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Structural dissection of helianthamide reveals the basis of its potent inhibition of human pancreatic #-amylase Christina Rose Tysoe, and Stephen G. Withers Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00825 • Publication Date (Web): 04 Sep 2018 Downloaded from http://pubs.acs.org on September 5, 2018
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Biochemistry
Structural dissection of helianthamide reveals the basis of its potent inhibition of human pancreatic α-amylase. Christina Tysoe† & Stephen G. Withers* AUTHOR ADDRESS Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia, V6T 1Z1, Canada AND Michael Smith Laboratories, 2125 East Mall, Vancouver, British Columbia, V6T 1Z4, Canada. KEYWORDS Diabetes; Alpha-amylase; Enzyme inhibition; Peptide synthesis; Site-directed mutagenesis Supporting Information Placeholder ABSTRACT: Helianthamide is a potent inhibitor of hu-
man pancreatic α-amylase (KI = 0.01 nM) produced by the Caribbean sea anemone Stichodactyla helianthus. Helianthamide was previously shown to be structurally homologous to the β -defensins, and represents a new structural class of protein inhibitors of α-amylase. In order to understand the source of this potent inhibition, site-directed mutagenesis studies were carried out on helianthamide-fusion proteins. A novel YIYH inhibitory motif that interacts with conserved active site residues was originally proposed as central to inhibitory activity based on the X-ray crystal structure of the PPAhelianthamide complex. However variants in which these polar residues were replaced, individually, by alanine or phenylalanine bound only 5- to 46-fold more weakly than wild-type helianthamide, suggesting modest contributions from these interactions. In contrast, individual replacement of helianthamide’s six cysteine residues with alanine resulted in much larger decreases in potency (up to a 1.3 x 104 increase in KI compared to the wildtype). In a complementary approach, a series of small peptides based on helianthamide’s sequence was synthesized and tested. Of these 19 synthetic peptides, only two showed any appreciable affinity for HPA, with inhibition constants of 141 and 396 µM, significantly higher than that of intact helianthamide. These results suggest that helianthamide’s potent HPA inhibition does not rely so much on the accumulation of individual polar contacts but rather its ability to form an extensive hydrophobic interface with the enzyme and occlude the active site cleft.
levels and its controlled inhibition has been identified as a viable means of controlling blood glucose levels towards the management of diabetes2. A number of organisms produce α-amylase inhibitors as a defense against predators, and many structural classes of α-amylase inhibitors have been previously identified3. The recently discovered helianthamide, from the Caribbean sea anemone Stichodactyla helianthus, represents a new class of α-amylase inhibitor. Helianthamide is a 44-residue peptide with three disulfide bonds in a 1-5, 2-4, 3-6 topology (Figure 1). Previous analysis of helianthamide’s 3D structure indicated that it was structurally homologous to the β-defensins, a class of small antimicrobial peptides. Helianthamide is a remarkably potent inhibitor of HPA with a KI = 0.01 nM, making it one of the most potent α-amylase inhibitors known to date4.
A
B
C6 C33 C16 C38 C39
C20
C ESGNSCYIYHGVSGICKASCAEDEKAMAGMGVCEGHLCCYKTPW
Human pancreatic α-amylase (HPA) carries out the endo-hydrolysis of ingested starches into maltose, maltotriose and related oligosaccharides1. HPA activity is positively correlated to post-prandial blood glucose
Figure 1. (A) Structure of helianthamide. β-sheets are depicted in orange, helices in red, loops in blue, and disulfide bonds in yellow. (B) Structure of PPA-helianthamide com-
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plex. PPA is shown in white and helianthamide is shown as blue spheres. (C) Helianthamide sequence. (PDB: 4X0N)
Previous attempts to co-crystallize the helianthamide with HPA were unsuccessful. However, the inhibitor did co-crystallize with porcine pancreatic α-amylase (PPA). PPA and HPA have 92% sequence similarity and identical folds, allowing for meaningful conclusions to be drawn from the porcine structure1. X-ray crystallographic analysis of helianthamide in complex with PPA showed that the inhibitor forms a 1:1 non-covalent complex with the enzyme3. Approximately 33% of helianthamide’s water-exposed surface area is buried upon formation of this complex4. Within the amylase active site, residues Y7, I8, Y9 and H10 of helianthamide formed the majority of the polar contacts with enzyme residues (Figure 2a). The localization of these interactions with the highly conserved catalytic carboxylates highlighted this region as a potentially novel inhibitory motif for α-amylase since this sequence could form the basis for smaller, simpler amylase inhibitors. In this report we describe two approaches to assess the potential of “mini”-helianthamides. In one approach the contributions of the individual side-chains in the YIYH sequence as well as two flanking serines that form interactions (Figure 2b), were assessed by construction of S5, Y7, Y9, H10 and S13 variants and measuring their affinity for HPA. In the other approach a series of peptides representing different sections of helianthamide was synthesized and their Ki values assessed. A
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ously explored as a means of producing active helianthamide, and this construct was used for our variant screen4. The barnase’-helianthamide fusion binds 50fold more weakly to HPA than does helianthamide itself, with a KI = 0.5 nM. Given the challenge of measuring inhibition constants of an extremely tight-binding inhibitor such as helianthamide, we used the weaker-binding bar’-hel fusion as the baseline for our site-directed mutagenesis study. While placement of the barnase at the N-terminus could interfere with interactions in that region, it was anticipated that the relative activities of the bar’-hel fusion variants should correlate to those of native helianthamide. S5A, Y7A, Y7F, Y9A, Y9F, H10A, and S13A variants of bar’-hel were expressed in Escherichia coli and tested as inhibitors of HPA using the Morrison method for analysis of tight-binding inhibitors5. This quadratic model accounts for the change in inhibitor and enzyme concentration as a result of the high affinity and describes the fractional initial reaction velocity as a function of [I]. Results for the kinetic analysis of the binding of Y7A, Y9A and H10A mutants are shown in Table 1. Overall, the effects of individual side chain replacements were small, ranging from a 5-fold decrease for Y7A to a 46-fold decrease for Y9A when compared to the wildtype. Cumulatively, the interactions of the Y7, Y9 and H10 side chains only contributed approximately 6 kcal/mol to binding affinity. Given the very high affinity of helianthamide, this suggests that other protein-ligand interactions must be making important contributions to inhibitor potency.
B
Table 1. Kinetic analysis of barnase’-helianthamide fusions against wild-type HPA and a T163R HPA variant.
Helianthamide Type
HPA KI (nM)
T163R HPA KI (nM)
Bar’-hel
0.5 ± 0.1
20 ± 7
Bar’-hel-Y7A
2.5 ± 0.1
107 ± 9
Bar’-hel-Y9A
23 ± 1
960 ± 88
Bar’-hel-H10A
9.0 ± 0.4
321 ± 57
C
Assays conducted at 30 degrees Celsius. Error determined through least mean squares method or
Figure 2. Highlighted areas of contact between helianthamide and PPA. Helianthamide is shown in blue while PPA is shown in grey. (A) Interactions between the YIYH motif of helianthamide and residues within the PPA active site. (B) Interactions between serines of helianthamide and a loop of high thermal motion in PPA. (C) Hydrophobic interface between helianthamide and V163 of PPA. Use of a T163R variant of HPA allowed analysis of the effects of disrupting this region. (PDB: 4X0N)
The recombinant expression of an N-terminal barnase’-helianthamide fusion (bar’-hel) had been previ-
through fit errors produced in Grafit
Indeed, the contribution of hydrogen bonding from these positions is particularly small, since the more sterically conservative Y7F and Y9F variants, which miss only their phenolic hydroxyl group, bound with significantly higher affinity than the alanine variants,Y9F exhibiting a KI = 2.2 nM and the Y7F variant binding with the same affinity as wild-type bar’-hel (KI = 0.5 nM). The S5A and S13A variants also bound with a KI = 0.5 nM suggesting that these interactions also contribute little to affinity. However, since S5 is so close to the N-
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Biochemistry
terminus, where the fusion point with barnase’ is located, it is possible that those interactions are already compromised. In addition to the polar contacts mentioned previously, there appears to be a major hydrophobic interface (~400 Å2) between the inhibitor and enzyme that contributes to helianthamide’s large buried surface area. Residues 162165 of PPA consist of the sequence LVGL, and this region forms contacts with residues I8, V12, Y40, T42 and W44 of helianthamide. In particular, helianthamide appears to form a pocket around V163 of PPA, which is T163 in HPA (Figure 2c). We wanted to explore the effects of perturbing interactions in this region through expression of a T163R variant of HPA. Kinetic analysis of T163R HPA with the chromogenic substrate CNPG-3 revealed similar kcat and KM values to those of the wildtype enzyme (T163R KM = 2.7 mM; T163R kcat = 17 s-1; WT KM = 3.2 mM; WT kcat = 21 s-1), indicating that this mutation does not result in significant perturbations of the HPA active site6. The T163R HPA mutant bound the bar’-hel fusion 40-fold more weakly than did wild-type HPA, with a new KI = 20 nM (Table 1). This loss of affinity allowed a second kinetic analysis of the helianthamide variants to be performed via the more commonly used Michaelis-Menten model. Quantification of inhibition of T163R HPA by the Y7A, Y9A, and H10A bar’-hel variants under these more traditional conditions revealed the same 40-fold decrease in affinity for each case (Table 1). This reinforced the trends observed in the original dataset and validates use of the Morrison method of absolute KI determination for our sub-nM inhibitors. In a second set of mutagenesis studies the importance of the disulfide bonds in helianthamide was probed by expressing and testing alanine variants of helianthamide’s six cysteine residues (Table 2). On average, mutating cysteines led to a much greater decrease in binding affinities when compared to the disruption of individual polar contacts. Table 2. Kinetic analysis of barnase’-helianthamide fusion cysteine variants.
Helianthamide Type
Morrison Method KI (µM)
MichaelisMenten KI (µM)
Bar’-hel
(0.5 ± 0.1) x 10-3
--
Bar’-hel-C6A
4.0 ± 0.3
5.3 ± 1.0
Bar’-hel-C16A
(5 ± 0.6) x 10
Bar’-hel-C20A
3.8 ± 0.6
-3
-1.9 ± 0.5
-3
Bar’-hel-C33A
(8 ± 0.4) x 10
--
Bar’-hel-C38A
3.5 ± 0.3
2.4 ± 0.6
Bar’-hel-C39A
6.0 ± 0.6
5.9 ± 2.0
The C6A, C20A, C38A, and C39A bar’-hel variants had inhibition constants in the µM-range, thus bound some 7000 – 13,000-fold more weakly than wild-type helianthamide. This substantial loss of potency associated with the disruption of disulfide bonding highlights the importance of helianthamide’s rigid three-dimensional structure for its potent inhibition of HPA. The C16A and C33A variants were still nM-range inhibitors, binding only 10 – 16 fold more weakly than the wild type inhibitor. This suggests that the C16-C33 disulfide bond plays a less important role in stabilization of helianthamide’s tertiary structure than the other two disulfides. Site-directed mutagenesis studies on the interactions between α-amylase and proteinaceous inhibitors have been conducted in a few other cases. A study of the large lectin-type αAI1 showed that replacing residues R74, W188 and Y190 led to a complete elimination of inhibitory activity against the closely homologous porcine amylase7. Structural analysis indicated that only Y190 formed contacts with the enzyme’s catalytic residues, while W188 formed stacking interactions in the active site cleft8. In the case of the cereal-type 0.19 inhibitor, catalytic variants of a bacterial α-amylase that showed no hydrolytic activity also lost all affinity for the inhibitor, but were still able to bind substrate molecules9. In both of these studies a much larger decrease in binding affinities was observed as a result of loss of individual polar contacts than we saw with helianthamide. However, unlike helianthamide, neither of these proteins forms such an extensive hydrophobic interface with the enzyme. To further probe the mode of helianthamide’s inhibition, a library of helianthamide-inspired peptides was assembled via solid phase peptide synthesis. Both linear and cyclic peptides were pursued. The cyclization of small peptides can introduce rigidity into the structure to potentially increase binding affinity; however it may also perturb the orientation of residues and have the opposite effect. Cyclic peptides were made by the incorporation of cysteine pairs and oxidation into disulfide bonds by overnight incubation in buffer (0.1 M NaHCO3, 3 mM cysteine, 0.3 mM cystine, 1 M GuHCl, pH 8.1)10. Synthetic peptides 1 – 11 were based on residues 1 – 16 of helianthamide, as the majority of the interactions with HPA occurred within this region (Figure 3 and Table 3). Peptides 12 – 19 were designed to include portions from both the N- and C-terminal regions. The Cterminal residues of helianthamide appear to contribute to the interface between inhibitor and enzyme. The C6C38 disulfide bond places the C- and N-terminal regions in close spatial proximity, inspiring us to synthesize peptides 12 – 19 with sequence elements from both.
Assays conducted at 30 degrees Celsius. Error determined through least-mean squares method or through fit errors produced by Grafit.
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19 A
B C16 I15 C6 E1
C6 C38 Y7
I8
Y9
W44
H10
(A) Peptides 1 to 11: ESGNSCYIYHGVSGICKASCAEDEKAMAGMGVCEGHLCCYKTPW (B) Peptides 12 to 19: ESGNSCYIYHGVSGICKASCAEDEKAMAGMGVCEGHLCCYKTPW
Figure 3. Regions of interest for the synthesis of small helianthamide inspired peptides, shown in blue as segments of the bound helianthamide structure and sequence. (A) Residues 1 – 16 of helianthamide formed the majority of the polar interactions in the α-amylase active site and were the basis of peptides 1 – 11. (B) Helianthamide’s C-terminal residues 38 – 44 also contributed to its interface with PPA and are directly connected to residues 6 – 15 through the C6-C38 disulfide bond. Synthetic peptides 12 – 19 were designed to include elements from both of these regions. (PDB: 4X0N) Helianthamide shown as blue sticks, PPA shown in grey. Table 3. Sequences of small helianthamide-based peptides.
Peptide
Sequence
1
YIYH (linear)
2
YIYHGV (linear)
3
YIYHGVS (linear)
4
YIYHGVSGI (linear)
5
CYIYHGVSGIC (cyclic)
6
ESGNSCYIYHGVSGIC (cyclic)
7
SYIYHGVSG (linear)
8
CYIYHGVSC (cyclic)
9
CYIYHGVSGC (cyclic)
10
ESGNSCYIYHGVSC (cyclic)
11
ESGNSCYIYHGVSGC (cyclic)
12
WPTKYCGGYIYHGVSGC (cyclic)
13
WPTKYCGYIYHGVSGC (cyclic)
14
CYIYHGVSGIGCSYKTPW (cyclic)
15
CYIYHGVSGIGCAYKTPW (cyclic)
16
CYIYHGVSGIPCSYKTPW (cyclic)
17
CYIYHGVSGIPCAYKTPW (cyclic)
18
CYIYHGVSGIGGCAYKTPW (cyclic)
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The peptides were tested in linear and cyclic forms where applicable. Despite containing the portions of helianthamide that formed the vast majority of the points of contact with the enzyme, only two of the 19 peptides showed any inhibitory activity towards HPA. Peptides 12 and 13 had IC50 values of 141 µM and 396 µM towards HPA, respectively, making them 7 orders of magnitude less potent than intact helianthamide. Similar studies have been conducted in the past involving the synthesis of peptides based on the potent HPA inhibitor tendamistat (KI = 0.009 – 0.2 nM). Tendamistat is a 74-residue protein and has a similar total buried surface area (~2400 Å2) to that of helianthamide (~1900 Å2) when bound to α-amylase. Most of the small tendamistat-inspired peptides exhibited µM-range inhibition constants against HPA8, 11, 12, and in one study researchers were able to synthesize an 11-residue peptide with an inhibition constant of 270 nM against HPA13. Tendamistat is larger and less rigid than helianthamide. Crystallographic analysis showed multiple points of contact with the enzyme through four discontinuous regions of the tendamistat sequence14. This mode of inhibition is more conducive to the synthesis of small peptide inhibitors, however it makes tendamistat susceptible to protease-mediated degradation15. On the other hand the knotted structure of helianthamide confers a greater level of stability but its potent inhibition is not easily replicated with small peptides4. This structural analysis of helianthamide indicates the importance of its intact structure and native disulfide bonding for potent inhibition of HPA. The formation of individual polar contacts with the enzyme does contribute to helianthamide’s potency to a degree, as observed in the results of the site-directed mutagenesis study on the YIYH motif and the mild inhibitory activity of synthetic peptides 12 and 13. However, the contribution of these polar contacts pales in comparison to the importance of helianthamide’s rigid three-dimensional structure and resulting ability to form an extensive and robust hydrophobic interface with the enzyme. With this knowledge in mind, the recombinant expression of intact helianthamide can be pursued towards the large-scale production of this highly potent HPA inhibitor.
ASSOCIATED CONTENT Supporting Information: Experimental Procedures; Sequences of barnase’-helianthamide gene, protein and primers; Purification data; Kinetic data; MALDI-TOF; HPLC data. The Supporting Information is available free of charge on the ACS Publications website (PDF).
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Biochemistry
AUTHOR INFORMATION Corresponding Author
* To whom correspondence should be addressed. Telephone: 604 822 3402 E-mail:
[email protected] Present Addresses
† Present address: Department of Chemistry, Pennsylvania State University, State College, Pennsylvania, 16801, USA. Author Contributions
The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. / Funding Sources
No competing financial interests have been declared. This research was supported through funding from the Canadian Institutes for Health Research (CIHR) and from the Canadian Glycomics network, GlycoNet, one of the Networks of Centres of Excellence of Canada.
ACKNOWLEDGMENT We thank Emily Kwan for assistance with purification of HPA.
ABBREVIATIONS HPA, human pancreatic α-amylase; PPA, porcine pancreatic α-amylase; CNPG-3, 2-chloro-4-nitrophenyl αmaltotrioside.
REFERENCES [1] Brayer, G. D., Luo, Y., and Withers, S. G. (1995) The structure of human pancreatic alpha-amylase at 1.8 A resolution and comparisons with related enzymes, Protein Sci 4, 1730-1742. [2] Numao, S., Damager, I., Li, C., Wrodnigg, T. M., Begum, A., Overall, C. M., Brayer, G. D., and Withers, S. G. (2004) In situ extension as an approach for identifying novel alpha-amylase inhibitors, J Biol Chem 279, 48282-48291. [3] Svensson, B., Fukuda, K., Nielsen, P. K., and Bønsager, B. C. (2004) Proteinaceous alpha-amylase inhibitors, Biochim Biophys Acta 1696, 145-156.
[4] Tysoe, C., Williams, L. K., Keyzers, R., Nguyen, N. T., Tarling, C., Wicki, J., Goddard-Borger, E. D., Aguda, A. H., Perry, S., Foster, L. J., Andersen, R. J., Brayer, G. D., and Withers, S. G. (2016) Potent Human α-Amylase Inhibition by the β-Defensin-like Protein Helianthamide, ACS Cent Sci 2, 154-161. [5] Morrison, J. F. (1969) Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors, Biochim Biophys Acta 185, 269-286. [6] Zhang, X., Caner, S., Kwan, E., Li, C., Brayer, G. D., and Withers, S. G. (2016) Evaluation of the Significance of Starch Surface Binding Sites on Human Pancreatic α-Amylase, Biochemistry 55, 6000-6009. [7] Mirkov, T. E., Evans, S. V., Wahlstrom, J., Gomez, L., Young, N. M., and Chrispeels, M. J. (1995) Location of the active site of the bean alpha-amylase inhibitor and involvement of a Trp, Arg, Tyr triad, Glycobiology 5, 45-50. [8] Bompard-Gilles, C., Rousseau, P., Rougé, P., and Payan, F. (1996) Substrate mimicry in the active center of a mammalian alphaamylase: structural analysis of an enzyme-inhibitor complex, Structure 4, 1441-1452. [9] Takase, K. (1994) Site-directed mutagenesis reveals critical importance of the catalytic site in the binding of alpha-amylase by wheat proteinaceous inhibitor, Biochemistry 33, 7925-7930. [10] Wu, Z., Hoover, D. M., Yang, D., Boulègue, C., Santamaria, F., Oppenheim, J. J., Lubkowski, J., and Lu, W. (2003) Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human beta-defensin 3, Proc Natl Acad Sci U S A 100, 8880-8885. [11] Etzkorn, F. A., Guo, T., Lipton, M. A., Goldberg, S. D., and Bartlett, P. A. (1994) Cyclic Hexapeptides and Chimeric Peptides as Mimics of Tendamistat Journal of American Chemical Society 116, 14. [12] Sefler, A. M., Kozlowski, M. C., Guo, T., and Bartlett, P. A. (1997) Design, Synthesis, and Evaluation of a Depsipeptide Mimic of Tendamistat, J Org Chem 62, 93-102. [13] Ono, S., Umezaki, M., Tojo, N., Hashimoto, S., Taniyama, H., Kaneko, T., Fujii, T., Morita, H., Shimasaki, C., Yamazaki, I., Yoshimura, T., and Kato, T. (2001) Cyclic and linear peptides derived from alpha-amylase inhibitory protein tendamistat, J Biochem 129, 783-790. [14] Wiegand, G., Epp, O., and Huber, R. (1995) The crystal structure of porcine pancreatic alpha-amylase in complex with the microbial inhibitor Tendamistat, J Mol Biol 247, 99-110. [15] Vértesy, L., Oeding, V., Bender, R., Zepf, K., and Nesemann, G. (1984) Tendamistat (HOE 467), a tight-binding alpha-amylase inhibitor from Streptomyces tendae 4158. Isolation, biochemical properties, Eur J Biochem 141, 505-512.
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For Table of Contents Use Only Structural dissection of helianthamide reveals the basis of its potent inhibition of human pancreatic αamylase Christina Tysoe & Stephen G. Withers.
C6 C33 C16 C38 C39
C20
ESGNSCYIYHGVSGICKASCAEDEKAMAGMGVCEGHLCCYKTPW
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