that the N- and C-domains are more closed than their counterparts in the crystal structures by 20–26° and 10–15°, respectively. The variability in the opening of CaM domains observed between the solution and crystal structures is congruent with another observation — the extent of opening that an individual domain experiences varies depending on the target. Together these observations highlight the importance of another aspect of the flexible nature of CaM: the adaptable helical packing finetunes the interactions with the wide range of CaM targets. Hydrophobic side chains at the recognition surfaces are also key elements that modulate target interactions, and the work by Chou et al.1 has made important contributions here as well. The amino acid sequence of CaM is extremely well conserved — identical in all vertebrates, and 90% identical for all multicellular eukaryotes15 — suggesting that the exact identity of each amino acid is very important. This implies that the spatial organization of CaM’s domains and/or the portion of the exposed surface of each domain varies in response to the requirements of different targets. Chou et al.1 demonstrate that the combination of RDCs and J-couplings, without traditional NOE analysis, can be used to characterize side chain structure and flexibility in a highly efficient manner. They found that a substantial percentage of the side chains in Ca2+–CaM occupy the same conformation in solution as in the crystal. Surprisingly, many side chains at the binding interface occupy
specific conformations, which indicates that these functional groups become less flexible even before the target is bound. In contrast, a high degree of flexibility was observed for Met residues, consistent with the widely held belief that they make critical contributions to plasticity at the binding interface16. NMR relaxation studies probing the local dynamics of the side chains17,18 have shown that the response to Ca2+ and target binding in many cases involves dramatic changes in the amplitudes of conformational fluctuations. The observation of a high degree of pre-organization of certain side chains and flexibility in others upon binding Ca2+ is intriguing, and suggests that residual side chain conformational entropy needs to be carefully evaluated as a significant contributor to target recognition and binding18. The present work reveals a difference between the single low-energy conformer in the crystal and the time- and ensembleaveraged conformer in solution. These data, together with previous CaM structures that show differences in the extent of domain opening, suggest that the individual domains of CaM undergo extensive dynamic averaging of various conformations in solution. The Ca2+-free C-terminal domain exchanges between a closed and a weakly populated open conformation in both intact CaM19 and the isolated domain20. Similar processes are observed in the Ca2+-saturated C-terminal domain mutant E140Q, which has greatly reduced Ca2+ affinity in the C-terminal EFhand21,22. In this context, it is interesting that B-factors observed in the crystal structure of Ca2+–CaM at 1 Å are anisotropic, reflecting scissor-type fluctuations of each EF-hand23. Are these types of motions at play in intact Ca2+–CaM in solution? The present results by Chou et al.1 rule out the possibility that the difference between the experimental dipolar couplings and those expected from the crystal structures could be caused by a transiently and weakly populated conformation (for example, a closed state) with very different interhelical angles. While the current data were not directly interpreted in terms of amplitudes of fluctuations, Chou et al.1 were able to put an upper limit of 20° for possible harmonic oscillations of the α-helices within a domain. There is clearly more work that lies ahead. One of the key questions is just how large are the conformational excursions within the globular N- and C- terminal domains of CaM? Exciting steps
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have recently been taken to address the technical challenges of assessing amplitudes and directions of motion of individual structural elements from RDCs24−26. Furthermore, to understand how conformational flexibility contributes to the biophysical properties and biological functions of the protein, it will also be necessary to determine the timescales and energetics of these excursions. Significant advances in the application of NMR to this problem have been made in this area as well27. The information on CaM’s flexibility and dynamics will then need to be analyzed in the context of the timescales and energetics of the Ca2+ and ligand binding events to assess the relevance of these particular conformational excursions to the biochemical activity of the protein. Most likely, both experimental and computational approaches will be needed to obtain the full picture. Are these properties unique to CaM, or does one find similar properties in other members of the EF-hand protein family? The conformational flexibility of CaM is viewed as essential to its ability to bind a range of cellular targets. Yet published studies of the structure and dynamic properties of troponin C (TnC)28, which has only a single target, and calbindin D9k (ref. 29), which apparently does not serve in directly transducing Ca2+ signals, show that there is a great deal of similarity in all three of these proteins. Additional studies to explore the conformational fluctuations within the globular domains of these proteins would contribute insights into whether the conformational flexibility found in free Ca2+–CaM is a uniquely critical to its biological function, or whether this is a general property of EFhand and other globular helical proteins. The paper by Chou et al.1 demonstrates one of the ways to utilize the complementarity between studies using X-ray crystallography and NMR spectroscopy. We believe this is an early example of the inevitable direction of structural biology research. High-resolution structure offers the biologist the possibility to think in novel ways about problems in biology and medicine. As X-ray crystallography, NMR spectroscopy and computation become increasingly automated, they will become more generally accessible and be used in combination to address difficult problems. Now more than ever, the onus is on the structural biologist to take advantage of the strengths of each of the three techniques and utilize their synergy to accelerate our understanding of how proteins function. 911
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Acknowledgments The authors thank M.R. Nelson and L. Mizoue for preparing the figure, A. Malmendal and J. Christodoulou for stimulating discussions about calmodulin’s structure, dynamics and function, and the Swedish Research Council and US National Institutes of Health for support of Ca2+-binding proteins research in our laboratories.
Mikael Akke is in the Department of Biophysical Chemistry, Lund University, Box 124, SE-221 00, Lund Sweden. Walter J. Chazin is in the Departments of Biochemistry and Physics and Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232-0146, USA. Correspondence should be addressed to M.A. email:
[email protected] or W.J.C. email:
[email protected] 1. Chou, J.J., Li, S., Klee, C.B. & Bax, A. Nature Struct. Biol. 8, 932–935 (2001).
2. Tjandra, N. & Bax, A. Science 278, 1111−1114 (1997). 3. Prestegard, J.H., Al-Hashimi, H.M. & Tolman, J.R. Q. Rev. Biophys. 33, 371−424 (2000). 4. Bax, A., Kontaxis, G. & Tjandra, N. Methods Enzymol. 339, 127−174 (2001). 5. Celio, M.R., Pauls, T. & Schwaller, B. In Guidebook to the calcium-binding proteins (eds Celio, M.R., Pauls, T. & Schwaller, B.) 15−20 (Oxford University Press, Oxford; 1996). 6. Henikoff, S. et al. Science 278, 609−614 (1997). 7. Herzberg, O., Moult, J. & James, N.G. J. Biol. Chem. 261, 2638−2644 (1986). 8. Babu, Y.S., Bugg, C.E. & Cook, W.J. J. Mol. Biol. 204, 191−204 (1988). 9. Heidorn, D.B. & Trewhella, J. Biochemistry 27, 909− 915 (1988). 10. Ikura, M. et al. Biochemistry 30, 9216−9228 (1991). 11. Barbato, G., Ikura, M., Kay, L.E., Pastor, R.W. & Bax, A. Biochemistry 31, 5269−5278 (1992). 12. Ikura, M., Clore, G.M., Gronenborn, A.M., Zhu, G. & Bax, A. Science 256, 632−638 (1992). 13. Meador, W.E., Means, A.R. & Quiocho, F.A. Science 262, 1718−1721 (1993). 14. Finn, B.E. et al. Nature Struct. Biol. 2, 777−783 (1995). 15. Moncrief, N.D., Kretsinger, R.H. & Goodman, M. J. Mol. Evol. 30, 522−562 (1990). 16. Vogel, H.J. Biochemistry and Cell Biology 72, 357−
376 (1994). 17. Siivari, K., Zhang, M.J., Palmer, A.G. & Vogel, H.J. FEBS Lett. 366, 104−108 (1995). 18. Lee, A.L., Kinnear, S.A. & Wand, A.J. Nature Struct. Biol. 7, 72−77 (2000). 19. Tjandra, N., Kuboniwa, H., Ren, H. & Bax, A. Eur. J. Biochem. 230, 1014−1024 (1995). 20. Malmendal, A., Evenäs, J., Forsén, S. & Akke, M. J. Mol. Biol. 293, 883−899 (1999). 21. Evenäs, J., Malmendal, A. & Akke, M. Structure 9, 185−195 (2001). 22. Evenäs, J., Forsén, S., Malmendal, A. & Akke, M. J. Mol. Biol. 289, 603−617 (1999). 23. Wilson, M.A. & Brünger, A.T. J. Mol. Biol. 301, 1237−1256 (2000). 24. Tolman, J.R., Flanagan, J.M., Kennedy, M.A. & Prestegard, J.H. Nature Struct. Biol. 4, 292−297 (1997). 25. Tolman, J.R., Al-Hashimi, H.M., Kay, L.E. & Prestegard, J.H. J. Am. Chem. Soc. 123, 1416−1424 (2001). 26. Meiler, J., Prompers, J.J., Peti, W., Griesinger, C. & Brüschweiler, R. J. Am. Chem. Soc. 123, 6098−6107 (2001). 27. Palmer, A.G., Kroenke, C.D. & Loria, J.P. Methods Enzymol. 339, 204−238 (2001). 28. Mercier, P., Spyracopoulos, L. & Sykes, B.D. Biochemistry. 40, 10063–10077 (2001). 29. Mäler, L., Blankenship, J., Rance, M. & Chazin, W.J. Nature Struct. Biol. 7, 245–250 (2000).
Protein dynamic studies move to a new time slot John Cavanagh and Ronald A. Venters Many proteins frequently undergo structural rearrangement to complete their functions. Ligand entry and binding are often associated with some degree of localized disorder. Indeed, low populations of disordered excited states may help drive such processes. Characterization of these states is vital to understanding the mechanisms of many biological functions.
Transitions from low energy, ground state conformations to higher energy, excited state conformations play a vital role in protein function. Consequently, to fully comprehend the biological mechanism of a protein, it is essential to obtain a full description of the energetics and kinetics of the processes involved in such transitions. The time-dependent conformational fluctuations of proteins, which generate excited states albeit with low populations can be exquisitely investigated by a powerful suite of relatively new heteronuclear NMR relaxation techniques1. Although initial NMR relaxation experiments and analyses concentrated on protein dynamics on the ps−ns timescale2,3 more recent methodologies have focused on µs−ms motions1. Why the growing interest in such slower movements? As more evidence accumulates, it becomes clear that many biological processes exhibit rates that coincide with this timescale. For example, rates for enzyme catalysis and product release4,5, 912
protein folding6,7 and allosteric transitions8 all occur in this time regime. Handin-hand with the continuing design and implementation of these novel NMR dynamics experiments has been the development of theoretical approaches for extracting thermodynamic and kinetic information from relaxation data9−12. In principle, therefore, we now have the capability to characterize both the thermodynamics and kinetics of the transition between the ground state and the excited state of a protein. NMR spectroscopy is unique in this regard, allowing the investigation of important biological events that are relatively inaccessible to other techniques. On page 932 of this issue of Nature Structural Biology, Kay, Dahlquist and coworkers describe one fine example of such characterization13. These researchers thoroughly investigated the transition of the L99A mutant of T4 lysozyme between its ground state conformation, which is inaccessible to ligand binding, and its
excited state, which allows for binding. By using recently developed relaxation dispersion NMR techniques performed at several magnetic field strengths and over a range of temperatures, Kay and coworkers have calculated the enthalpic and entropic contributions to the interconversion between states. In addition, they present evidence to bolster the suggestion that ligand binding to this mutant is related to the slower timescale dynamics of the protein. The partial ‘unfolding’ or ‘local disorder’ that the protein dynamics confer in the binding region of L99A is thought to be important for ligand recognition/binding in a variety of other proteins that target small hydrophobic molecules. The ‘hole’ story Why are the dynamics of this particular lysozyme mutant interesting? Why the myriad of crystallographic and NMR studies? The answer lies at the core of this protein. Most proteins possess a core of well-packed hydrophobic residues that
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