Sweetness and Sweeteners - American Chemical Society

Chapter 7

Crystal Structures of the Sweet Protein MNEI: Insights into Sweet Protein-Receptor Interactions 1

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Jeanette R. Hobbs , Steven D. Munger , and Graeme L. Conn

Downloaded by FUDAN UNIV on April 19, 2017 | http://pubs.acs.org Publication Date: March 4, 2008 | doi: 10.1021/bk-2008-0979.ch007

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Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, Manchester M l 7DN, United Kingdom Department of Anatomy and Neurobiology, University of Maryland School of Medicine, 20 Penn Street, Baltimore, MD 21201

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Monellin is one of a small number of highly potent, sweet-tastingproteins. X-ray crystal structures of a wild-type single chain monellin (MNEI) and a 10-fold less sweet mutant (G16A) were determined in order to understand the cause of this reduction in sweetness and gain insight into sweet protein-receptorinteractions. Comparison of the two structures reveals little change to the global protein fold. However, alterations of amino acid side chain position and exposure adjacent to the site of mutation result in a reorganization of key functional groups on the surface of M N E I . This finding supports the idea that an extensive surface of monellin is involved in binding the T1R2:T1R3 receptor.

© 2008 American Chemical Society Weerasinghe and DuBois; Sweetness and Sweeteners ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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110 The protein monellin is a highly potent sweet stimulus: on a molar basis, it is many thousands of times sweeter than sucrose (1). It is sweet to humans and some Old World primates, but is not preferred by other mammals. Natural monellin from the African 'Serendipity Berry* (Dioscoreophyllum cumminsii) is composed of two chains, A and B , of 44 and 50 amino acids respectively. Single chain monellin proteins were created that enhance its thermal and chemical stability; the two natural chains (B-A) are either directly connected (SCM) (2) or joined by a dipeptide linker (MNEI) (3). Despite extensive characterization of the sweet protein (4-12), little is known about the interaction of monellin with the T1R2.T1R3 sweet taste receptor. The concept that sweet proteins might share a common structural motif (13), a so-called 'sweet finger', that in some way mimics the binding of small molecular weight ligands has been largely discarded as no such motif has been identified (14). To date, modeling studies using a T1R2:T1R3 model based on the mGluRl receptor ectodomain (15), have provided the best route to understanding sweet protein-receptor interactions. Such modeling studies suggest that the major binding site for several sweet proteins resides within the T1R amino terminal domains (NTDs) (16, 17), as has been demonstrated experimentally for some small molecule ligands (18). However, the interaction surface may be more extensive than for small molecule sweeteners and such a recognition mechanism, with the high affinity it suggests, can readily provide an explanation for the high potency and persistent aftertaste of sweet proteins. Experimentally, much less has been determined to date. Activation of the T1R2:T1R3 receptor by the sweet protein brazzein is dependent upon the cysteine-rich linker between the transmembrane domain and the Venus Flytrap Module ( V F T M ) of T1R3 (19), in addition to any interactions with the V F T M itself. Very recently, mutants of M N E I with altered charges on the protein surface provided some initial experimental validation of the 'wedge model (16). These results suggest that surface and charge complementarity are important components of the MNEI-receptor interaction. We sought to learn more about how M N E I binds the sweet receptor by examining the structure of mutant M N E I proteins with known reductions in sweet taste. 1

Wild-type and G16A Mutant MNEI Crystal Structures The crystal structures of wild-type M N E I and G16A mutant were determined by molecular replacement. The wild-type structure has been refined to the highest resolution (1.15 A ) of any monellin structure available to date (20). Electron density maps for both proteins showed well-defined connected density but with some indications of disorder for G16A revealed by broken density around the more dynamic regions of the protein (i.e. loop regions). The mutation at position 16 was clearly visible in a F - F difference density map. 0

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Weerasinghe and DuBois; Sweetness and Sweeteners ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

Ill Monellin has a secondary structure consisting of five β-strands that form an antiparallel β-sheet (βΐ to β5), and a 17-residue α-helix ( a l ) cradled in the concave face of strands β2-β5 (Figure 1A). In single chain monellins, such as MNEI, the β-strands β2 and β3 are joined by the engineered loop L 3 (residues 47 to 56). The polypeptide chain ends with a short sequence containing four proline residues; three of these, Pro94-96 form a 3 polyproline II helix. Both structures are fully refined and of high quality; Ramachandran plots (21) indicate all 96 residues are in the 'Favorable' or 'Allowed' regions for both proteins. 2


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Figure 1. Crystal structure of wild-type MNEI.

A Monomeric Crystal Form of Monellin (MNEI) Previous natural and single chain monellin structures (4-6) have invariably contained the protein packed in such a way as to suggest a homodimeric complex may be present (in the case of natural monellin comprising two copies of both Chain A and Chain B). This observation led to the suggestion that this might be the functional form of the protein. However, this was contradicted by native gel analysis (22) and other solution studies (7, 23) that argued monellin exists as a monomer. While the G16A MNEI crystal shows the typical monellin dimer, generated by rotation of the protein about a crystallographic two-fold axis, the wild-type crystal is remarkably different. Although in a space group (P2|) previously observed for monellin (5) this crystal has a markedly different crystal packing arrangement: a single monellin molecule is contained in the asymmetric unit and no dimer interface is observed (Figure 1B). Light scattering y


112 measurements on protein samples used for crystallization yielded molecular weights of ~11.8 kDa, corresponding to the M N E I monomer, for both wild-type and G16A M N E I proteins. Thus the two proteins, one of which crystallizes as a monomer and the other as a dimer, both exist as monomelic proteins in solution.

The MNEI Protein Fold is Unaffected by the G16A Mutation


The wild-type and G16A M N E I crystal structures are globally very similar to each other and to other monellin structures (Figure 2), indicating that the G16A mutation has little effect on the protein fold.

Figure 2. The G16A mutation does not affect the global fold of MNEI but does cause sidechain alterations that extend across the protein surface.

This was confirmed using pairwise superpositions of the C a atoms of residues 1-46 and 57-96 (residues located on L23 were excluded, as these are intrinsically flexible). This confirmed that the wild-type and G16A polypeptide backbones are very similar to each other (r.m.s.d. 0.65 Â), and to natural monellin (3MON) (5), orthorhombic natural monellin (4MON) (6), and S C M (1MOL) (5), with r.m.s.d. values < 0.86 Â for each possible alignment. Comparison of both our crystal structures to the solution N M R structure of wildtype M N E I (1FA3) (7) was similarly favorable, with an r.m.s.d. for alignment of approximately 1.4 A for both. In contrast, the solution structure of G16A-MNEI (1M9G) (24) gave considerably larger r.m.s.d. values for alignment to both wildtype (r.m.s.d. 4.57 A) and G16A M N E I (r.m.s.d. 4.46 A) crystal structures. Our G16A M N E I crystal structure indicates that this wild-type backbone fold is an energetically favorable one for the mutant protein. However, we note that the


113 large differences observed in the G16A M N E I solution structure, due to an apparently greater degree of flexibility (24), may reflect an important property of the mutant protein that contributes to a reduced binding affinity for T1R2:T1R3 and therefore sweetness.


Changes Due to the G16A Mutation Extend Across the Surface of MNEI The G16A mutation is located in the inside of the α-helix, opposite the short P2a strand. The addition of the methyl group directly perturbs the surrounding residues, in particular, VaI37 directly opposite the site of mutation. The terminal methyl groups on Val37 are rotated away from A l a l 6 and displaced by approximately 0.6 Â to avoid a steric clash. Several residues important for monellin sweet taste (Table I) located in the vicinity of A l a l 6 and Val37 are also found in different conformations, apparently affected by the mutation. One helical turn above the mutation site, Gin 13 adopts an alternate rotamer. In the wild-type M N E I structure, the amine group of Gin 13 points towards the core of the protein, hydrogen bonding to the backbone of Val37, while the carbonyl group is exposed on the surface of the helix. In the mutant structure, the head of the Gin 13 sidechain is rotated, such that both the amine and carbonyl groups are exposed on the surface, roughly parallel with the helical axis. Two other important residues, Phe34 and Lys36, are located near Val37. Again, each is found in a different rotamer in the G16A mutant structure and has a significantly altered side chain position. Beyond the N-terminus of the α-helix, a change is also observed in the position of Asp7. In the wild-type structure, Asp7 forms a salt bridge with Arg39 on the surface of the protein; this interaction is maintained in the mutant but Asp7 is rotated to a roughly orthogonal orientation (Arg39 also adopts an alternate rotamer). As a result, the functional groups of Asp7 are significantly altered in orientation and now point downwards in the direction of the helix.

Table I. The Effect of the G16A Mutation on Other Residues Important for MNEI Sweetness Residue Asp 7 Gin 13 Phe34

Fold reduction in sweetness" >200 8 7-10

Alignment r.ms.d (Af 2.10(1.01) 1.96(0.10) 1.74(0.48)

Surface Exposure Change (%) 9.4 42.1 4.9

'Values from various studies (8, 9, 12). Some mutations were to nonnatural amino acids. Values for sidechain and backbone atoms (in parenthesis). b


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Implications for MNEI Sweet Taste and Receptor Binding The G16A mutation causes negligible change to the protein fold and, as a buried position, is unlikely to directly contribute to the reduction in sweetness observed for the mutant protein (25). We sought to quantitate the observed alterations in amino acid sidechain conformations surrounding G16A, including several that are important for sweet taste. The wild-type and G16A M N E I structures were aligned with all sidechain atoms included in the calculation (but excluding residues 47-57 as before). The calculation indicated that the positions of several important residues were altered in the mutant. As shown in Table I, each of the residues around the site of mutation, Asp7, Gin 13, and Phe34, has a sidechain r.m.s.d. value at least two times greater than that for the mainchain atoms. In addition to sidechain position/ conformation, a major influence on the interaction of M N E I with the T1R2:T1R3 receptor will arise from the accessibility of these important residues on the protein surface. We therefore measured the solvent exposed surface area and protein volume of each structure. Two residues, Gin 13 and Lys36, are significantly more exposed on the G16AM N E I surface due to the changes in their conformation. Overall, the changes are small as expected given the similarity of the protein backbones: G16A M N E I has a slightly reduced total surface area (-2.4 %) but, appropriately for addition of a bulkier side chain in the protein core, an increased protein volume (-1.0 %). Thus, major changes in conformation and surface exposure due to the G16A mutation are only present at the level of individual residues, several of which are important for monellin sweet taste. Differences in crystallization solution conditions and crystal packing could potentially cause similar alterations in surface amino acid conformations. However, here the protein samples were prepared in the same buffer and crystallized at the same p H . Furthermore, other crystal forms of monellin indicate that Asp7 is invariably found in the same conformation that we observe in our wild-type M N E I structure, regardless of crystallization conditions or crystal packing. Phe34 is found in several similar positions but its position in the G16A structure is at one extreme of the positions observed. Gin 13 is found in the same conformation as for our wild-type M N E I structure in all other structures except one, the structure of S C M (5). Interestingly, here the position of Val37 also aligns perfectly in S C M and our G16A mutant structure. While in the case of S C M it is not clear what is the cause of the movement in Val37 (there is no steric clash in the protein core as for G16A), the resulting effect on the key residue Gin 13 is the same. Together, these observations indicate that the differences observed between wild-type M N E I and G16A-MNEI in key residues for M N E I sweetness across the surface of the protein can be attributed to the G16A mutation.


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Conclusions Sweet proteins have great potential as sweeteners and could be particularly beneficial to individuals such as diabetics who must control sugar intake. For this potential to be realized, a full understanding of the interaction between sweet proteins such as monellin and the T1R2.T1R3 sweet receptor is needed. We determined high resolution crystal structures of the sweet protein M N E I and a 10-fold less sweet mutant. The G16A mutation does not directly cause the loss of sweetness: there is no major change in protein structure nor a dramatic alteration to any one critical determinant for binding T1R2:T1R3. Instead, more subtle alterations in sidechain conformation and accessibility extend across the surface of M N E I affecting several key residues for sweetness. Modeling studies suggest that sweet proteins bind the receptor NTD(s) through an extended surface. This idea is supported by our observation that an extended surface of M N E I is affected by the G16A mutation. It is not yet possible to distinguish the contribution of key residues within this extended surface to binding, the induction of changes in receptor conformation and/ or receptor activation. This will require further detailed analysis of TIR-monellin interactions such has recently been begun for low molecular weight sweeteners (18).

Acknowledgements X-ray diffraction data were collected at the European Synchrotron Radiation Facility, Grenoble (ID14-2). Work in the Conn and Munger laboratories is generously supported by grants from NIH (ROI DC05786) and The Wellcome Trust.

References 1. 2. 3. 4. 5. 6.

Morris, J. Α.; Cagan, R. H . , Biochim. Biophys. Acta 1972, 261, 114-122. K i m , S. H . ; Kang, C. H . ; Kim, R.; Cho, J. M.; Lee, Y. B.; Lee, T. K . Protein Eng. 1989, 2, 571-575. Tancredi, T.; Iijima, H . ; Saviano, G . ; Amodeo, P.; Temussi, P. A . FEBS Lett. 1992, 310, 27-30. Ogata, C.; Hatada, M.; Tomlinson, G . ; Shin, W. C.; K i m , S. H . Nature 1987, 328, 739-742. Somoza, J. R.; Jiang, F.; Tong, L . ; Kang, C. H . ; Cho, J. M.; Kim, S. H . J. Mol. Biol. 1993, 234, 390-404. Bujacz, G.; Miller, M.; Harrison, R.; Thanki, N.; Gilliland, G . L.; Ogata, C. M . ; Kim, S. H . ; Wlodawer, A . Acta Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 713-719.


116 7.

8. 9. 10.


11.

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

Spadaccini, R.; Crescenzi, Ο.; Tancredi, T.; De Casamassimi, N.; Saviano, G.; Scognamiglio, R.; D i Donato, Α.; Temussi, P. A. J. Mol. Biol. 2001, 305, 505-514. Ariyoshi, Y.; Kohmura, M. J. Synth. Org. Chem. Jpn. 1994, 52, 359-369. Kohmura, M.; Nio, N.; Ariyoshi, Y . Biosci. Biotechnol. Biochem. 1992, 56, 1937-1942. Kohmura, M.; Nio,N.;Ariyoshi, Y. Biosci. Biotechnol. Biochem. 1992, 56, 472-476. Niccolai, N.; Spadaccini, R.; Scarselli, M.; Bernini, Α.; Crescenzi, Ο.; Spiga, Ο.; Ciutti, Α.; D i Maro, D.; Bracci, L . ; Dalvit, C.; Temussi, P. A . Protein Sci. 2001, 10, 1498-1507. Somoza, J. R.; Cho, J. M.; Kim, S. H . Chem. Senses 1995, 20, 61-68. Kim, S. H.; Kang, C. H.; Cho, J. M. ACS Symp. Ser. 1991, 450, 28-40. Tancredi, T.; Pastore, Α.; Salvadori, S.; Esposito, V . ; Temussi, P. A . Eur. J. Biochem. 2004, 271, 2231-2240. Kunishima, N.; Shimada, Y.; Tsuji, Y . ; Sato, T.; Yamamoto, M.; Kumasaka, T.; Nakanishi, S.; Jingami, H.; Morikawa, K. Nature 2000, 407, 971-977. Temussi, P. A . FEBS Lett. 2002, 526, 1-4. Morini, G.; Bassoli, Α.; Temussi, P. A . J. Med. Chem. 2005, 48, 5520-5529. Nie, Y.; Vigues, S.; Hobbs, J. R.; Conn, G . L . ; Munger, S. D. Curr. Biol. 2005, 15, 1948-1952. Jiang, P. H.; Ji, Q. Z.; Liu, Z.; Snyder, L . Α.; Benard, L . M. J.; Margolskee, R. F.; Max, M. J. Biol. Chem. 2004, 279, 45068-45075. Hobbs, J. R.; Munger, S. D.; Conn, G . L . Acta Crystallogr., Sect. F (In press). Ramachandran, G . N., Sasisekharan, V . Adv. Protein Chem. 1968, 23, 283437. Morris, J. Α.; Martenso, R.; Deibler, G.; Cagan, R. H . J. Biol. Chem. 1973, 248, 534-539. Lee, S. Y . ; Lee, J. H . ; Chang, H . J.; Cho, J. M.; Jung, J. W.; Lee, W. T. Biochemistry 1999, 38, 2340-2346. Spadaccini, R.; Trabucco, F.; Saviano, G . ; Picone, D.; Crescenzi, O.; Tancredi, T.; Temussi, P. A . J. Mol. Biol. 2003, 328, 683-692. Iijima, H.; Morimoto, K . Ann. NY. Acad. Sci. 1995, 750, 62-65.


Sweetness and Sweeteners - American Chemical Society

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