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Solid state NMR: new tools for insight into enzyme function
Solid state NMR: new tools for insight into enzyme function Ann McDermott1 and Tatyana Polenova2 NMR has had considerable impact in enzymology, probing evidence for ionization states, conformational ‘strain’, compressed interactions, electronically unusual species, and conformational dynamics of enzymes. Solid-state NMR is becoming increasingly important in studying enzymes because of a number of recent tools for analysis of proteins by SSNMR, and because of the growing ability to isolate the species of interest for analysis. Here, we review recent studies of a Michaelis complex, of the dynamic functioning of membrane-associated enzymes, and initial studies of several enzymes with redox-active and paramagnetic centers. Addresses 1 Columbia University, Department of Chemistry, New York, NY 10027, United States 2 University of Delaware, Department of Chemistry and Biochemistry, Newark, DE 19716, United States Corresponding author: McDermott, Ann ([email protected]) and Polenova, Tatyana ([email protected])
Current Opinion in Structural Biology 2007, 17:617–622 This review comes from a themed issue on Biophysical methods Edited by Keith Moffat and Wah Chiu
0959-440X/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2007.10.001
Introduction Although understanding the basis of catalysis by enzymes remains a significant challenge, its study has already brought many rewards; the growing knowledge of the enzyme mechanisms has not only had considerable intellectual impact in basic science, but has also guided design of novel catalysts and inhibitors including potential therapeutics and diagnostics. In contrast to the initial focus on the elusive transition state, the extent of differential binding of the transition state has been recently debated [1], and meanwhile, the importance of probing the experimentally accessible ground state has been emphasized [2,3]. Computational studies have illustrated the powerful ability of optimal alignment of the substrate with respect to key active site residues to accelerate chemistry, as illustrated in Figure 1. Enzyme active sites in complex with substrates exhibit compression [4–6], where key reactive groups have particularly short non-bonded distances and are expected to show unusual dynamics. www.sciencedirect.com
These unusual structures are incisively studied through their NMR and vibrational signatures [7]. Chemically detailed information inherent to NMR, together with the opportunity to stabilize species at lower temperatures or even study catalysis in its native biological environment [8] makes SSNMR especially powerful for this goal. Furthermore, the recent technical advances and emerging methods in SSNMR now allow analysis of extensively labeled systems [9,10], very high resolution structures [11], and dynamics of the enzymes [12,13,14,15]. Recent advances in accessing SSNMR signatures for the reactive states of enzymes include new tools for observing waters in biological systems, observing metals including quadrupolar nuclei and paramagnetic systems, and characterizing conformational and chemical dynamics. Thus it is an opportune time to review the study of enzymes by SSNMR. SSNMR has many other exciting application areas in structural and mechanistic biology, besides enzymology, and we point the reader to some corresponding reviews: [16–20].
SSNMR of enzymes: a new range of applications Studies of a Michaelis complex
Triosephosphate isomerase (TIM), a central glycolytic enzyme, accelerates by over 10 orders of magnitude the interconversion of dihydroxyacetone phosphate and glyceraldehydes phosphate via proton transfers (Figure 2), and offers an unusual opportunity to characterize a bona fide Michaelis complex. Quantum computational investigations [21,22], and the considerable structural and spectroscopic literature on this system, emphasize the role of the general base Glu165 in abstracting the proton on C1 of DHAP and C2 of GAP and His95, in rearranging the alcohol protons for the key enediol intermediate. Loops 6 and 7 (particularly the amidic NH of Gly 171, 173 and 232) are essential for binding of the substrate’s phosphate group. The nature of the nascent prereacctive complex can be conveniently studied in this system for the simple reason that it is an isomerase with the uphill direction being of particular biological significance. Recent atomic-resolution X-ray crystal structures of the Michaelis complex [5] exhibit a compressed and bifurcated hydrogen bond network where the carboxylate group of Glu165 indicates intriguing conformational disorder. While very revealing in terms of the binding environment, these data were ambiguous regarding the chemical form of the substrate. The chemical nature and populations of the substrate intermediate and product have been recently probed using variable-temperature NMR solution and 13C Current Opinion in Structural Biology 2007, 17:617–622
618 Biophysical methods
Figure 1
Two limiting hypotheses about enzyme rate enhancements. Compared to the uncatalyzed reaction (black) the catalyzed reaction can be faster because of transition state stabilization (green), or through ground state destabilization (red).
solid-state NMR spectroscopy [25]. In contrast to prior predictions from detailed isotope effect studies, DHAP was the only chemical form observed on the enzyme both in solution and in the crystals. The discrepancy with prior analysis might be related to a previously unappreciated kinetic step, the opening of the loop on the reaction product. In fact, TIM’s reaction kinetics provided some evidence for motion of these loops during the reaction, and the rate of loop opening was therefore probed by solid state NMR [24], solution NMR [25–27], and T-jump fluorescence spectroscopy [28]. In contrast to a number of prior predictions, these studies showed that the loopopening rate is on the order of the turnover rate, and is somewhat sensitive to the details of the ligand [24,25,28]. Solid state NMR studies altered our concept of the key
intermediates and essential rate constants rates for TIM, establishing the importance of protein motion on this enzyme’s reactive pathway as well as the ability to probe these key motions by solid state NMR methods. Membrane enzymes
An important frontier for structural biology is the understanding the structures function and dynamics of membrane proteins. SSNMR studies have had significant impact into understanding of membrane channel function. It is possible to access alternative states of channels under SSNMR conditions, including states where it might be difficult to obtain crystallographic data [29]. The principles underlying enzyme function, for example stabilization by unusually strong hydrogen bond motifs
Figure 2
The proposed mechanism and reaction rates of the isomerization reaction catalyzed by triosephosphate isomerase [23]. Current Opinion in Structural Biology 2007, 17:617–622
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Solid state NMR: new tools for insight into enzyme function McDermott and Polenova 619
and distributed hydrogen bond motifs, also extend to channels, as beautifully illustrated in the M2 H+ channel from influenza A. The heart of this conductance mechanism is the hydrogen bonding interactions in the homotetrameric set of four histidine side chains. NMR experiments have been used to show that protonation of one of these imidazoles coincides with acid activation of this transmembrane channel, so that at physiological pH, the channel is closed by two imidazole-imidazolium dimers, each sharing a low-barrier hydrogen bond [30]. Thereby a pair of charges is distributed over four rings and stabilized in the low dielectric membrane environment. SSNMR has offered insights into how guanine nucleotide-binding proteins utilize transformation from GTPbound and GDP-bound states, with pronounced conformational changes, to act as molecular switches. In solution additional conformational states in the GDP as well as in the GTP forms coexist, which were not detected by X-ray crystallography [31]. The conformational changes observed by liquid-state NMR dynamics measurements were related to X-ray crystallographic structures, in that the SSNMR data clearly indicated the existence of two different conformations of the molecule in the crystalline state under some conditions [32,33]. 2H SSNMR probed the packing and molecular dynamics of ras lipid chains connected to a ras fragment. Compared to the host DMPC matrix, the ras lipids are extremely flexible, which has interesting possible consequences for function [34]. Conformational dynamics of a full length ABC multidrug efflux pump LmrA from Lactococcus lactis in lipid membranes has been investigated by deuterium SSNMR. Motional freedom is restricted upon ATP binding, and then returns to full flexibility in the posthydrolysis, vanadate-trapped state [35]. The recent appearance of many studies assigning instrinsic membrane systems, for example the initial resonance assignments have been reported for the ‘heart’ ATP synthase, the c subunit [36], will pave the way for detailed mechanistic work on this and other intrinsic membrane enzymes. Paramagnetic, redox and metal binding systems
A very large number of enzymes utilize metals for stabilization, redox facilitation and catalysis, and arguably some of the most powerful catalysts rely on metals to carry out their function. An explosion of studies of enzymes and enzyme-related proteins with metals and/or paramagnetic centers has appeared very recently, setting the stage for further detailed analysis. Matrix metalloproteins are important drug targets, where flexibility is clearly an essential aspect in relation to function [37]. SSNMR studies have been reported with wonderfully resolved lines, affording an independent probe of this important class of proteins [38]. Detailed analysis of chemical shifts has been reported recently for www.sciencedirect.com
thioredoxin, which is involved in a number of interesting redox related biochemical pathways [39,40]. Spectral analyses of several paramagnetic metalloenzymes have been conducted recently, representing an opening of a new field and a new class of systems for enzyme study. Moreover, this kind of spectroscopy has the promise of providing valuable structural constraints, just as it has for solution NMR of paramagnetic proteins. The spectral assignment of human dimeric oxidized Cu(II)–Zn(II) superoxide dismutase (SOD in the solid state has been recently reported [41]. SSNMR studies of microcrystalline cobalt(II)-substituted matrix metalloproteinase 12 (MMP-12, 17 kDa) gave 250 pseudocontact shifts, including nuclei up to more than 20 A˚ from the metal. These data were found to be in very good agreement with calculated shifts based on structure [42]. These studies have also been extended to systems of much higher molecular weight, through a combination of fortuitous resolution and selective labeling strategies. SSNMR was used to probe ligand binding and conformational state in cytrochrome P450, a system of considerable complexity [43]. This approach has the potential to access the dynamics of the enzymes in catalytically important states. Studies of the active site of other heme proteins clarified the degree of disorder in ligands proximal to the heme group through deuterium spectroscopy [44,45]. Initial SSNMR investigations have also been reported for a 144 kDa integral membrane protein, Escherichia coli cytochrome bo(3) oxidase [46]. The unique chemical shifts of the cofactors, for example flavin rings, makes SSNMR an effective way to probe larger redox enzymes [47]. Metals whose nuclei have an electric quadrupole moment represent very important players in biochemistry, but their NMR detection remained a challenge area until recent advances in spectroscopic detection of these sites. Direct detection of half-integer quadrupolar metals in proteins, even for nuclei with large quadrupolar moments, has also been achieved in several cases, offering considerable insights into the active site geometry. Detailed studies of the Zn2+ in human carbonic anhydrase (CAII), including comparison of predicted quadrupolar couplings with the experimental values, suggested the presence of hydroxide as a fourth ligand at pH 5–8.5. This observation suggests that a new mechanism for CAII may be needed [48]. The quadrupole coupling tensor for 63Cu in the active site of bovine erythrocyte (Cu/Zn) superoxide dismutase, has been measured, and comparison with the results of ab initio calculations has implications for the site symmetry [49]. A field-cycling pure quadrupole resonance experiment indicated that the environment of boron is tetrahedral in two peptide boronic acid inhibitors bound to alpha-lytic protease [50]. Direct detection of 43 Ca signal in Ca2+ calbindin allowed to distinguish the Current Opinion in Structural Biology 2007, 17:617–622
620 Biophysical methods
Figure 3
[52], and will offer insight on substrate specificity in haloperoxidases. Probing hydration and dynamics of crystalline proteins
51
V experimental (a) and simulated (b) spectrum of vanadium chloroperoxidase (c), yielding protonation environment of the resting state vanadate cofactor depicted in (d) [52].
two calcium sites [51]. 51V SSNMR of the 67.5-kDa vanadium chloroperoxidase yielded the quadrupole coupling and chemical shielding anisotropy tensors (Figure 3), which revealed the protonation states of the vanadate cofactor in the resting form, unavailable from the X-ray structure or any spectroscopic measurements
The importance of conformational plasticity of enzymes has been underscored in a recent comprehensive review of NMR studies of enzyme dynamics [53]. Typically, enzymes access multiple conformational states, including a Michaelis complex with the reactant and an open, ligated state. Their facile interconversion on the microor milli-second timescale is a crucial requirement, allowing for rapid binding and release. Knowledge of the kinetics and thermodynamics of these states has benefited greatly from NMR methodological advances [54]. Rapid thermal fluctuations surrounding the Michaelis state structure, are also of interest, because they can elicit strongly enhanced reactivity [55,56]. Improved tools to characterize dynamics have emerged that are predicated on the recent tools for assignment and structural characterization of uniformly isotopically enriched proteins. Orientation-dependent dipolar measurements [57] have been applied to probe fast molecular motions in AX/AX2/ AX3 spin systems, in small molecules and in ubiquitin [14], illustrated in Figure 4. Exciting advances have also appeared for deuterium lineshapes as a probe of protein dynamics [15,58]. Other methods are promising on slower timescales [13,59,60]. Clearly, changes in hydration of enzyme active sites care connected to thee conformatonal exchange events and have been somewhat elusive experimentally up to now. Emerging studies of water in enzyme surfaces and cavities by solid state NMR
Figure 4
Order parameters for fast limit motion were determined in the solid state for the protein ubiquitin, illustrating enhanced mobility in loops and turns [14]. Current Opinion in Structural Biology 2007, 17:617–622
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Solid state NMR: new tools for insight into enzyme function McDermott and Polenova 621
[61,62,63,64], together with increased use of high-resolution spectra of protons [65,66], are expected to be indispensable in analyzing enzyme active sites and their unusual electronic environments, making SSNMR an ever more attractive tool for enzymology. Unlike the prior generation of tools for studying dynamics by SSNMR, these emerging tools efficiently characterize many sites on the protein simultaneously and offer far more insight into function. They have already been used to demonstrate a high degree of motion is crystalline systems, and are very promising for future studies of membrane systems in their native lipid bilayer environments.
Conclusion In the past few years, new SSNMR tools for studying enzymes and reaction pathway intermediates in biology have emerged and been demonstrated on a number of interesting protein systems. Insights into strain, hydration, and conformational and chemical exchange from these methods will enhance our understanding of the remarkable function of enzymes.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Kraut DA, Sigala PA, Pybus P, Liu CW, Ringe D, Petsko GA, Herschlag D: Testing electrostatic complementarity in enzyme catalysis: Hydrogen bonding in the ketosteroid isomerase oxyanion hole. Plos Biol 2006, 4:501.
an amyloid fibril determined by magic angle spinning NMR spectroscopy. Proc Natl Acad Sci U S A 2004, 101:711. 12. Giraud N, Blackledge M, Goldman M, Bockmann A, Lesage A, Penin F, Emsley L: Quantitative analysis of backbone dynamics in a crystalline protein from nitrogen-15 spin-lattice relaxation. J Am Chem Soc 2005, 127:18190. Site specific and extensive measurements of nitrogen-15 longitudinal relaxation times in MAS spectra of microcrystalline proteins are analyzed assuming a diffusion in a cone model. 13. Krushelnitsky A, Reichert D: Solid-state NMR and protein dynamics. Progr Nucl Magn Reson Spectrosc 2005, 47:1. 14. Lorieau JL, McDermott AE: Conformational flexibility of a microcrystalline globular protein: Order parameters by solidstate NMR spectroscopy. J Am Chem Soc 2006, 128:11505. Order parameters describing angular motions of CH bond vectors are measured for a microcrystalline protein. 15. Reif B, Xue Y, Agarwal V, Pavlova MS, Hologne M, Diehl A, Ryabov YE, Skrynnikov NR: Protein side-chain dynamics observed by solution- and solid-state NMR: Comparative analysis of methyl H-2 relaxation data. J Am Chem Soc 2006, 128:12354. Methyl 2H relaxation rates are measured in the crystalline and liquid samples of an SH3 domain, and compared using a model-free approach. 16. Baldus M: Molecular interactions investigated by multidimensional solid-state NMR. Curr Opin Struct Biol 2006, 16:618. 17. Tycko R: Molecular structure of amyloid fibrils: insights from solid-state NMR. Quart Rev Biophys 2006, 39:1. 18. Goobes G, Stayton PS, Drobny GP: Solid-state NMR studies of molecular recognition at protein-mineral interfaces. Progr Nucl Magn Reson Spectrosc 2007, 50:71. 19. Ramamoorthy A, Wei YF, Lee DK: PISEMA solid-state NMR spectroscopy. Annu Rep NMR Spectrosc 2004, 52:1. 20. De Angelis AA, Jones DH, Grant CV, Park SH, Mesleh MF, Opella SJ: NMR experiments on aligned samples of membrane proteins. Methods Enzymol 2005, 394:350-382.
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21. Cui Q, Karplus M: Quantum mechanics/molecular mechanics studies of triosephosphate isomerase-catalyzed reactions: Effect of geometry and tunneling on proton-transfer rate constants. J Am Chem Soc 2002, 124:3093.
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Bruice TC: Computational approaches: Reaction trajectories, structures, and atomic motions. Enzyme reactions and proficiency. Chem Rev 2006, 106:3119.
22. Guallar V, Jacobson M, McDermott A, Friesner RA: Computational modeling of the catalytic reaction in triosephosphate isomerase. J Molec Biol 2004, 337:227.
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Fodor K, Harmat V, Neutze R, Szilagyi L, Graf L, Katona G: Enzyme: Substrate hydrogen bond shortening during the acylation phase of serine protease catalysis. Biochemistry 2006, 45:2114.
23. Knowles JR: Enzyme catalysis: not different, just better. Nature 1991, 350:121.
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Jogl G, Rozovsky S, McDermott AE, Tong L: Optimal alignment for enzymatic proton transfer: Structure of the Michaelis complex of triosephosphate isomerase at 1.2-angstrom resolution. Proc Natl Acad Sci U S A 2003, 100:50.
24. Rozovsky S, McDermott AE: The time scale of the catalytic loop motion in triosephosphate isomerase. J Molec Biol 2001, 310:259. 25. Rozovsky S, McDermott AE: Substrate product equilibrium on a reversible enzyme, triosephosphate isomerase. Proc Natl Acad Sci U S A 2007, 104:2080.
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Radisky ES, Lee JM, Lu CJK, Koshland DE: Insights into the serine protease mechanism from atomic resolution structures of trypsin reaction intermediates. Proc Natl Acad Sci U S A 2006, 103:6835.
26. Kempf JG, Jung JY, Ragain C, Sampson NS, Loria JP: Dynamic requirements for a functional protein hinge. J Molec Biol 2007, 368:131.
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Anderson VE: Quantifying energetic contributions to ground state destabilization. Arch Biochem Biophys 2005, 433:27.
27. Massi F, Wang CY, Palmer AG: Solution NMR and computer simulation studies of active site loop motion in triosephosphate isomerase. Biochemistry 2006, 45:14232.
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Cegelski L, Schaefer J: NMR determination of photorespiration in intact leaves using in vivo (CO2)-C-13 labeling. J Magn Reson 2006, 178:1.
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Igumenova TI, McDermott AE, Zilm KW, Martin RW, Paulson EK, Wand AJ: Assignments of carbon NMR resonances for microcrystalline ubiquitin. J Am Chem Soc 2004, 126:6720.
10. Pauli J, Baldus M, van Rossum B, de Groot H, Oschkinat H: Backbone and side-chain C-13 and N-15 signal assignments of the alpha-spectrin SH3 domain by magic angle spinning solid-state NMR at 17.6 tesla. Chembiochem 2001, 2:272. 11. Jaroniec CP, MacPhee CE, Bajaj VS, McMahon MT, Dobson CM, Griffin RG: High-resolution molecular structure of a peptide in www.sciencedirect.com
28. Desamero R, Rozovsky S, Zhadin N, McDermott A, Callender R: Active site loop motion in triosephosphate isomerase: T-jump relaxation spectroscopy of thermal activation. Biochemistry 2003, 42:2941. 29. Mo Y, Cross TA, Nerdal W: Structural restraints and heterogeneous orientation of the gramicidin A channel closed state in lipid bilayers. Biophys J 2004, 86:2837. 30. Hu J, Fu R, Nishimura K, Zhang L, Zhou HX, Busath DD, Vijayvergiya V, Cross TA: Histidines, heart of the hydrogen ion channel from influenza A virus: Toward an understanding of conductance and proton selectivity. Proc Natl Acad Sci U S A 2006, 103:6865. Current Opinion in Structural Biology 2007, 17:617–622
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31. Spoerner M, Wittinghofer A, Kalbitzer HR: Perturbation of the conformational equilibria in Ras by selective mutations as studied by P-31 NMR spectroscopy. FEBS Lett 2004, 578:305. 32. Iuga A, Spoerner M, Kalbitzer HR, Brunner E: Solid-state P-31 NMR spectroscopy of microcrystals of the Ras protein and its effector loop mutants: Comparison between crystalline and solution state. J Molec Biol 2004, 342:1033. 33. Spoerner M, Prisner TF, Bennati M, Hertel MM, Weiden N, Schweins T, Kalbitzer HR: Conformational states of human Hras detected by high-field EPR, ENDOR, and P-31 NMR spectroscopy. Magn Reson Chem 2005, 43:S74. 34. Vogel A, Katzka CP, Waldmann H, Arnold K, Brown MF, Huster D: Lipid modifications of a Ras peptide exhibit altered packing and mobility versus host membrane as detected by 2H solidstate NMR. J Am Chem Soc 2005, 127:12263. 35. Siarheyeva A, Lopez JJ, Lehner I, Hellmich UA, van Veen HW, Glaubitz C: Probing the molecular dynamics of the ABC multidrug transporter LmrA by deuterium solid-state nuclear magnetic resonance. Biochemistry 2007, 46:3075. 36. Kobayashi M, Matsuki Y, Yumen I, Fujiwara T, Akutsu H: Signal assignment and secondary structure analysis of a uniformly [C-13, N-15]-labeled membrane protein, H+-ATP synthase subunit c, by magic-angle spinning solid-state NMR. J Biomolec NMR 2006, 36:279.
48. Lipton AS, Heck RW, Ellis PD: Zinc solid-state NMR spectroscopy of human carbonic anhydrase: Implications for the enzymatic mechanism. J Am Chem Soc 2004, 126:4735. 49. Liao MY, Subramanian R, Yung RL, Harbison GS: Two and three dimensional nuclear quadrupole resonance in the investigation of structure and bonding. Zeitschrift Fur Naturforschung Section A-A J Phys Sci Jan-Feb, 2000, 55:29. 50. Ivanov D, Bachovchin WW, Redfield AG: Boron-11 pure quadrupole resonance investigation of peptide boronic acid inhibitors bound to alpha-lytic protease. Biochemistry 2002, 41:1587. 51. Ellis PD: Abstracts, 48th Experimental Nuclear Magnetic Resonance Conference; Daytona Beach, Florida: 2007. 52. Pooransingh-Margolis N, Renirie R, Hasan Z, Wever R, Vega AJ, Polenova T: 51V solid-state magic angle spinning NMR spectroscopy of vanadium chloroperoxidase. J Am Chem Soc 2006, 128:5190. 53. Boehr DD, Dyson HJ, Wright PE: An NMR perspective on enzyme dynamics. Chem Rev 2006, 106:3055. 54. Kern D, Eisenmesser EZ, Wolf-Watz M: Enzyme dynamics during catalysis measured by NMR spectroscopy. Methods Enzymol 2005, 394:507-524.
37. Rush TS, Powers R: The application of X-ray, NMR, and molecular modeling in the design of MMP inhibitors. Curr Top Med Chem 2004, 4:1311.
55. Antoniou D, Basner J, Nunez S, Schwartz SD: Effect of enzyme dynamics on catalytic activity. Adv Phys Org Chem 2006, 41:315-362.
38. Balayssac S, Bertini I, Falber K, Fragai M, Jehle S, Lelli M, Luchinat C, Oschkinat H, Yeo KJ: Solid-state NMR of matrix metalloproteinase 12: An approach complementary to solution NMR. Chembiochem 2007, 8:486.
56. Antoniou D, Schwartz SD: Internal enzyme motions as a source of catalytic activity: Rate-promoting vibrations and hydrogen tunneling. J Phys Chem B 2001, 105:5553.
39. Marulanda D, Tasayco ML, Cataldi M, Arriaran V, Polenova T: Resonance assignments and secondary structure analysis of E-coli thioredoxin by magic angle spinning solid-state NMR spectroscopy. J Phys Chem B 2005, 109:18135. 40. Marulanda D, Tasayco ML, McDermott A, Cataldi M, Arriaran V, Polenova T: Magic angle spinning solid-state NMR spectroscopy for structural studies of protein interfaces. Resonance assignments of differentially enriched Escherichia coli thioredoxin reassembled by fragment complementation. J Am Chem Soc 2004, 126:16608. 41. Pintacuda G, Giraud N, Picrattelli R, Bockmann A, Bertini I, Emsley L: Solid-state NMR spectroscopy of a paramagnetic protein: Assignment and study of human dimeric oxidized CuII-Zn-II superoxide dismutase (SOD). Angewandte Chemie-Int Ed 2007, 46:1079. 42. Balayssac S, Bertini I, Lelli M, Luchinat C, Maletta M: Paramagnetic ions provide structural restraints in solid-state NMR of proteins. J Am Chem Soc 2007, 129:2218. Microcrystalline cobalt(II)-substituted matrix metalloproteinase 12 was studied by SSNMR, demonstrating that the strong paramagnetism of the high spin ion can lead to useful structural insights about this enzyme. 43. Jovanovic T, McDermott AE: Observation of ligand binding to cytochrome P450-BM-3 by means of solid-state NMR spectroscopy. J Am Chem Soc 2005, 127:13816.
57. Hong M, Yao XL, Jakes K, Huster D: Investigation of molecular motions by Lee-Goldburg cross-polarization NMR Spectroscopy. J Phys Chem B 2002, 106:7355. 58. Hologne M, Chen ZJ, Reif B: Characterization of dynamic processes using deuterium in uniformly H-2, C-13, N-15 enriched peptides by MAS solid-state NMR. J Magn Reson 2006, 179:20. 59. deAzevedo ER, Bonagamba TJ, Schmidt-Rohr K: Pure-exchange solid-state NMR. J Magn Reson 2000, 142:86. 60. Ge´rardy-Montouillout V, Malveau C, Tekely P, Olender Z, Luz Z: ODESSA, a new 1D NMR exchange experiment for chemically equivalent nuclei in rotating solids. J Magn Reson Ser A 1996, 123:7. 61. Bockmann A, Juy M, Bettler E, Emsley L, Galinier A, Penin F, Lesage A: Water-protein hydrogen exchange in the microcrystalline protein Crh as observed by solid state NMR spectroscopy. J Biomolec NMR Jul, 2005, 32:195. 62. Chevelkov V, Faelber K, Diehl A, Heinemann U, Oschkinat H, Reif B: Detection of dynamic water molecules in a microcrystalline sample of the SH3 domain of a-spectrin by MAS solid-state NMR. J Biomolec NMR 2005, 31:295.
44. Lee H, de Montellano PRO, McDermott AE: Deuterium magic angle spinning studies of substrates bound to cytochrome P450. Biochemistry 1999, 38:10808.
63. Lesage A, Bockmann A: Water-protein interactions in microcrystalline Crh measured by H-1-C-13 solid-state NMR spectroscopy. J Am Chem Soc 2003, 125:13336. Heteronuclear double-quantum (DQ) filtered correlation experiments and NOE measurements are used to detect water-protein interactions, and show residence times of solvent molecules faster than 104 s 1.
45. Liu K, Williams J, Lee HR, Fitzgerald MM, Jensen GM, Goodin DB, McDermott AE: Solid-state deuterium NMR of imidazole ligands in cytochrome c peroxidase. J Am Chem Soc 1998, 120:10199.
64. Lesage A, Emsley L, Penin F, Bockmann A: Investigation of dipolar-mediated water-protein interactions in microcrystalline Crh by solid-state NMR spectroscopy. J Am Chem Soc 2006, 128:8246.
46. Frericks HL, Zhou DH, Yap LL, Gennis RB, Rienstra CM: Magicangle spinning solid-state NMR of a 144 kDa membrane protein complex: E coli cytochrome bo(3) oxidase. J Biomolec NMR 2006, 36:55.
65. Chevelkov V, Rehbein K, Diehl A, Reif B: Ultrahigh resolution in proton solid-state NMR spectroscopy at high levels of deuteration. Angewandte Chemie-Int Ed 2006, 45:3878.
47. Koder RL, Walsh JD, Pometun MS, Dutton PL, Wittebort RJ, Miller AF: N-15 solid-state NMR provides a sensitive probe of oxidized flavin reactive sites. J Am Chem Soc 2006, 128:15200.
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Current Opinion in Structural Biology 2007, 17:617–622
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Current Opinion in Structural Biology 2007, 17:617–622 This review comes from a themed issue on Biophysical methods Edited by Keith Moffat and Wah Chiu
0959-440X/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2007.10.001
Introduction Although understanding the basis of catalysis by enzymes remains a significant challenge, its study has already brought many rewards; the growing knowledge of the enzyme mechanisms has not only had considerable intellectual impact in basic science, but has also guided design of novel catalysts and inhibitors including potential therapeutics and diagnostics. In contrast to the initial focus on the elusive transition state, the extent of differential binding of the transition state has been recently debated [1], and meanwhile, the importance of probing the experimentally accessible ground state has been emphasized [2,3]. Computational studies have illustrated the powerful ability of optimal alignment of the substrate with respect to key active site residues to accelerate chemistry, as illustrated in Figure 1. Enzyme active sites in complex with substrates exhibit compression [4–6], where key reactive groups have particularly short non-bonded distances and are expected to show unusual dynamics. www.sciencedirect.com
These unusual structures are incisively studied through their NMR and vibrational signatures [7]. Chemically detailed information inherent to NMR, together with the opportunity to stabilize species at lower temperatures or even study catalysis in its native biological environment [8] makes SSNMR especially powerful for this goal. Furthermore, the recent technical advances and emerging methods in SSNMR now allow analysis of extensively labeled systems [9,10], very high resolution structures [11], and dynamics of the enzymes [12,13,14,15]. Recent advances in accessing SSNMR signatures for the reactive states of enzymes include new tools for observing waters in biological systems, observing metals including quadrupolar nuclei and paramagnetic systems, and characterizing conformational and chemical dynamics. Thus it is an opportune time to review the study of enzymes by SSNMR. SSNMR has many other exciting application areas in structural and mechanistic biology, besides enzymology, and we point the reader to some corresponding reviews: [16–20].
SSNMR of enzymes: a new range of applications Studies of a Michaelis complex
Triosephosphate isomerase (TIM), a central glycolytic enzyme, accelerates by over 10 orders of magnitude the interconversion of dihydroxyacetone phosphate and glyceraldehydes phosphate via proton transfers (Figure 2), and offers an unusual opportunity to characterize a bona fide Michaelis complex. Quantum computational investigations [21,22], and the considerable structural and spectroscopic literature on this system, emphasize the role of the general base Glu165 in abstracting the proton on C1 of DHAP and C2 of GAP and His95, in rearranging the alcohol protons for the key enediol intermediate. Loops 6 and 7 (particularly the amidic NH of Gly 171, 173 and 232) are essential for binding of the substrate’s phosphate group. The nature of the nascent prereacctive complex can be conveniently studied in this system for the simple reason that it is an isomerase with the uphill direction being of particular biological significance. Recent atomic-resolution X-ray crystal structures of the Michaelis complex [5] exhibit a compressed and bifurcated hydrogen bond network where the carboxylate group of Glu165 indicates intriguing conformational disorder. While very revealing in terms of the binding environment, these data were ambiguous regarding the chemical form of the substrate. The chemical nature and populations of the substrate intermediate and product have been recently probed using variable-temperature NMR solution and 13C Current Opinion in Structural Biology 2007, 17:617–622
618 Biophysical methods
Figure 1
Two limiting hypotheses about enzyme rate enhancements. Compared to the uncatalyzed reaction (black) the catalyzed reaction can be faster because of transition state stabilization (green), or through ground state destabilization (red).
solid-state NMR spectroscopy [25]. In contrast to prior predictions from detailed isotope effect studies, DHAP was the only chemical form observed on the enzyme both in solution and in the crystals. The discrepancy with prior analysis might be related to a previously unappreciated kinetic step, the opening of the loop on the reaction product. In fact, TIM’s reaction kinetics provided some evidence for motion of these loops during the reaction, and the rate of loop opening was therefore probed by solid state NMR [24], solution NMR [25–27], and T-jump fluorescence spectroscopy [28]. In contrast to a number of prior predictions, these studies showed that the loopopening rate is on the order of the turnover rate, and is somewhat sensitive to the details of the ligand [24,25,28]. Solid state NMR studies altered our concept of the key
intermediates and essential rate constants rates for TIM, establishing the importance of protein motion on this enzyme’s reactive pathway as well as the ability to probe these key motions by solid state NMR methods. Membrane enzymes
An important frontier for structural biology is the understanding the structures function and dynamics of membrane proteins. SSNMR studies have had significant impact into understanding of membrane channel function. It is possible to access alternative states of channels under SSNMR conditions, including states where it might be difficult to obtain crystallographic data [29]. The principles underlying enzyme function, for example stabilization by unusually strong hydrogen bond motifs
Figure 2
The proposed mechanism and reaction rates of the isomerization reaction catalyzed by triosephosphate isomerase [23]. Current Opinion in Structural Biology 2007, 17:617–622
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Solid state NMR: new tools for insight into enzyme function McDermott and Polenova 619
and distributed hydrogen bond motifs, also extend to channels, as beautifully illustrated in the M2 H+ channel from influenza A. The heart of this conductance mechanism is the hydrogen bonding interactions in the homotetrameric set of four histidine side chains. NMR experiments have been used to show that protonation of one of these imidazoles coincides with acid activation of this transmembrane channel, so that at physiological pH, the channel is closed by two imidazole-imidazolium dimers, each sharing a low-barrier hydrogen bond [30]. Thereby a pair of charges is distributed over four rings and stabilized in the low dielectric membrane environment. SSNMR has offered insights into how guanine nucleotide-binding proteins utilize transformation from GTPbound and GDP-bound states, with pronounced conformational changes, to act as molecular switches. In solution additional conformational states in the GDP as well as in the GTP forms coexist, which were not detected by X-ray crystallography [31]. The conformational changes observed by liquid-state NMR dynamics measurements were related to X-ray crystallographic structures, in that the SSNMR data clearly indicated the existence of two different conformations of the molecule in the crystalline state under some conditions [32,33]. 2H SSNMR probed the packing and molecular dynamics of ras lipid chains connected to a ras fragment. Compared to the host DMPC matrix, the ras lipids are extremely flexible, which has interesting possible consequences for function [34]. Conformational dynamics of a full length ABC multidrug efflux pump LmrA from Lactococcus lactis in lipid membranes has been investigated by deuterium SSNMR. Motional freedom is restricted upon ATP binding, and then returns to full flexibility in the posthydrolysis, vanadate-trapped state [35]. The recent appearance of many studies assigning instrinsic membrane systems, for example the initial resonance assignments have been reported for the ‘heart’ ATP synthase, the c subunit [36], will pave the way for detailed mechanistic work on this and other intrinsic membrane enzymes. Paramagnetic, redox and metal binding systems
A very large number of enzymes utilize metals for stabilization, redox facilitation and catalysis, and arguably some of the most powerful catalysts rely on metals to carry out their function. An explosion of studies of enzymes and enzyme-related proteins with metals and/or paramagnetic centers has appeared very recently, setting the stage for further detailed analysis. Matrix metalloproteins are important drug targets, where flexibility is clearly an essential aspect in relation to function [37]. SSNMR studies have been reported with wonderfully resolved lines, affording an independent probe of this important class of proteins [38]. Detailed analysis of chemical shifts has been reported recently for www.sciencedirect.com
thioredoxin, which is involved in a number of interesting redox related biochemical pathways [39,40]. Spectral analyses of several paramagnetic metalloenzymes have been conducted recently, representing an opening of a new field and a new class of systems for enzyme study. Moreover, this kind of spectroscopy has the promise of providing valuable structural constraints, just as it has for solution NMR of paramagnetic proteins. The spectral assignment of human dimeric oxidized Cu(II)–Zn(II) superoxide dismutase (SOD in the solid state has been recently reported [41]. SSNMR studies of microcrystalline cobalt(II)-substituted matrix metalloproteinase 12 (MMP-12, 17 kDa) gave 250 pseudocontact shifts, including nuclei up to more than 20 A˚ from the metal. These data were found to be in very good agreement with calculated shifts based on structure [42]. These studies have also been extended to systems of much higher molecular weight, through a combination of fortuitous resolution and selective labeling strategies. SSNMR was used to probe ligand binding and conformational state in cytrochrome P450, a system of considerable complexity [43]. This approach has the potential to access the dynamics of the enzymes in catalytically important states. Studies of the active site of other heme proteins clarified the degree of disorder in ligands proximal to the heme group through deuterium spectroscopy [44,45]. Initial SSNMR investigations have also been reported for a 144 kDa integral membrane protein, Escherichia coli cytochrome bo(3) oxidase [46]. The unique chemical shifts of the cofactors, for example flavin rings, makes SSNMR an effective way to probe larger redox enzymes [47]. Metals whose nuclei have an electric quadrupole moment represent very important players in biochemistry, but their NMR detection remained a challenge area until recent advances in spectroscopic detection of these sites. Direct detection of half-integer quadrupolar metals in proteins, even for nuclei with large quadrupolar moments, has also been achieved in several cases, offering considerable insights into the active site geometry. Detailed studies of the Zn2+ in human carbonic anhydrase (CAII), including comparison of predicted quadrupolar couplings with the experimental values, suggested the presence of hydroxide as a fourth ligand at pH 5–8.5. This observation suggests that a new mechanism for CAII may be needed [48]. The quadrupole coupling tensor for 63Cu in the active site of bovine erythrocyte (Cu/Zn) superoxide dismutase, has been measured, and comparison with the results of ab initio calculations has implications for the site symmetry [49]. A field-cycling pure quadrupole resonance experiment indicated that the environment of boron is tetrahedral in two peptide boronic acid inhibitors bound to alpha-lytic protease [50]. Direct detection of 43 Ca signal in Ca2+ calbindin allowed to distinguish the Current Opinion in Structural Biology 2007, 17:617–622
620 Biophysical methods
Figure 3
[52], and will offer insight on substrate specificity in haloperoxidases. Probing hydration and dynamics of crystalline proteins
51
V experimental (a) and simulated (b) spectrum of vanadium chloroperoxidase (c), yielding protonation environment of the resting state vanadate cofactor depicted in (d) [52].
two calcium sites [51]. 51V SSNMR of the 67.5-kDa vanadium chloroperoxidase yielded the quadrupole coupling and chemical shielding anisotropy tensors (Figure 3), which revealed the protonation states of the vanadate cofactor in the resting form, unavailable from the X-ray structure or any spectroscopic measurements
The importance of conformational plasticity of enzymes has been underscored in a recent comprehensive review of NMR studies of enzyme dynamics [53]. Typically, enzymes access multiple conformational states, including a Michaelis complex with the reactant and an open, ligated state. Their facile interconversion on the microor milli-second timescale is a crucial requirement, allowing for rapid binding and release. Knowledge of the kinetics and thermodynamics of these states has benefited greatly from NMR methodological advances [54]. Rapid thermal fluctuations surrounding the Michaelis state structure, are also of interest, because they can elicit strongly enhanced reactivity [55,56]. Improved tools to characterize dynamics have emerged that are predicated on the recent tools for assignment and structural characterization of uniformly isotopically enriched proteins. Orientation-dependent dipolar measurements [57] have been applied to probe fast molecular motions in AX/AX2/ AX3 spin systems, in small molecules and in ubiquitin [14], illustrated in Figure 4. Exciting advances have also appeared for deuterium lineshapes as a probe of protein dynamics [15,58]. Other methods are promising on slower timescales [13,59,60]. Clearly, changes in hydration of enzyme active sites care connected to thee conformatonal exchange events and have been somewhat elusive experimentally up to now. Emerging studies of water in enzyme surfaces and cavities by solid state NMR
Figure 4
Order parameters for fast limit motion were determined in the solid state for the protein ubiquitin, illustrating enhanced mobility in loops and turns [14]. Current Opinion in Structural Biology 2007, 17:617–622
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Solid state NMR: new tools for insight into enzyme function McDermott and Polenova 621
[61,62,63,64], together with increased use of high-resolution spectra of protons [65,66], are expected to be indispensable in analyzing enzyme active sites and their unusual electronic environments, making SSNMR an ever more attractive tool for enzymology. Unlike the prior generation of tools for studying dynamics by SSNMR, these emerging tools efficiently characterize many sites on the protein simultaneously and offer far more insight into function. They have already been used to demonstrate a high degree of motion is crystalline systems, and are very promising for future studies of membrane systems in their native lipid bilayer environments.
Conclusion In the past few years, new SSNMR tools for studying enzymes and reaction pathway intermediates in biology have emerged and been demonstrated on a number of interesting protein systems. Insights into strain, hydration, and conformational and chemical exchange from these methods will enhance our understanding of the remarkable function of enzymes.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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