proteins STRUCTURE O FUNCTION O BIOINFORMATICS

Structural insights into the aggregation behavior of Murraya koenigii miraculin-like protein below pH 7.5 Purushotham Selvakumar, Nidhi Sharma, Prabhat Pratap Singh Tomar, Pravindra Kumar, and Ashwani Kumar Sharma* Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247 667, India

ABSTRACT Murraya koenigii miraculin-like protein (MKMLP) gradually precipitates below pH 7.5. Here, we explore the basis for this aggregation by identifying the aggregation-prone regions via comparative analysis of crystal structures acquired at several pH values. The prediction of aggregation-prone regions showed the presence of four short peptides either in beta sheets or loops on surface of the protein. These peptides were distributed in two patches far apart on the surface. Comparison of crystal structures of MKMLP, determined at 2.2 A˚ resolution in pH 7.0 and 4.6 in the present study and determined at 2.9 A˚ in pH 8.0 in an earlier reported study, reveal subtle conformational differences resulting in gradual exposure of aggregation-prone regions. As the pH is lowered, there are alterations in ionic interactions within the protein interactions of the chain with water molecules and exposure of hydrophobic residues. The analysis of symmetry-related molecular interfaces involving one patch revealed shortening of nonpolar intermolecular contacts as the pH decreased. In particular, a decrease in the intermolecular distance between Trp103 of the aggregation-prone peptide WFITTG (103–108) unique to MLPs was observed. These results demonstrated that aggregation occurs due to the cumulative effect of the changes in interactions in two aggregation-prone defined regions. Proteins 2014; 82:830–840. C 2013 Wiley Periodicals, Inc. V

Key words: acidic pH; aggregation-prone peptides; crystal structure; conformational changes; hydrophobic interactions.

INTRODUCTION Understanding the underlying reasons for protein aggregation, both in vivo and in vitro, is a major challenge for protein biochemists. Protein aggregation results in a number of human pathologies including Alzheimer’s, Parkinson’s, and Creutzfeldt-Jakob diseases; and the systemic amyloidoses associated with immunoglobulin light chain, transthyretin, lysozyme, and Beta-2 microglobulin.1 There are specific regions of amino acid sequence, termed “aggregation prone,” which plays a major role in determining the tendency of proteins to aggregate.2 These “aggregation-prone” regions are not exposed in a native protein. However, proteins can be destabilized by heat, pH, denaturants, etc. exposing these regions and leading to aggregation.3–5 Miraculin-like proteins (MLPs) exhibit significant sequence identity (39%–55%) to miraculin protein, a 24.6 kDa plant protein purified from red berries of Richadella dulcifica.6,7 Both proteins belong to Kunitz

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superfamily and have sequence similarity (30%) to soybean Kunitz family trypsin inhibitors.8 Murraya koenigii miraculin-like protein (MKMLP), a 21.4 kDa protein with trypsin inhibitory activity, was purified and characterized from seeds of Murraya koenigii belonging to Rutaceae family.9 Despite being a member of Kunitz superfamily, MKMLP demonstrated some distinct features. It formed a distinct cluster with MLPs in phylogenetic Additional Supporting Information may be found in the online version of this article. Abbreviations: MKMLP, Murraya koenigii miraculin-like protein; MLPs, Miraculin-like proteins; STI, soybean Kunitz inhibitor; PQS, Protein Quaternary Structure server; ANS, 8-anilino-1-naphthalene sulfonate. Grant sponsor: Department of Science and Technology, Government of India; Grant number: SR/SO/BB-002/2007. *Correspondence to: Ashwani Kumar Sharma, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247 667, India. E-mail: [email protected] Received 30 May 2013; Revised 3 October 2013; Accepted 21 October 2013 Published online 22 November 2013 in Wiley Online Library (wileyonlinelibrary. com). DOI: 10.1002/prot.24461

C 2013 WILEY PERIODICALS, INC. V

Insights into Aggregation Behavior of MKMLP

analyses and showed major differences at primary and secondary specificity sites in reactive loop when compared with classical Kunitz inhibitors like soybean Kunitz inhibitor (STI). The conventional Arg/Lys at P1 position in MKMLP has been replaced by an Asn residue suggesting that the protein may not act as a typical substrate-like inhibitor because of the absence of a residue essential for trypsin specificity.10 The crystal structure of MKMLP determined at 2.9 A˚ exhibited a classical b-trefoil fold similar to Kunitz family inhibitors with major conformational differences limited to loop regions. The unique features of the MKMLP structure was the presence of three disulfide bridges and two short 310 helices.10 The MKMLP, native and heat treated, was found to be highly stable against proteolysis due to the disulfide bridges. Unlike classical Kunitz inhibitors, MKMLP was functionally unstable at higher temperature.11 The reason for this loss in inhibitory activity has been attributed to the absence of stabilization of reactive loop conformation where Asn13, which plays an important role in stabilizing the reactive loop conformation in STI, is replaced by Ala.12 MKMLP shows bioinsecticidal activities and possess an N-terminal signal sequence with possible plasma membrane-spanning motif for plasma membrane indicating the translocation of protein from the site of synthesis.10,13 Another important unique feature of MKMLP, as compared with most Kunitz members which are stable over a broad range of pH,14,15 is its solubility properties at acidic pH. The protein gradually precipitates below pH 7.5 with an increasing rate of precipitation as pH is lowered.9 The present study explores the basis for this aggregation by identifying the aggregation-prone regions and analyzing the subtle conformation changes by comparative crystal structure analysis in different pH conditions. Here we report crystal structures of MKMLP at 2.2 A˚ resolution determined at pH 7.0 and 4.6, and also compare the three structures determined at pH 8 (previously reported), 4.6, and 7 to unravel the molecular basis of aggregation behavior.

MATERIALS AND METHODS Purification, crystallization and data collection

Purification of MKMLP was carried out as described earlier9,16 Briefly, MKMLP was purified by crushing of M. koenigii seeds and soaking overnight at 4 C in 30 mL of 50 mM Tris–HCl buffer, pH 7.5. The homogenate was cleared by centrifugation at 12,000g for 1 h and the supernatant was used for purification. The protein was purified by combination of anion exchange and size exclusion chromatography. Also, affinity chromatography using Cibacron blue 3GA was done for single step purification. The protein was crystallized by the sitting-drop vapor diffusion method under two pH conditions. The

precipitant solutions were 4M ammonium acetate, 0.1M BIS-TRIS propane, pH 7.0 and 4M ammonium acetate, 0.1M sodium acetate trihydrate, pH 4.6. Drops were prepared by mixing 1 mL protein solution with 1 mL precipitant solution and were equilibrated against 50 mL reservoir solution. For cryoprotection, crystals briefly exposed to well solution containing 20% glycerol were mounted in cryoloops prior to collection of X-ray diffraction data. Data were collected on a MAR 345dtb image-plate system using Cu Ka radiation generated by a Bruker Microstar-H rotating-anode generator operated at 45 kV and 60 mA, and equipped with Helios optics. The crystals belonged to the monoclinic space group C121, with unit-cell parameters a 5 101.51 A˚, b 5 45.69 A˚, c 5 38.78 A˚ for crystal grown at pH 7 and a 5101.62 A˚, b 5 45.42 A˚, c 5 38.79 A˚ for crystal grown at pH 4.6. The crystals contained one molecule in asymmetric unit. Diffraction was observed to 2.2 A˚ resolution (Fig. S1, Supporting Information). The diffraction data were processed and scaled with iMOSFLM and SCALA program in CCP4i suite.17 Structure solution and refinement

The structure was solved by the molecular replacement method using MOLREP17 with the structure of the MKMLP [PDB ID: 3IIR] as the search model. The initial model obtained with molecular replacement was refined using REFMAC 5.217 first as a rigid body, and subsequently, it was refined using restrained refinement. Model building was conducted in manual mode in Coot,18 followed by refinement in REFMAC 5.2. The alternate cycles of refinement and model building were performed for all the data in the resolution range 50.5–2.2 A˚. The water molecules were added according to the criteria that each water molecule must make at least one stereochemically reasonable hydrogen bond, that it should be well defined in (2mjfobsj–Djfcalcj) and (mjfobsj–Djfcalcj) electron density maps. The water molecules were added and removed in subsequent refinement and model building cycles as per the above criteria. The stereochemistry of final model was analyzed by PROCHECK19 and MOLPROBITY.20 The protein structures were examined using molecular visualization software Coot18 and PyMOL.21 The atomic coordinates have been deposited in the Protein Data Bank. Prediction of aggregation-prone regions

Aggregation-prone regions were predicted using different programs employing empirical and structure-based algorithms such as Tango,22 FoldAmyloid,23 Aggrescan,24 Waltz,25 Zyggregator,26 and Pasta.27 Accession numbers

The atomic coordinates and structure factors have been deposited into the Protein Data Bank under the PROTEINS

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following accession codes: 3ZC8 (pH 7 structure) and 3ZC9 (pH 4.6 structure).

were higher and the residues included in six loops L1 (1–16), L5 (63–74, reactive loop), L7 (94–104), L9 (128– 134), L10 (140–151), and L11 (162–163) were found to be less compared with average value.

RESULTS Three-dimensional structure of MKMLP at pH 7.0 and pH 4.6 Quality of the model

The refinement data in Table I shows that both models are well refined with excellent stereochemistry and crystallographic R-factor values. The deviations in bond lengths and angles are within reasonable limits from ideal values. The electron density is well defined in both structures except at the C-terminal end residues 183–190 so they were not used for model building. In pH 7 structure, the temperature factors of residues in five loops namely L2 (24–30), L3 (35–43), L4 (48–57), L6 (80–89), and L12 (168–174) were found to be higher than the average value. For the residues involving loops L1 (1–16), L5 (63–74, reactive loop), L7 (94–104), L8 (110–114), L9 (128–134), L10 (140–151), and L11 (162– 163) the temperature factors were less than the average value. For pH 4.6 structure, the temperature factors of residues in six loops namely L2 (22–30), L3 (35–43), L4 (48–57), L6 (80–89), L8 (110–114), and L12 (168–175) Table I Crystal Parameters, Data Collection, and Structure Refinement Crystal data and intensity statistics Space group Unit-cell parameters () a b c Resolution range () Completeness (%) Rmergea(%) Multiplicity Mean I/sigma (I) Refinement and model statistics Total no. of reflections No. of reflections (used) Percentage observed Wilson B-factor (2) Crystallographic R-factor (%) Free R-factor (%) Average B factor (2) RMSD bonds () RMSD angles ( ) Validation by MOLPROBITY Ramachandran plot Favored (%) Allowed (%) Outliers (%) PDB code

pH 7

pH 4.6

C121

C121

101.51 45.69 38.78 50.5–2.2 96.3 (74.9) 0.09 (0.27) 3.3 (3.1) 10.3 (4.4)

101.61 45.42 38.79 50.5–2.2 90.0 (69.0) 0.07 (0.21) 3.5 (3.3) 13.7 (5.7)

28,501 8605 99.6 20.9 19.5 24.2 16.6 0.01 1.3

28,271 8647 92.1 24.6 19.4 23.8 19.1 0.01 1.2

95.5 4.5 0 3zc8

93.5 5.5 1 3zc9

The values in parentheses refer to statistics in the highest bin. a Rmerge 5 RhklRijIi(hkl) – j/RhklRiIi(hkl), where Ii(hkl) is the intensity of an observation and is the mean value for its unique reflection; summations are overall reflections.

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Overall structure

The three-dimensional structures of MKMLP, determined at 2.2 A˚ resolution, at both pH values are similar with a few exceptions. The pH 7 structure was predicted to be monomeric but the pH 4.6 structure was predicted to be dimeric by the Protein Quaternary Structure server (PQS).28 The superposition of core region gave RMSD of 1.971 A˚ for 110 Ca atoms between two MKMLP structures. The overall crystal structure consists of 12 antiparallel b-strands, loops connecting the b-strands, one a-helix, and a short 310 helix (Fig. 1). In both structures, except for first and last b-strands, the corresponding residues forming b-strands are same. b-strand 1 is shortened by two residues (residues 17–21) and b-strand 12 by one residue (176–180) in pH 4.6 structure as compared with pH 7.0 structure (residues 17–23 and 175– 180). The corresponding loop regions are also changed in two structures where residues 24–30 (L1), 168–174 (L12) form loop region in pH 7.0 structure and residues 22–30 (L2), 168–175 (L12) form loop region in pH 4.6 structure. Both MKMLP structures, like Kunitz family inhibitor structure, exhibits a typical b-trefoil fold with six of the strands arranged in a barrel structure and other six forms a triangular lid on the barrel. A pseudothreefold internal symmetry with symmetry axis roughly parallel to barrel axis divides the structure into three repeating units. Each unit consists of approximately 60 amino acids arranged in four b-strands. In addition to one a-helix and a short 310 helix, the presence of short stretches of distorted helices within loops was observed. In pH 7 structure, these are present in loops L1 (residues 6–8 and 13–15), L2 (26–28), L4 (residues 51–53), L5 (residues 63–65), L6 (residues 84–87), and L10 (residues 144–147). In pH 4.6 structure, six short stretches of distorted helices were observed similar to pH 7 structure except for the L2 (residues 26–28). The presence of helices which constitutes almost 6% of the structure is a unique feature of MKMLP and substantiates our earlier CD results which demonstrated a, b pattern for the protein. The residues forming sheets, helices, and loops are indicated in Table II. The crystal structure of MKMLP at higher resolution of 2.2 A˚ showed remarkable improvement. The diffraction data was collected at room temperature in case of 2.9 A˚. Although the overall three-dimensional structures of MKMLP determined at 2.2 A˚ in two pH conditions were similar to the earlier reported structure of MKMLP at 2.9 A˚ in pH 8.0 condition, a few noticeable differences were observed. The superposition of core regions of 2.2 and 2.9 A˚ structures gave an RMSD of 0.322 A˚ (Fig. 2).

Insights into Aggregation Behavior of MKMLP

Figure 1 Overall structure of MKMLP. Part figures (a) and (c) represent cartoon models at pH 7 and 4.6 showing b-sheets, a-helices, loops, and putative reactive loop indicated in yellow, red, blue, and magenta, respectively. The disulfide bridges are shown in stick in blue (C41–C85, C144–C147, and C140–151). The structures were submitted in PDB database (PDB ID code: 3ZC8 for pH 7 and 3ZC9 for pH 4.6 structures). Part figures (b) and (d) represent the final electron density map around the region (Tyr63–His 70) in the MKMLP pH 7 and 4.6 structure, respectively. The map was calculated using 50.56–2.24 A˚ data and contoured at 1.0r. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

The major changes include a longer extended helix from five to eight residues (residues 115–122 as compared with 118–122) and conformational changes at b-strand 1 and loop 2 involving residues 17–30. The orientation and length of b-strand 1 and loop 2 is quite different in two structures solved at different resolutions. The electron density was well defined in both cases. One reason could be that data were collected in different conditions. Interestingly, the conformational differences in two high resolution structures determined in pH 7.0 and 4.6 conditions involve the same region. The conformational changes at b-strand 1 and loop 2 involving residues 17– 30, therefore, could be attributed to different pH conditions. In 2.9 A˚ resolution structure at pH 8, residues 18– 25 form b-strand 1 and residues 26–27 form the loop 2 while in 2.2 A˚ resolution structures, residues 17–23 form b-strand 1 and residues 24–30 form loop 2 at pH 7.0 and residues 17–21 form b-strand 1 and residues 22–30 form loop 2 at pH 4.6. It is to be noted that b-strand 2 (residues 31–34) is shortened in 2.2 A˚ resolution compared with 2.9 A˚ resolution structures (residues 28–34) because of increased length of loop 2. A significant change in conformation in this region is seen. Superposi-

tion of residues 17–30 between structures at pH 4.6 and 7, pH 7 and 8, and pH 4.6 and 8 gave RMSD of 0.142, 1.246, and 1.236 A˚ respectively [Fig. 3(a–c)]. Also, the differences in the presence of distorted helices were observed. The structures at 2.2 A˚ showed the presence of distorted helices L2 (residues 26–28) and L6 (residues 84–87), and absence of distorted helices L3 (residues 37– 39) and L7 (residues 98–101) which are found in 2.9 A˚ structure. Reactive loop

The exposed reactive site loop of MKMLP (P4–P40 ) adopts a characteristic canonical conformation found in classical Kunitz inhibitors like STI.10 The lower B-factors and well defined electron density for residues of reactive site loop were observed in 2.2 A˚ structures indicating conformational stability of the region. Like in 2.9 A˚ structure at pH 8.0, the reactive loop in both high resolution structures exhibits a well defined electron density with a typical canonical conformation. A well defined unoccupied electron density at Asn64 confirms the presence of glycan moieties. Only MLPs possess this PROTEINS

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Table II Secondary structural element details of MKMLP structures at pH 7 and 4.6 conditions pH 7 MKMLP structure Loops

Sheets

Helices Disulfide bridges

4.6 MKMLP structure 1-16 24-30 35-43 48-57 63-74 80-89 94-104 110-114 128-134 140-151 162-163 168-174 9 17223 > > > > > 31234 = Subdomain A 44247 > > > > > ; 58262 9 75279 > > > > > 90293 = Subdomain B 1052109 > > > > > ; 1242127 9 1352139 > > > > > 1522156 = Subdomain C 1642167 > > > > > ; 1752180 115-122 158-161 Cys41-Cys89, Cys140-Cys151 Cys144-Cys147

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 b1 b2 b3 b4

Loops

Sheets

b5 b6 b7 b8 b9 b10 b11 b12 a1 a2

Helices Disulfide bridges

1-16 22-30 35-43 48-57 63-74 80-89 94-104 110-114 128-134 140-151 162-163 168-175 9 17221 > > > > > 31234 = Subdomain A 44247 > > > > > ; 58262 9 75279 > > > > > 90293 = Subdomain B 1052109 > > > > > ; 1242127 9 1352139 > > > > > 1522156 = Subdomain C 1642167 > > > > > ; 1762180 115-122 158-161 Cys41-Cys89, Cys140-Cys151 Cys144-Cys147

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 b1 b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 a1 a2

glycosylation motif at active site loop and even miraculin lacks the same. The superposition of Ca atoms of reactive loop when compared with pH 7 and 8, pH 4.6 and 8, and pH 7 and 4.6 gave an RMSD of 0.329, 0.328, and 0.091, respectively [Fig. 3(d)]. The orientation of P2 residue Asn64 is changed in both pH 7 and 4.6 structure compared with pH 8 structure. Also there is slight change in orientation of P30 residue Ile68 in pH 4.6 structure. Crystallographic symmetry analysis

Figure 2 Structural superimposition of Ca atoms of three MKMLP structures. The structures at pH 8, 7, and 4.6 showing similar overall fold are represented in green, blue, and red ribbons, respectively. The glycine rich loop undergoing conformational change is shown in box. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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The pH 7 and 4.6 structures had single molecule in the asymmetric unit whereas pH 8 structure had two. The symmetry-related molecule interface area shows that in all structures it involved region 145–152-KSCVFLCN (Fig. 4). In pH 7 and 4.6 structures, the symmetryrelated molecules generated are within 4 A˚ contain this region. The loop region L7 (94–104) and a1 helix region (115–122) are also in close contact with KSCVFLCN (145–152) peptide region. The distance between two

Insights into Aggregation Behavior of MKMLP

regions in MKMLP compared with other members. The analysis of these regions in three-dimensional structure determined in different conditions would seem to be a reasonable approach for understanding the aggregation behavior of MKMLP. For aggregation to happen there should be more than one region for intermolecular contact on a protein molecule. In the crystal structure determined at pH 4.6, the purified MKMLP used for crystallization was in Tris buffer at pH 7.5 and precipitant sodium acetate was at pH 4.6. The protein solution and precipitant were mixed in equal proportions. Therefore, structure represented here may not be a true structure at pH 4.6 but it is definitely a structure below pH 7. Peptide sequences of MKMLP predicted to be involved in aggregation

Figure 3 Superimposition involving residues 17–30 and putative reactive loop in three MKMLP structures at different pH exhibiting variation in secondary structure elements are shown. The structures at (a) pH 8, (b) pH 7, and (c) pH 4.6 are represented in green, blue, and red cartoons, respectively. (d) Superimposition of putative reactive loop residues Ala62 (P4) to Ile69 (P40 ) of three MKMLP structures at different pH conditions showing variation in conformation of Asn64 (P2) at pH 8 structure (green) compared with pH 7 (blue) and pH 4.6 (red sticks). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Trp103 residues in loop region (94–104) in adjacent symmetry molecules decreases with pH. In pH 8 structures, the distance between two Trp103 residues in adjacent symmetry molecules is 4.0 A˚, in pH 7 structure it is 3.7 A˚, and in pH 4.6 structure it is 3.5 A˚ (Fig. 5).

DISCUSSION Analysis of MKMLP aggregation

The pH-induced reversible aggregation of MKMLP is unique among Kunitz family members. Most of the classical members, like STI, are stable at a broad range of pH and temperature, and there are no reports about aggregation at low pH. The reason for this unique behavior may lie in the differences in the primary structure compared with other classical Kunitz members. Many studies have revealed that certain peptides in protein sequences initiate or mediate aggregation. These regions have also been successfully predicted. There must be differences in the number of aggregation-prone

Six programs were used for prediction of aggregationprone regions of MKMLP. Since these programs employ various parameters for prediction so there were subtle differences in the results (Fig. S2, Supporting Information). We have considered consensus from all the results and have found four regions involved in aggregation distributed across the MKMLP sequence. These regions involve peptides YYLVSVI (17–23), WFITTG (103–108), SCVFLCN (146–152), and VFGVVIV (173–179). Tango algorithm predicts the b-aggregation propensity in sequences considering physico-chemical parameters such as pH, temperature, and ionic strength. MKMLP was assessed from pH 4 to 8. Among predicted regions by Tango there are no charge residues. At pH 8, Tango predicts that three peptides, YYLVSVIG (17–24), WFITTGGV (103–110), and VFGVVIVP (173–180) are involved in aggregation. Prediction at pH 7, 6, 5, and 4 reveals another aggregation region, CVFLC (147–151) with scores highest at pH 6 and pH 5 (Fig. 6; and Table S1, Supporting Information). These results indicate increased aggregation tendency of MKMLP below pH 7.5. Sequence comparisons reveal that other related members lack predicted consensus aggregation-prone peptides found in MKMLP sequence. Compared with MKMLP, results for STI showed only two aggregation prone peptide regions, TYYILS (16–20) and LKFDSFAVIMLCVG (74–88). STI lacks the WFITTG (103–108) peptide and a peptide containing hydrophilic amino acids (DDKCG) is present in STI as compared with the corresponding peptide SCVFLCN (146–152) in MKMLP (Fig. 7). Structural insights into the aggregation behavior of MKMLP

We hypothesize that the aggregation of MKMLP below pH 7.5 is driven by the exposure of aggregation-prone regions. Aggregation can be seen as an anomalous type of protein–protein interaction. Tertiary protein structure is governed mainly by electrostatic and hydrophobic interactions and proteins are most likely to aggregate at PROTEINS

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Figure 4 Crystallographic symmetry-related molecular interface at different pH conditions involving KSCVFLCN (145–152). (a) At pH 8, (b) pH 7, and (c) pH 4.6. Black sticks represent KSCVFLCN (145–152) loop region which forms interface residues in all structures. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

their isoelectric points, where they bear no net charges. Hydrophobic interactions are the main mode through which nonpolar patches of the protein surface are shielded by water molecules arranged in an ordered structure. When two nonpolar patches come together, the water molecules are expelled, increasing their entropy. This increase is the main driving force for protein association.29 Crystal structures of MKMLP determined at pH 8.0, 7.0, and 4.6 were analyzed for subtle conformational differences that can provide clues about aggregation below pH 7.5. Most of the predicted four aggregation-prone peptides are involved in native b-sheet formation, except for SCVFLCN (146–152) and residues 103–108 of WFITTG, which are in surface loops. In

MKMLP the predicted aggregation-prone peptides form two patches/regions (Fig. 8). Patch 1 involves the predicted aggregation prone peptides YYLVSVI (17–23), VFGVVIV (173–179), and the glycine rich loop region GGAGGGG (24–30). Patch 2 involves peptides WFITTG (103–108) and SCVFLCN (146–152). These two patches are far from each other and present in slightly opposite orientations with respect to each other. The data demonstrated the potential of these peptides to form intermolecular b-sheets that are not seen in native structure. Most importantly SCVFLCN (146–152) showed baggregation propensity only below pH 8, consistent with the biochemical data showing decrease in solubility below pH 7.5.11 The conformational changes in these

Figure 5 Crystallographic symmetry-related molecule interface showing distances between two Trp103 at different pH involving WFITTG (103–108). Location of Trp103, which is part of WFITTG (103–108), is shown in blue sticks. The distance between two symmetrical Trp103 of molecules gets decreased from (a) pH 8, (b) pH 7, and (c) pH 4.6. The red loop region indicates SCVFLCN (146–152). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Insights into Aggregation Behavior of MKMLP

Figure 6 Plots of b-aggregation propensity for MKMLP. At all pH values three peptides are predicted to be aggregation prone. An additional peptide CVFLC (147–151) is found only below pH 8. Peaks in brown (pH 8), green (pH 7.5), purple (pH 7), yellow (pH 6), and blue (pH 5) signifies increase in b-aggregation score. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

two regions and the resulting alteration in electrostatic or hydrophobic interaction will be mainly responsible for promoting aggregation below pH 7.5. The analysis of crystal structures determined at three pH conditions showed a pattern in conformational changes as the pH was lowered. Conformational changes were observed at aggregation prone peptides YYLVSVI (17–23), SCVFLCN (146–152), and WFITTG (103–108). In Patch 1, the two main structural alterations were observed around the glycine-rich region involving the aggregation-prone peptide YYLVSVI (17–23). First, the change in orientation of

glycine rich loop when pH 8 and 7, and pH 4.6 structures are compared (Fig. 3). Second, a shortening of b1 and b2 strands and lengthening of loop L2. The comparison of the conformation in this region in three structures showed that b1 strand shortened from 18–25 at pH 8.0 to 17–23 at pH 7.0 to 17–21 at pH 4.6 structure. This observation implies that Val22 in aggregation-prone peptide and Ile23 are not involved in native b-sheet formation in the pH 4.6 structure and exists in loop region, suggesting partial unfolding in region which, in turn, exposes aggregation-prone peptide YYLVSVI (17–23). Likewise, b2 strand is shortened in structures determined at pH 7.0 and 4.6. Thus, the conformational changes at low pH lead to partial unfolding, exposing aggregationprone regions and thereby promoting hydrophobic interprotein interactions. These interactions would promote intermolecular associations leading to aggregation. In Patch 2, there are changes in the interaction within the molecule and at the interface of the crystallographic symmetry-related molecule. The SCVFLCN (146–152) peptide formed the interface in all structures (Fig. 4). In pH 7 structure, Val148 forms one hydrogen bond with water and Phe149 forms two hydrogen bonds with water. Leu150 forms hydrogen bond with main chain of His139. None of these interactions are seen in pH 4.6 structure [Fig. 9(a)]. Also, WFITTG (103–108) is in close contact with this interface. Interesting results were seen on analyzing the distances between two adjacent symmetrical Trp103 residues. The distance decreases when compared with pH 8 to pH 4.6 structures (Fig. 5). Taken together these results demonstrate the alterations that are seen in pH 4.6 structure: ionic interactions are lost and hydrophobic interactions become more prominent. These changes are a prerequisite for intermolecular association. The differences among the interactions involved in two patches at pH 4.6 and 7 provided important

Figure 7 Multiple sequence alignment of MKMLP, miraculin, and STI. The predicted aggregation-prone regions in MKMLP are shown and underlined in red. Boxes indicate comparative critical amino acid sequence changes and deletions. MKMLP shows presence of more hydrophobic residues compared with miraculin and STI. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Figure 8 Surface view of MKMLP structure showing location of two aggregation prone patches. Patch 1 region shown in blue includes residues 17–30 and 173–179. Patch 2 region shown in red consists of residues 103–108 and 146–152.

observations. For Patch 1, the pH 4.6 structure lost critical hydrogen bonding interactions compared with the pH 7 structure. These include interactions between Ileu23–Gly25, Gly175, Gly24–158Tyr, Gly25–Gln47, and Gly28. Also certain hydrogen bonding interactions are gained at pH 4.6 compared with pH 7. These include interactions between Gly24–Gly175, Ala26–Arg162, and Val179–Ala181. Superimposition of Ile23 at both pH conditions reveals a flip of the carbonyl in the pH 4.6 structure. Due to this flip, Ile23 cannot hydrogen bond

Figure 9 Ionic and hydrogen bonding interactions in MKMLP structures at pH 7 and 4.6. (a) Water molecule and ionic interactions of hydrophobic residues Val148, Phe149, and Leu150 residues of SCVFLCN (146–152) of Patch 2 region of MKMLP at pH 7 are shown. Red spheres indicate water molecules shown with electron density contoured at 1.0 r. (b) Superimposition of Ile23 residue of Patch 1 region for pH 7 and 4.6 show displacement of carboxyl group. (c) In pH 7 structure Ile23 forms hydrogen bond to Gly175. (d) In pH 4.6 structure Ile23 cannot interact with Gly175 as distance between them increased due to the flip. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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to Gly175 because distance between them increases from 2.8 to 5.1 A˚ [Fig. 9(b–d)]. For Patch 2, interactions with water molecules involving the hydrophobic residues Val148, Phe149, and Cys151 were lost in the pH 4.6 structure. The details of the above interactions are described in the Table S2 of the Supporting Information. The results suggest that partial unfolding and changes in interactions are responsible for aggregation behavior. Clearly, these alterations in the two patches results in intermolecular contacts that lead to visible precipitation. Peptide WFITTG (103–108) is unique to MLPs and may be responsible for initiation, particularly W103, which plays a crucial role in promoting hydrophobic interaction between two molecules at low pH. When symmetryrelated molecules are analyzed the distance between adjacent W103 sidechains are decreased from 3.9 A˚ at pH 8 to 3.5 A˚ at pH 4.6. These signify dominance of hydrophobic interactions, and this slight shift in intramolecular associations might trigger two similar molecules to associate leading to aggregation (Fig. 10). Also, the biological assembly of pH 7 structure was predicted to be monomeric and pH 4.6 structure as dimeric by PQS, which uses crystal symmetry matrices to generate symmetryrelated copies of the chains and by considering the buried surface area between pairs of chains. 8-Anilino-1-naphthalene sulfonate (ANS) fluorescence studies demonstrated a linear increase in fluorescence intensity with increasing temperatures with no substantial blue shift indicating that conformational changes does not significantly expose hydrophobic pockets. However, there is sharp increase in fluorescence intensity below pH 5 indicating relaxation.11,30 Strong evidences exist that translocation of proteins across a variety of membranes and membrane insertion involves non-native or denatured states and sometimes molten globule structures.31 In some cases conformational changes are seen at low pH conditions.32,33 The reversible nature of aggregation can be explained by the presence of three disulfide bonds in MKMLP one more than classical Kuntiz inhibitors, a unique conserved feature seen only in MLPs. The two cysteines involved in the extra disulfide are found in SCVFLCN (146–152) which borders the exposed hydrophobic patch VFL residues. These two cysteines provide structural constraint and prevent further aggregation by not allowing adjacent residues to form intermolecular b-sheets. These features suggest that apart from protease inhibition there is a possibility of additional functions linked to MKMLP associated with the aggregation process because clearly there is a route to prevent amyloid formation.

CONCLUSIONS The overall three-dimensional structures of MKMLP determined at 2.2 A˚ resolution were similar to the earlier

Insights into Aggregation Behavior of MKMLP

Figure 10 The ribbon diagram showing proposed MKMLP aggregation model involving Patch 1 (red) and Patch 2 (blue) regions involved in intermolecular associations. A total of six MKMLP molecules are represented and aggregation-prone patches form the interface between two molecules. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

reported structure at 2.9 A˚. However, a remarkable improvement in the model was observed and certain key structural details were revealed in the high resolution structures. The presence of increased helix content (residues 115–122) is seen, which is consistent with earlier circular dichroism studies. The presence of helices is a unique feature found in MLPs as compared with classical Kunitz inhibitors. Also, flexibility in the glycine rich region (GGAGGGG—24–30) is evident where drastic alteration in conformation is observed. The comparison of crystal structures grown in three different pH conditions (pH 8.0, 7.0, and 4.6) provided the structural basis for the aggregation of MKMLP below pH 7.5. The analysis of aggregation-prone regions revealed four aggregation-prone peptides distributed in two patches present far apart on the surface of the protein. The subtle pH-dependent conformational changes resulted in alterations to electrostatic and hydrophobic interactions. A gradual exposure of aggregation-prone peptides in two patches was observed. In Patch 1, a partial unfolding due to the shortening of b-strand of aggregation-prone peptide and change in orientation of glycine-rich loop is observed in low pH structure. Comparison of the three crystal structures at the symmetry-related molecular interface involving Patch 2, revealed increased hydrophobic interactions due to the juxtaposition closing in of two symmetry-related molecule as the pH decreased. The distance between Trp103 in aggregation-prone peptide WFITTG (103–108) in Patch 2 decreased when the symmetry-related molecular interface was compared in three structures. The peptide WFITTG (103–108) is unique to MLPs and, therefore, W103 may be responsible for initiating aggregation. The results indicate that the aggregation in MKMLP below pH 7.5 is a result of subtle conformational change accompanied by alteration in electrostatic and hydrophobic interaction, and thereby exposing the aggregation-prone peptides in two patches as pH decreases. The exposure of aggregation-prone regions

results into increased nonpolar intermolecular contacts where Patch 1 and Patch 2 of one molecule interact with Patch 1 and Patch 2 of adjacent molecules, respectively. The aggregation results from the cumulative effect of these hydrophobic interactions between the two defined respective patches among adjacent MKMLP molecules as pH is lowered.

ACKNOWLEDGMENTS The crystal structure analysis was performed at Macromolecular Crystallographic Unit, IIC at IIT Roorkee. We acknowledge Ms Sonali Dhindwal for the technical help. P. Selvakumar, N. Sharma, and P.P.S. Tomar thank, DBT and CSIR, Government of India for financial support, respectively. REFERENCES 1. Stefani M, Dobson CM. Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J Mol Med 2003;81(11):678–699. 2. Tartaglia GG, Pawar AP, Campioni S, Dobson CM, Chiti F, Vendruscolo M. Prediction of aggregation-prone regions in structured proteins. J Mol Biol 2008;380(2):425–436. 3. Colon W, Kelly JW. Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro. Biochemistry 1992;31(36): 8654–8660. 4. Calamai M, Chiti F, Dobson CM. Amyloid fibril formation can proceed from different conformations of a partially unfolded protein. Biophys J 2005;89(6):4201–4210. 5. Kelly JW. The alternative conformations of amyloidogenic proteins and their multi-step assembly pathways. Curr Opin Struct Biol 1998;8(1):101–106. 6. Theerasilp S, Kurihara Y. Complete purification and characterization of the taste-modifying protein, miraculin, from miracle fruit. J Biol Chem 1988;263(23):11536–11539. 7. Hirai T, Sato M, Toyooka K, Sun H-J, Yano M, Ezura H. Miraculin, a taste-modifying protein is secreted into intercellular spaces in plant cells. J Plant Physiol 2010;167(3):209–215.

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