Neoculin as a New Sweet Protein with Taste ... - ACS Publications

Chapter 35

Neoculin as a New Sweet Protein with Taste-Modifying Activity: Purification, Characterization, and X-ray Crystallography

Downloaded by IOWA STATE UNIV on April 19, 2017 | http://pubs.acs.org Publication Date: March 4, 2008 | doi: 10.1021/bk-2008-0979.ch035

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A. Shimizu-Ibuka, Y. Morita , K. Nakajima , T. Asakura , T. Terada, T. Misaka , and K. Abe 1

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Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Laboratory of Food Science, Atomi Junior College, Bunkyo-ku, Tokyo 112-8687, Japan 2

The majority of sweet compounds are of low-molecular-weight, but several proteins elicit sweet taste responses in humans. The fruit of Curculigo latifolia has been known to contain a protein that has both sweetness and a taste-modifying activity to convert sourness to sweetness. Recently, we have purified and re-identified the active component to reveal that it is a heterodimeric protein named "neoculin". The result of X-ray crystallographic analysis has indicated that the overall structure of neoculin is similar to those of monocot mannose-binding lectins, while there is little structural similarity between neoculin and structure-solved sweet proteins. Direct interaction between neoculin and human sweet taste receptor hT1R2-hT1R3 has been indicated by response of HEK293T

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© 2008 American Chemical Society

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

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cells expressing this receptor, and by the inhibition of neoculin activity with lactisole, a hT1R2-hT1R3 blocker. Combining the results of molecular dynamics simulations and docking model generation between neoculin and hT1R2-hT1R3, we propose a hypothesis that neoculin is in dynamic equilibrium between "open" and "closed" states, and that the addition of an acid shifts the equilibrium to the "open" state for easier fitting to the receptor.

Taste is a primal sense that enables organisms to accept sweet nutrients in foods and reject bitter environmental poisons. The majority of sweet molecules are of low-molecular-weight, but six proteins—brazzein, thaumatin, monellin, curculin, mabinlin and pentadin—have been shown to elicit a sweet taste response in humans (/). Previously identified sweet proteins and taste-modifying proteins have different molecular lengths, from the 54 residues of brazzein to the 202 residues of thaumatin, with no significant similarities in their amino acid sequences (7). Curculin, occurring in the fruit of Curculigo latifolia that grows in West Malaysia, is the only known protein that both elicits a sweet response and has taste-modifying activity to convert sourness to sweetness (2). Acids taste sweet in the presence of curculin. This type of taste-modifying activity is also evoked by miraculin, which has no sweetness by itself (5). Curculin was initially regarded as a homodimer consisting of two identical subunits, although the recombinant homodimer was devoid of any taste-modifying activity (2). In this chapter, we report the purification, re-identification, and crystallographic analysis of the active component, named "neoculin". We also obtained the results that indicate direct interaction between neoculin and the human sweet taste receptor h T l R 2 - h T l R 3 . These results, together with the results of molecular dynamics simulations and docking model generation, offer insights into a possible mechanism of taste-modifying activity.

Purification and Re-identification Though the protein curculin was regarded as a homodimer consisting of two identical subunits, no successful expression of the recombinant curculin with taste-modifying activity had been reported. Why does the recombinant curculin have no activity? To answer this question, we have purified and re-identified the active component contained in Curculigo latiforia. When the purified active component was subjected to SDS-PAGE and stained with C B B , it gave a main band of about 20 kDa under non-reducing



548 condition, and a main band of about 11 kDa and a faint band of about 13 kDa under reducing conditions. This active fraction was then submitted to twodimentional electrophoresis in denaturing and reducing condition, resulting in the 13 kDa and 11 kDa fractions appearing at pi 4-6 and pi 7.5-9.5, respectively (data not shown). These data indicated that the active component was a 20 kDa heterodimer consisting of 13 kDa and 11 kDa subunits connected by disulfide bond(s). We named this heterodimeric protein "neoculin (NCL)". The 13 kDa fraction was referred to as "neoculin acidic subunit (NAS)" and the 11 kDa fraction as "neoculin basic subunit (NBS)". The results of N-terminal protein sequencing indicated that the N B S had an N-terminal sequence identical to that of curculin, while N A S was an apparently new polypeptide sharing 80% amino acid identity with N B S in this N-terminal region (Figure 1). From the analysis of proteinase-digested N A S with protein sequencer, and from the nucleotide sequences of cDNA, we determined the whole N A S sequence as shown in Figure 1 (4). N A S and N B S shared 77% amino acid identity. These neoculin subunits exhibit high degrees of amino acid sequence similarity to monocot mannose-binding lectins, such as those from garlic, daffodil and snowdrop, showing 42-46% amino acid identity. It suggests that they share a common architecture in their three-dimensional structures, though there is no known functional similarity between neoculin and these molecules. In fact, we confirmed that neoculin had no hemagglutinin activity.

X-ray crystallographic analysis The structural basis of sweetness has been studied extensively for thaumatin, monellin and brazzein and amino acid residues important for sweetness of these proteins have been reported previously (5-77). However, no common structural features have been identified among these proteins. Since little structural information is available for the taste-modifying proteins, we have tried to analyze the three-dimensional structure of neoculin. The neoculin crystal structure was solved by molecular replacement and refined at 2.76 Â (Figure 2A) (72). Eight polypeptide chains from A through H form four crystallographically independent heterodimers, A B , C D , E F and G H , respectively, in the asymmetric unit. The chains A , C, Ε and G correspond to N A S , while the chains B , D , F and H correspond to N B S . The structures of the two subunits, N A S and N B S , are very similar to each other and are superimposable (Figure 2B). Richness of disulfide bridges is common to some sweet proteins. Thaumatin and brazzein have eight and four disulfide bonds, respectively (5,75). In the neoculin molecule, all eight cysteine residues participate in the formation of disulfide bridges, and thus there are four disulfide bonds in the heterodimer. Two



Figure I. Sequence alignment of neoculin subunits and monocot mannose-binding lectins. The residues conserved in all of the molecules are shown in black and those conserved mainly in neoculin subunits are shown in grey. The N-terminal amino acid sequenses analyzed with protein sequencer are double-underlined. N-glycosylation site in NAS is asterisked.



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Figure 2. Ribbon diagram of neoculin. Neoculin acidic subunit (NAS) is shown in white and neoculin basic subunit (NBS) in grey. A) Overall structure of neoculin heterodimer. The β-strands of each subunit are labeledfrom Bl to Β12. The cysteine reisdues forming disulfide bonds and the sugar molecule bound to AsnSl ofΝ AS are shown as ball-and-stick model. B) Superposition of the two subunits, NAS and NBS. (Reproduced with permissionfromref 12 with minor modification.)



551 are intra-subunit disulphide bonds, between Cys29 and Cys52 in each subunit, while the other two are inter-subunit disulphide bridges, between Cys77 of one subunit and Cysl09 of another (Figure 2A). Intra-subunit S-S bonds are also observed in the mannose-binding lectins, while inter-subunit S-S bonds are not. Neoculin and the sweet proteins with known three-dimensional structures, namely monellin, thaumatin and brazzein, show no obvious similarity in their tertiary structures, although there is broad similarity in their richness of β-sheet structures. Meanwhile, as predicted from the high degree of amino acid sequence homology between neoculin and the mannose-binding lectins, the crystal structure of neoculin shows striking similarity to those of mannose-binding lectins, having the overall same structural topology (Figure 3A). The most pronounced structural difference between neoculin and the lectins is observed in the C-terminal regions of both subunits (Figure 3B). In both N A S and N B S , the 12th β-strand Β12 is composed of only three or four residues from 100 to 102 or 103, and a subsequent large turn is fixed by an inter-subunit disulfide bond between Cysl09 and Cys77. In the lectins, the corresponding regions stretch straight over the surface of another subunit. Such differences in the C-terminal regions effect the subunit-subunit interactions. The interface of two subunits is mainly composed of N-terminal β-strand B l and C-terminal β-strands from BIO to B l 1 or Β12 in both neoculin and the lectins. The buried surface area between two subunits of neoculin is smaller than that of the lectins, suggesting weaker interaction between the two subunits in neoculin. The shape of the protein surface and the electrostatic potential on the surface are also significantly different between neoculin and the lectins. These differences reflect the fact that the residues protruding at the surface of the molecules are those which are less conserved between neoculin and the lectins. It is characteristic that the distribution of basic residues is non-homogeneous, especially in N B S . In N B S , there are 13 basic residues, consisting of seven arginines, three lysines, and three histidines. Six of them, H i s l 1, His 14, Lys28, Arg47, Arg48, and Arg53 compose a large cluster of basicity on the surface of N B S (Figure 4). Such a large cluster of charged residues is not observed in the lectins. Since it is suggested that the basicity of broad surface regions plays a significant role in the elicitation of sweetness in some sweet proteins(0,/0,/4), this cluster of basic amino acids in N B S may contribute to the sweetness or tastemodifying activity of neoculin. This basic cluster includes two histidine residues, with pKa of 6.0. Since neoculin elicits only slight sweetness at neutral p H but strong sweetness at acidic pH, these histidine residues might be essential to the sweetness and/or taste-modifying activity of neoculin. The structure also shows that three out of four basic residues located at the dimer interface of neoculin are unique to neoculin. Such location of basic amino acids is not observed in the lectins and may be the cause of the pH-dependent conformational change.



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Figure 3. Comparison between neoculin and monocot mannose-binding lectins. A) Superposition of neoculin (dark grey), garlic lectin (light grey), snowdrop lectin (white). Methyl-a-D-mannose molecules bound to the snowdrop lectin are shown as ball-and-stick models. B) Superposition of neoculin and the lectins viewedfromthe NBS side. NAS and corresponding subunits in the lectins are drawn with light grey. (Reproduced with permission from reference 12 with minor modification.)

Figure 4. Polar residues on the surface of neoculin. A large basic patch is indicated with an oval dotted line.


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Possible p H effect on the neoculin structure The crystal structure of neoculin solved in this study only represents the structure at neutral pH, since the protein was crystallized at p H 7.4. The neoculin heterodimer has 16 aspartates, 5 histidines, with no glutamate residues contained. Judging from the crystal structure, the titratable groups of these residues are unprotonated at neutral pH. Furthermore, it can be assumed that all of them are protonated at acidic p H (around 2.5), since they are exposed to the solvent. The results of constant-pH molecular dynamics simulations were consistent with this assumption. At neutral pH, N A S and N B S carries - 3 and +3 charges, respectively, whereas they carry +8 and +15 as a result of protonation of the titratable groups and the C-termini. Such a shift in charge profile causes a drastic change in the electrostatic interaction between the monomers. We speculated about whether the strong repulsion between the positive charges might alter the structure of neoculin and about a possible correlation between its strong sweet taste at acidic p H and the neoculin structure. We have performed molecular dynamics simulations on the protonated and unprotonated states of neoculin to estimate the extent of the effect of the p H on the neoculin structure(72). Figures 5 shows representative structures obtained from the simulations on the unprotonated (i.e. neutral pH) and protonated (i.e. acidic pH) states, respectively. These suggest that the protonated neoculin would have a tendency to take a widely "open" conformation, while unprotonated neoculin is in a "closed" conformation, similar to the crystal structure. Together with the observation that neoculin elicits slight sweetness even when dissolved in pure water (pH 7.0), we propose that the neoculin structure is in equilibrium between "open" and "closed" states. The equilibrium may be shifted to the "closed" state at neutral p H and to the "open" state at acidic pH, with only the neoculin molecules in the "open" state eliciting strong sweetness.

Interaction with the human sweet-taste receptor h T l R 2 - h T l R 3 Most sweet molecules are thought to interact with the sweet taste receptor, h T l R 2 - h T l R 3 , which is known to mediate the recognition of diverse natural and synthetic sweeteners, including sweet proteins(/5). Based on the docking of the sweet proteins into the models of h T l R 2 - h T l R 3 , it has been hypothesized that these proteins elicit sweet taste through binding to a large cavity of the receptor(/6,/7). Direct interaction between neoculin and h T ! R 2 - T l R 3 has been indicated by two experiments. Firstly, a response to neoculin was observed when h T l R 2 , h T l R 3 , and promiscuous G protein (G16/gust25) were coexpressed in HEK-293 cells(75). Most of the neoculin-responsive cells responded also to the application of low molecular-weight sweeteners such as aspartame and saccharin (Figure 6).



Figure 5. Representative neoculin structures under unprotonated state (left) and under protonated state (right), generated by molecular dynamics simulations.



555 Secondly, sweet-taste response to neoculin was inhibited in the presence of lactisole, a h T l R 2 - h T l R 3 blocker (18) (Figure 6). In addition, the result of human sensory analysis showed the inhibition of taste-modifying activity of neoculin by lactisole (Figure 7). Therefore, we hypothesized that the target of neoculin, as well as of the other sweeteners, is the T1R2-T1R3 receptor and that we could gain insight into how neoculin elicits sweetness and taste-modifying activity through docking with the receptor. There is a contradiction of pH values where the sweetness is elicited between in vivo and in vitro results. This contradiction might be caused by following two factors. First, in the cell-based assay (Figure 6), the mode of receptor activation would be somehow different (i.e. G-protein coupling and additional membrane protein with supporting function, etc) from that of in vivo. Second, in human sensoiy test (Figure 7), we could not precisely measure the in situ p H at the taste pore. Therefore we did not compare the pH values directly.

The putative mechanism of taste-modifying activity Prior to the generation of the docking models, we modelled the tertiary structures of the hTlR2-hTlR3 receptor based on sequence similarity to the N terminal region of the metabotropic glutamate receptor (mGluR)(/9), as has been performed in previous sUxd\es(l6,17,20,21). At present, two different forms of the mGluR structure are available. One is the inactive or resting open-open form of mGluR (PDB entry: 1EWT) and the other is the active closed-open form of mGluR (PDB entry: ÎEWV), whose structure is almost identical to that in complex with glutamate (PDB entry: 1EWK). Here, we modelled all possible forms of the h T l R 2 - h T l R 3 receptor, following the method of Morini et al.(77), i.e. four models designated R o o A B (T1R2 modelled on chain A and T1R3 modelled on chain Β of the resting open-open form), R o o B A (T1R2 modelled on chain Β and T1R3 modelled on chain A), A o c A B (T1R2 modelled on chain A and T1R3 modelled on chain Β of the active open-close form), and A o c J B A (T1R2 modelled on chain Β and T1R3 modelled on chain A). The representative structures obtained from the simulations on unprotonated and protonated states were docked into each model of the receptor. About 10,000 docking models were generated for each pair and we selected the best candidates on the assumption that the interaction surface area reaches a maximum when the cavities of neoculin and one of the subunits of the hTlR2-hT!R3 receptor face each other with the long axis of neoculin vertical to the long axis of the receptor. From the calculations with the neoculin model in the protonated state, nine preferable solutions were obtained. Among these solutions, neoculin was accommodated in the large cavity of h T l R 2 in six solutions and in that of h T l R 3 in three solutions. In contrast, no preferable solution was obtained from the calculations with the neoculin model in the unprotonated state(/2). These results


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MSG NCL

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Time (sec) Figure 6. Line trace of the ratiometric value changes for the representative HEK293Tcell expressing hTlR2/TlR3. Black arrows indicate the time of the application of 10 mM monosodium glutamate (MSG), 20 μΜ neoculin (NCL), 20 μΜ NCL containing 2.5 mM lactisole (NCL+lac), 10 mM aspartame (Asp), and 10 mM saccharin (Sac).

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PH Figure 7. Evaluation of the taste-modifying activity of neoculin (NCL) in weak acidic buffer. Panelists tasted aspartame solutions at three different concentrations (0.1, 0.5, and 2.0 mM) and rated the sweetness on a scalefrom1 to 7: 7for >2.0, 6for 2.0, 5for 0.5-2.0, 4for 0.5, 3for0.1-0.5, 2for 0.1, and 1 for

Neoculin as a New Sweet Protein with Taste ... - ACS Publications

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