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proteinsSTRUCTURE O FUNCTION O BIOINFORMATICS
Structural insights into the aggregationbehavior of Murraya koenigii miraculin-like proteinbelow pH 7.5Purushotham 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 inter-
faces 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 inter-
actions in two aggregation-prone defined regions.
Proteins 2014; 82:830–840.VC 2013 Wiley Periodicals, Inc.
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 chal-
lenge for protein biochemists. Protein aggregation results
in a number of human pathologies including Alzhei-
mer’s, Parkinson’s, and Creutzfeldt-Jakob diseases; and
the systemic amyloidoses associated with immunoglobu-
lin 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
superfamily and have sequence similarity (�30%) to soy-
bean Kunitz family trypsin inhibitors.8 Murraya koenigii
miraculin-like protein (MKMLP), a 21.4 kDa protein
with trypsin inhibitory activity, was purified and charac-
terized from seeds of Murraya koenigii belonging to Ruta-
ceae 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 Quater-
nary 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
830 PROTEINS VVC 2013 WILEY PERIODICALS, INC.
analyses and showed major differences at primary and sec-
ondary specificity sites in reactive loop when compared
with classical Kunitz inhibitors like soybean Kunitz inhibi-
tor (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 conforma-
tional differences limited to loop regions. The unique fea-
tures 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 function-
ally 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 indicat-
ing the translocation of protein from the site of synthe-
sis.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 low-
ered.9 The present study explores the basis for this aggre-
gation 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 datacollection
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 purifi-
cation. 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 pre-
pared by mixing 1 mL protein solution with 1 mL precipi-
tant 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 dif-
fraction 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 proc-
essed 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 subse-
quently, it was refined using restrained refinement. Model
building was conducted in manual mode in Coot,18 fol-
lowed 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 visual-
ization software Coot18 and PyMOL.21 The atomic coor-
dinates have been deposited in the Protein Data Bank.
Prediction of aggregation-prone regions
Aggregation-prone regions were predicted using differ-
ent programs employing empirical and structure-based
algorithms such as Tango,22 FoldAmyloid,23 Aggres-
can,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
Insights into Aggregation Behavior of MKMLP
PROTEINS 831
following accession codes: 3ZC8 (pH 7 structure) and
3ZC9 (pH 4.6 structure).
RESULTS
Three-dimensional structure of MKMLP atpH 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 crys-
tallographic 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 struc-
tures 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)
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.
Overall structure
The three-dimensional structures of MKMLP, deter-
mined 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 struc-
tures, except for first and last b-strands, the correspond-
ing 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 com-
pared 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 pseudo-
threefold 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 dis-
torted helices were observed similar to pH 7 structure
except for the L2 (residues 26–28). The presence of heli-
ces 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 pro-
tein. 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 diffrac-
tion 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).
Table ICrystal Parameters, Data Collection, and Structure Refinement
Crystal data and intensity statistics pH 7 pH 4.6
Space group C 1 2 1 C 1 2 1Unit-cell parameters (�)a 101.51 101.61b 45.69 45.42c 38.78 38.79Resolution range (�) 50.5–2.2 50.5–2.2Completeness (%) 96.3 (74.9) 90.0 (69.0)Rmerge
a(%) 0.09 (0.27) 0.07 (0.21)Multiplicity 3.3 (3.1) 3.5 (3.3)Mean I/sigma (I) 10.3 (4.4) 13.7 (5.7)Refinement and model statisticsTotal no. of reflections 28,501 28,271No. of reflections (used) 8605 8647Percentage observed 99.6 92.1Wilson B-factor (�2) 20.9 24.6Crystallographic R-factor (%) 19.5 19.4Free R-factor (%) 24.2 23.8Average B factor (�2) 16.6 19.1RMSD bonds (�) 0.01 0.01RMSD angles (�) 1.3 1.2Validation by MOLPROBITYRamachandran plotFavored (%) 95.5 93.5Allowed (%) 4.5 5.5Outliers (%) 0 1PDB code 3zc8 3zc9
The values in parentheses refer to statistics in the highest bin.aRmerge 5 RhklRijIi(hkl) – <I(hkl)>j/RhklRiIi(hkl), where Ii(hkl) is the intensity
of an observation and <I(hkl)> is the mean value for its unique reflection; sum-
mations are overall reflections.
P. Selvakumar et al.
832 PROTEINS
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 elec-
tron 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 con-
ditions 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 condi-
tions. 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 com-
pared 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 reso-
lution structures exhibits a well defined electron density
with a typical canonical conformation. A well defined
unoccupied electron density at Asn64 confirms the pres-
ence of glycan moieties. Only MLPs possess this
Figure 1Overall 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, andC140–151). 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 wascalculated 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.]
Insights into Aggregation Behavior of MKMLP
PROTEINS 833
glycosylation motif at active site loop and even miraculin
lacks the same. The superposition of Ca atoms of reac-
tive 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 resi-
due 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
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 symmetry-
related 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
Table IISecondary structural element details of MKMLP structures at pH 7 and 4.6 conditions
pH 7 MKMLP structure 4.6 MKMLP structure
Loops 1-16 L1 Loops 1-16 L124-30 L2 22-30 L235-43 L3 35-43 L348-57 L4 48-57 L463-74 L5 63-74 L580-89 L6 80-89 L6
94-104 L7 94-104 L7110-114 L8 110-114 L8128-134 L9 128-134 L9140-151 L10 140-151 L10162-163 L11 162-163 L11168-174 L12 168-175 L12
Sheets 17223
31234
44247
58262
9>>>>>=>>>>>;
Subdomain A
b1 Sheets 17221
31234
44247
58262
9>>>>>=>>>>>;
Subdomain A
b1b2 b2b3 b3b4 b4
75279
90293
1052109
1242127
9>>>>>=>>>>>;
Subdomain B
b5 75279
90293
1052109
1242127
9>>>>>=>>>>>;
Subdomain B
b5b6 b6b7 b7b8 b8
1352139
1522156
1642167
1752180
9>>>>>=>>>>>;
Subdomain C
b9 1352139
1522156
1642167
1762180
9>>>>>=>>>>>;
Subdomain C
b9b10 b10b11 b11b12 b12
Helices 115-122 a1 Helices 115-122 a1158-161 a2 158-161 a2
Disulfide bridges Cys41-Cys89, Disulfide bridges Cys41-Cys89,Cys140-Cys151 Cys140-Cys151Cys144-Cys147 Cys144-Cys147
Figure 2Structural superimposition of Ca atoms of three MKMLP structures.
The structures at pH 8, 7, and 4.6 showing similar overall fold are rep-resented 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 atwileyonlinelibrary.com.]
P. Selvakumar et al.
834 PROTEINS
Trp103 residues in loop region (94–104) in adjacent sym-
metry 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 clas-
sical 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 struc-
ture compared with other classical Kunitz members.
Many studies have revealed that certain peptides in pro-
tein sequences initiate or mediate aggregation. These
regions have also been successfully predicted. There must
be differences in the number of aggregation-prone
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 con-
tact on a protein molecule. In the crystal structure deter-
mined at pH 4.6, the purified MKMLP used for
crystallization was in Tris buffer at pH 7.5 and precipi-
tant sodium acetate was at pH 4.6. The protein solution
and precipitant were mixed in equal proportions. There-
fore, structure represented here may not be a true struc-
ture at pH 4.6 but it is definitely a structure below pH 7.
Peptide sequences of MKMLP predicted tobe involved in aggregation
Six programs were used for prediction of aggregation-
prone regions of MKMLP. Since these programs employ
various parameters for prediction so there were subtle
differences in the results (Fig. S2, Supporting Informa-
tion). We have considered consensus from all the results
and have found four regions involved in aggregation dis-
tributed 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 pre-
dicts 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, Support-
ing Information). These results indicate increased aggrega-
tion tendency of MKMLP below pH 7.5.
Sequence comparisons reveal that other related mem-
bers lack predicted consensus aggregation-prone peptides
found in MKMLP sequence. Compared with MKMLP,
results for STI showed only two aggregation prone pep-
tide 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 pep-
tide SCVFLCN (146–152) in MKMLP (Fig. 7).
Structural insights into the aggregationbehavior 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
Figure 3Superimposition involving residues 17–30 and putative reactive loop inthree MKMLP structures at different pH exhibiting variation in second-
ary 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, respec-
tively. (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 fig-ure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
Insights into Aggregation Behavior of MKMLP
PROTEINS 835
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 pro-
tein association.29 Crystal structures of MKMLP deter-
mined 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 pre-
dicted 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 demon-
strated the potential of these peptides to form intermo-
lecular b-sheets that are not seen in native structure.
Most importantly SCVFLCN (146–152) showed b-
aggregation 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 4Crystallographic 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.]
Figure 5Crystallographic symmetry-related molecule interface showing distances between two Trp103 at different pH involving WFITTG (103–108). Loca-tion 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.]
P. Selvakumar et al.
836 PROTEINS
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 pep-
tide YYLVSVI (17–23). First, the change in orientation of
glycine rich loop when pH 8 and 7, and pH 4.6 struc-
tures are compared (Fig. 3). Second, a shortening of b1
and b2 strands and lengthening of loop L2. The compar-
ison of the conformation in this region in three struc-
tures 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 for-
mation 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 aggregation-
prone regions and thereby promoting hydrophobic inter-
protein 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 sym-
metrical 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 6Plots 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 theonline issue, which is available at wileyonlinelibrary.com.]
Figure 7Multiple sequence alignment of MKMLP, miraculin, and STI. The predicted aggregation-prone regions in MKMLP are shown and underlined inred. Boxes indicate comparative critical amino acid sequence changes and deletions. MKMLP shows presence of more hydrophobic residues com-
pared with miraculin and STI. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Insights into Aggregation Behavior of MKMLP
PROTEINS 837
observations. For Patch 1, the pH 4.6 structure lost criti-
cal 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
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 symmetry-
related molecules are analyzed the distance between adja-
cent W103 sidechains are decreased from 3.9 A at pH 8
to 3.5 A at pH 4.6. These signify dominance of hydropho-
bic interactions, and this slight shift in intramolecular
associations might trigger two similar molecules to associ-
ate leading to aggregation (Fig. 10). Also, the biological
assembly of pH 7 structure was predicted to be mono-
meric and pH 4.6 structure as dimeric by PQS, which
uses crystal symmetry matrices to generate symmetry-
related 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 substan-
tial blue shift indicating that conformational changes
does not significantly expose hydrophobic pockets. How-
ever, 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 struc-
tures.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 Kun-
tiz 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 cys-
teines 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 addi-
tional 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
Figure 9Ionic and hydrogen bonding interactions in MKMLP structures at pH 7
and 4.6. (a) Water molecule and ionic interactions of hydrophobic resi-
dues Val148, Phe149, and Leu150 residues of SCVFLCN (146–152) ofPatch 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 formshydrogen 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 atwileyonlinelibrary.com.]
Figure 8Surface view of MKMLP structure showing location of two aggregationprone 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.
P. Selvakumar et al.
838 PROTEINS
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 (resi-
dues 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 con-
formational 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 ori-
entation of glycine-rich loop is observed in low pH struc-
ture. 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 respec-
tive patches among adjacent MKMLP molecules as pH is
lowered.
ACKNOWLEDGMENTS
The crystal structure analysis was performed at Macro-
molecular 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.
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