Molecular

Omics
View Article Online
RESEARCH ARTICLE View Journal
Published on 13 May 2019. Downloaded by MTA Research Centre for Natural Science on 5/27/2019 2:40:11 PM.

Is protein context responsible for

peptide-mediated interactions?†
Cite this: DOI: 10.1039/c9mo00041k
Peng Zhou, *abc Qingqing Miao,ab Fugang Yan,ab Zhongyan Li,ab Qianhu Jiang,b
Li Wenb and Yang Mengb

Received 12th March 2019,
Accepted 10th May 2019
DOI: 10.1039/c9mo00041k
rsc.li/molomics
Many cell signaling pathways are orchestrated by the weak, transient, and reversible protein–protein
interactions that are mediated by the binding of a short peptide segment in one protein (parent protein)
to a globular domain in another (partner protein), known as peptide-mediated interactions (PMIs).
Previous studies normally had an implicit hypothesis that a PMI is functionally equivalent or analogous to
the protein–peptide interaction (PTI) involved in the PMI system, while ignoring parent context contribu-
tion to the peptide binding. Here, we perform a systematic investigation on the reasonability and applic-
ability of the hypothesis at structural, energetic and dynamic levels. It is revealed that the context
impacts PMIs primarily through conformational constraint of the peptide segments, which can (i) reduce
the peptide flexibility and disorder in an unbound state, (ii) help the peptide conformational selection to
fit the active pocket of partner proteins, and (iii) enhance the peptide packing tightness against the part-
ners. Long, unstructured and/or middle-located peptide segments seem to be more vulnerable to their
context than short, structured and/or terminal ones. The context is found to moderately or considerably
improve both the binding affinity and specificity of PMIs as compared to their PTI counterparts; with the
context support a peptide segment can contribute to B30–60% total binding energy of the whole PMI
system, whereas the contribution is reduced to B5–50% when the context constraint is released. In
addition, we also observe that peptide selectivity is largely impaired or even reversed upon stripping of
their parent context (global selectivity decreases from 34.2 to 1.7-fold), by examining the crystal
structures of full-length Src family kinases in an autoinhibitory state. Instead of the direct interaction and
desolvation that are primarily concerned in traditional studies, peptide flexibility and the entropy penalty
should also play a crucial role in the context effect on PMIs. Overall, we suggest that the context factor
should not be ignored in most cases, particularly those with peptide segments that are long, highly
disordered, and/or located at the middle region of their parent proteins.

Introduction in another.2 Such PPIs are also known as peptide-mediated

It is widely appreciated that protein–protein interactions (PPIs)
play a fundamental role in various aspects of biological pro-
cesses in living cells.1 Many PPIs, particularly those that are
weak, transient and/or related to post-translational modifica-
tion events like phosphorylation, are mediated by the binding
of a short peptide segment in one protein to a globular domain
a
Center for Informational Biology, University of Electronic Science and Technology
of China (UESTC), Qingshuihe Campus, No. 2006, Xiyuan Ave West Hi-Tech Zone,
Chengdu 611731, China. E-mail: p_zhou@uestc.edu.cn; Fax: +86 28 61830654;
Tel: +86 28 61830670
b
School of Life Science and Technology, University of Electronic Science and
Technology of China (UESTC), Chengdu 610054, China
c
Center for Information in BioMedicine, University of Electronic Science and
Technology of China (UESTC), Chengdu 611731, China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9mo00041k
interactions (PMIs), which are enriched in cell signaling net-
works and ideal for mediating interactions that are easily
formed and disrupted, and required as a fast response to
stimuli.3 Neduva and Russell estimated that up to 40% of the
PPIs in the human interactome are directly or indirectly
mediated by short peptide segments presented at the inter-
action sites, and nearly 60% of the PPIs in cell signaling
pathways involve at least one PMI event.4
The functional peptide region in a PMI is usually a small
segment on the surface of its parent protein, which lacks a
definite structure in solution and binds to its partner protein in
a folding-upon-binding manner.5,6 However, peptide motifs do
not only interact with their protein partners via the coupled
folding-to-binding mechanism. Instead, the phenomenon of
fuzziness has also been observed in many PMIs, which repre-
sents polymorphism and structural disorder in PPIs and forms
so-called fuzzy complexes.7,8 Previously, a number of studies

This journal is © The Royal Society of Chemistry 2019 Mol. Omics


Research Article
Published on 13 May 2019. Downloaded by MTA Research Centre for Natural Science on 5/27/2019 2:40:11 PM.
Fig. 1 There is a doubt whether PMI is functionally equivalent or analogous
(e.g. affinity, specificity, conformation and binding mode, etc.) to the PTI
involved in the PMI? (A) A peptide segment is protruded on the surface of its
parent protein and binds to its partner protein to form a PMI, and during the
process the segment is within the context of its parent protein. (B) The
segment is split from its parent protein to obtain an isolated peptide ligand,
which then binds to its partner protein to form a PTI. The PTI can be
regarded as a reduced counterpart version of the PMI.
have been addressed on the PPI/PMI fuzziness phenomenon to,
for example, explore their thermodynamics basis,9 compare
with folding coupled binding mechanisms10 and develop bio-
informatics tools and databases to annotate them.11 Never-
theless, due to the large flexibility and high independence of
the peptide segment, previous studies normally had an implicit
hypothesis that a PMI is functionally equivalent or analogous
to the protein–peptide interaction (PTI) involved in the PMI
system, while ignoring the protein context contribution to the
peptide binding. Surely, such a hypothesis can considerably
simplify the PMI system since only a short peptide is used to
represent its large parent protein. For example, many existing
crystallographic analyses and binding affinity assays of PMIs
only adopted the reduced PTI versions (but not the full-length
PMI protein–protein interactions) to conduct the investiga-
tions, but the obtained conclusions were directly applied to
explain the PMI events. However, one would doubt whether
the hypothesis is reasonable and which conditions it can
(or cannot) work in (Fig. 1).
In fact, there were some studies that had shed light on the
importance of protein context in PMIs. For instance, Fuxreiter
and coworkers found that peptide linear motifs (LMs) are
usually constructed by grafting a few specificity-determining
residues favoring structural order on a highly flexible carrier
region. They also established a connection between LMs and
molecular recognition elements of intrinsically disordered
proteins (IDPs), which realize a non-conventional mode of
partner binding mostly in regulatory functions.12 Later, the
contribution of context-specific evolutionary conservation to
LM recognition and the context dependence of fuzzy interaction
were revealed, implicating the context role in PMIs.13,14 Recently,
Konrat et al. showed how structural unfolding compensates for
entropic losses through ligand binding in IDPs and elucidated the
interplay between structure and thermodynamics of rapid substrate-
binding and -release events in IDP interaction networks.15
Mol. Omics
View Article Online
Molecular Omics
They also observed correlated long-range motions and anti-
correlated fluctuations involved in IDP recognition and bind-
ing, indicating a loosening of structural compaction upon the
binding.16 These studies could help to establish a plastic
concept for PMIs. In addition, the binding dynamics and
thermodynamics of the disordered N-tails of Antp and NK-2
homeodomains to their DNA partners have also been charac-
terized in detail by Tóth-Petróczy et al. using coarse-grained
dynamics simulations and found a disorder-to-order transition
of the disordered N-tails upon interacting with DNA. This is very
similar to the folding-upon-binding phenomenon of PMIs.17
The canonical binding mode of the peptide-recognition PDZ
domain to its target motifs involves a small interface that
is unlikely to fully account for the PDZ–target interaction
behavior; sequence context surrounding the motifs has been
suggested to introduce structural diversity, to impact the
stability and solubility of the constructs, and to influence the
binding affinity and specificity of the interaction.18 The context
information has also been successfully combined with a
peptide sequence pattern to improve the prediction of the
14-3-3 domain-mediated PMI interactome in S. cerevisiae and
H. sapiens.19 More importantly, Stein and Aloy have system-
atically dissected 3D structural data matching the known
eukaryotic linear motifs (ELMs) and found that the protein
structural context contributes to B20% PMI binding events
and may play an important role in determining the binding
specificity, by either improving affinity with the native partner
or impeding non-native interactions.20
Recently, we have examined a special PMI phenomenon in
proto-oncogene tyrosine kinase c-Src, termed as self-binding
peptide (SBP),21 which is an intramolecular interaction between
the SH3 domain and PPII peptide of the kinase. We found that
the PPII lacks the standard PxxP motif that is normally required
for SH3 recognition, and thus can only interact effectively with
SH3 domains when it is integrated into the full-length c-Src
kinase protein; splitting of the PPII peptide segment from the
intact protein would considerably impair SH3 affinity by
increasing the entropy penalty upon domain–peptide binding,
revealing that the protein context plays an essential role in the
SBP biological function.22 On the other hand, during a compu-
tational design of antiangiogenic peptibody PbHRH by fusing
an HRH peptide agent to human IgG1 Fc fragment we found
that the HRH is structurally and functionally independent of
the Fc fragment in the designed peptibody; integration of HRH
peptide into the peptibody does not alter the peptide inter-
action with its target protein substantially. Instead, the
designed peptibody may indirectly help to improve the phar-
macokinetic profile and bioavailability of HRH.23 This finding
suggests that the protein context may not be crucial for
every PMI event, and in some cases the peptide segment can
work independently of its parent context to interact with its
partner protein.
Here, we performed a deep investigation of the molecular
mechanism of the protein context contribution to diverse PMIs,
attempting to answer questions like whether the context is
responsible for PMIs, how the context works in PMIs, and what
This journal is © The Royal Society of Chemistry 2019

Molecular Omics
kinds of contexts contribute significantly or insignificantly to
PMIs. First, we systematically compared the crystal conforma-
tions of peptide ligands in complex with their protein receptors
and the same peptide segments in their parent proteins.
Second, twenty-third representative PMI systems were curated
and subjected to atomistic molecular dynamics simulations to
Published on 13 May 2019. Downloaded by MTA Research Centre for Natural Science on 5/27/2019 2:40:11 PM.
characterize the structural dynamics of the peptide segments
with or without the support from their parent context. Thirdly,
the PMI binding energy was also derived, analyzed and decom-
posed in detail to examine the context contribution to different
energetic components involved in the PMIs. This work would
establish a complete profile for the structural basis, energetic
property and dynamic behavior of the context effect on PMI
intermolecular recognition and interaction, and may help to
correct the inappropriate applications of PTI succedaneums in
future PMI studies.
Materials and methods
Crystal structure survey
The current deposition (Sept. 2018) of the RCSB Protein Data
Bank (PDB) database24 was systematically surveyed using the
following criteria implemented with an advanced search tool
provided by the database:
(i) Macromolecule types: only protein entities, no nucleic
acids.
(ii) Experimental method: X-ray diffraction.
(iii) X-ray resolution: r3 Å.
(iv) Number of chains in biological assembly: Z2.
(v) Length of at least one chain: 5–30.
(vi) Sequence identity: r30%.
The criteria were applied for filtering the entire PDB database
to obtain the high-resolution, non-homologous crystal structure
data of the PTI complexes. Consequently, in total 6497 records
meeting the criteria were retrieved from the database, which
represents a distinct panel of PTI complex crystal structures
(listed in ESI,† Table S1). We manually excluded some invalid
samples from the panel, such as those that were largely incom-
plete and highly modified. Next, the peptide sequences were
extracted from these collected complexes, which were then one-
by-one searched back against the database to identify the crystal
structures that contain the same peptides as a segment of their
parent proteins. The crystal structures are solved in high resolu-
tion (r3 Å) and their peptide segment regions should be intact
(e.g. no backbone breaking and/or side-chain missing).
Definition, curation, and setup of PMI systems and their PTI
counterparts
A PMI system is defined as a binary or multi-PPI complex that
consists of two members M1 and M2. The M1 is termed as the
parent protein that contains a functional peptide segment as the
core sequence element to mediate M1–M2 interaction; the M2
is designated as the partner protein that recognizes and inter-
acts with the peptide segment of M1. The M1 is commonly a
monomeric protein, whereas the M2 can be either a protein
This journal is © The Royal Society of Chemistry 2019
View Article Online
Research Article
monomer or a multiprotein complex. Most residues in the
peptide segment belong to the interfacial residues of M1–M2
interaction, although a few non-interfacial residues are allowed
to discretely distribute over peptide sequence to keep the
peptide continuity. We considered two residues separately
coming from M1 and M2 to be ‘‘in contact’’ (and thus as
interfacial residues) if there was a hydrogen bond, a water-
mediated hydrogen bond, a van der Waals interaction, or at
least one pair of contacting nonhydrogen atoms (r4.5 Å)
between them. This strategy was modified from our previous
work on protein–nucleic acid interactions.25 The hydrogen
bonds, water-mediated hydrogen bonds and van der Waals
interactions were detected using HBPLUS26 and PROBE27
programs, respectively.
Here, we only chose the PMI systems that have high-
resolution crystal structures (r3 Å) and also the structures
should be intact in and around their peptide-mediated inter-
action site. In addition, the PMI should be a typical single
peptide-mediated interaction whose interaction site only
presents a sole successive peptide segment and other inter-
facial residues, if present, out of the segment in the parent
protein are very few (r1/5 of the peptide residues). According
to the above rule, a total of 23 representative PMI systems
were curated from a number of literature reports.20,28,29 The
collected PMIs are diverse in terms of their biological functions
as well as the size, location, structural type and amino acid
composition of their core peptide segments (Table 1). Some
examples of these PMI structural architectures are shown in
ESI,† Fig. S1. The crystal structures of the PMI complexes were
used to derive their respective PTI counterparts. As shown in
Fig. 2, the PMI system of subtilisin–chymotrypsin inhibitor 2
complex (PDB: 2SNI_I:E) is mediated by the intermolecular
interaction between subtilisin (partner protein) and a loop
peptide segment of chymotrypsin inhibitor 2 (parent protein).
The peptide bonds separately at the N- and C-termini of the
peptide segment are manually broken and then capped by
acetyl (–Ac) and amide (–NH2) moieties to eliminate the formal
charges at the free N- and C-termini of the generated peptide
ligand, respectively, while rest of the parent protein (protein
context) is discarded.
Molecular dynamics simulation
The 23 representative PMIs and their PTI counterparts (Table 1)
were subjected to atomistic molecular dynamics (MD) simula-
tions to examine the context effect on the structural dynamics
of peptide segments involved in these PMIs. All simulations were
carried out using the AMBER ff03 force field30 implemented with
the sander module of the Amber package.31 According to our
previous studies,32–34 dynamics may be sensitive to the combi-
nation of parameter settings, solvent conditions, forcefield
versions and so on for different systems. Here, we have per-
formed pre-simulations to optimize the combination, although
the optimization procedure is empirical and intuitive, and no
general/objective rule is applicable for guiding it. Briefly, a
truncated octahedral box of TIP3P water molecules35 was added
with a 12 Å buffer around the simulated system. Counterions of
Mol. Omics
 Published on 13 May 2019. Downloaded by MTA Research Centre for Natural Science on 5/27/2019 2:40:11 PM.

Mol. Omics
Research Article

Table 1 The 23 representative PMI systems

Peptide segment Parent protein Partner protein

a b c d e
PBD Sequence Size Structural type Location Name Length Name Lengthe
486
1AK4_D:A VHAGPIAP493 8-mer, short Loop Middle HIV-1 capsid p17 145 Cyclophilin 545
54
2SNI_I:E GTIVTMEYRI63 10-mer, short Loop Middle Chymotrypsin inhibitor 2 64 Subtilisin 275
34
1AY7_B:A LDALWDCLTG43 10-mer, short Helix Middle Barstar 89 RNase Sa 96
51
2C7M_A:B QIQEDWELAERLQREE66 16-mer, long Helix Middle Rabex-5 58 Ubiquitin 74
11
3FP6_I:E TGPCKARII19 9-mer, short Loop Middle Pancreatic trypsin inhibitor 58 Anionic trypsin-2 230
640
3L4F_D:ABC AWDETNL646 7-mer, short Strand C-terminus Shank PDZ 107 bPIX 183
283
1UEL_B:A EEEQIAYAMQMSLQGA298 16-mer, long Helix Middle Proteasome subunit S5a 48 RAD23 homolog B 95
99
1GH6_A:B NEENLFCSEEMP110 12-mer, midsize Helix/loop C-terminus SV40 large T antigen 114 Rb tumor suppressor 394
106
2HLE_B:A QDIKFTIKFQEFSPNLWGLE125 20-mer, long Strand Middle EphrinB2 140 EphA4 178
19
2FD6_A:U CVSNKYFSNIHWCN32 15-mer, midsize Strand N-terminus Urokinase plasminogen activator 122 Urokinase receptor 276
11
1D6R_I:A QCACTKSNPPQ21 11-mer, midsize Strand/loop N-terminus Bowman-Birk inhibitor 58 Trypsinogen 230
81
1GHQ_B:A YKIRGSTP88 8-mer, short Strand/loop Middle Complement receptor 2 129 Complement C3d 307
204
1E6J_P:HL ALGPAATLEEMMTA217 14-mer, midsize Helix/loop C-terminus HIV-1 capsid P24 210 Fab13B5 antibody 429
21
1E96_A:B ISYTTNAFPGE31 11-mer, midsize Helix/loop Middle Rac.GTP 178 P67Phox Tpr 185
86
2I25_N:L ESRYGSYD93 8-mer, short Loop Middle IgNAR 114 Lysozyme C 129
561
1AVX_B:A PYRIRF566 6-mer, short Loop Middle Kunitz-type trypsin inhibitor 177 Trypsin 230
933
2OOB_A:B DAKIAKLMG941 9-mer, short Helix N-terminus E3 ubiquitin ligase Cbl-b 44 Ubiquitin 72
203
1A2K_E:B VAQTTALPDE212 10-mer, short Helix/loop C-terminus GTPase Ran 205 Nuclear transport factor 2 124
514
7CEI_B:A SRNNNDRMKVGKAPKTRT531 18-mer, long Helix/loop Middle Colicin-E7 127 Immunity protein Im7 87
49
1KTZ_B:A SITSICE55 7-mer, short Strand Middle TGF-b type II receptor 106 TGF-b3 100
67
2MTA_C:A GKIGPGLNDAYWT79 13-mer, midsize Loop Middle Cytochrome c551i 147 Amicyanin 105
346
4MLF_D:B PKPQSHNDGDFEEIPEEYLQ365 20-mer, long Loop C-terminus Hirudin 65 Thrombin 232
65
4JLR_S:HL SELLSKINDMPITNDQKKLMSNDVL89 25-mer, long Helix Middle RSV immunogen 104 Motavizumab 424
a
PDB_M1:M2: the PDB code of a PMI crystal structure and the complex members (M1 and M2) involved in the PMI. The first member M1 is the parent protein of the peptide segment, and the
second member M2 is the partner protein of the peptide segment. The partner protein can consist of one or more protein chains. b Size: short peptide: r10-mer, midsize peptide: 410-mer and
r15-mer, long peptide: 415-mer. c Structural type: helix: a-helix and helical hairpin, strand: b-strand, b-sheet and b-hairpin, loop: loop, coil and disordered region. d Location: N-terminus,
C-terminus and middle: the peptide segment is located at (or close to) the N-terminus, C-terminus and middle region of its parent protein, respectively. e Length: the sequence length of (parent
or partner) protein in investigated crystal structure.
View Article Online

This journal is © The Royal Society of Chemistry 2019

Molecular Omics

Molecular Omics
Published on 13 May 2019. Downloaded by MTA Research Centre for Natural Science on 5/27/2019 2:40:11 PM.
Fig. 2 Schematic representation of modeling PTI counterparts from PMI systems. The subtilisin–chymotrypsin inhibitor 2 complex (PDB: 2SNI_I:E) is a
PMI system mediated by the intermolecular interaction between subtilisin (partner protein) and a loop peptide segment of chymotrypsin inhibitor 2
(parent protein). The peptide bonds separately at the N- and C-termini of the peptide segment are manually broken and then capped by acetyl (–Ac) and
amide (–NH2) moieties to eliminate the formal charges at the free N- and C-termini of the split peptide ligand, respectively.
Na+ or Cl were placed based on the coulombic potential to keep
the whole systems electroneutral. Initially, the system was energy
minimized in two steps. First, only the water molecules and ions
were minimized in 5000 steps while keeping the protein struc-
ture restricted by weak harmonic constraints of force constant
2 kcal mol1 Å2. Second, a 3000 steps minimization with
steepest descent (500 steps) and conjugate gradient (2500 steps)
methods on the whole system was performed to remove bad
atomic contacts and bond distortions involved in the crude
structure. After minimizations the system was heated in the
NVT ensemble from 0 to 300 K over 50 ps followed by constant
temperature equilibration at 300 K for 300 ps. Subsequently, MD
equilibrium simulations were run for 50 ns in the NPT ensemble
with periodic boundary conditions. An integration step of 2 fs
was set for the simulations and the particle mesh Ewald (PME)
method36 was employed to analyze the long-range electrostatic
energy of a unit cell in a macroscopic lattice of repeating images.
A cut-off distance of 10 Å was considered to calculate the short-
range electrostatics and van der Waals interactions. The SHAKE
strategy was used to restrain all covalent bonds involving
hydrogen atoms.37 Temperature regulation was carried out using
a Langevin thermostat with a collision frequency of 2 ps1.38
Snapshot collection, conformational clustering and energetics
analysis
The conformational space of the investigated PMI/PTI systems
(i.e. PMI protein–protein interaction/PTI protein–peptide inter-
action) during MD simulations was characterized using a
clustering data analysis technique. A total of 1000 conforma-
tional snapshots (frames) of each investigated system were
saved evenly over the equilibrium trajectory of MD production
simulations.39 The average-linkage algorithm40 was employed
to produce a number of clusters using the pairwise root-mean-
square-deviations between frames as a metric comparing all the
atoms from residues of the peptide segment (for PMI system) or
peptide ligand (for PTI system). PDB files were dumped for the
average and representative structures from the clusters.41 In
addition, for examining the sole binding behavior of a peptide
segment in an intact PMI system (i.e. PMI protein–peptide
This journal is © The Royal Society of Chemistry 2019
View Article Online
Research Article
interaction), the trajectory data of only the segment section
were extracted from the MD simulations of the whole PMI
system to perform the energetics analysis, where the N- and
C-termini of the segment did not need to be capped by –Ac or
–NH2 groups, since the extraction did not really break the
peptide segment from its parent protein, which just used the
dynamics trajectory of the segment section to analyze its sole
binding behavior in the whole PMI system.
The conformational snapshots were also used to derive
binding free energy DGttl for the system:42
D E
ðiÞ ðiÞ
DGttl ¼ DEint þ DGslv  TDS ðiÞ
D    E
ðiÞ ðiÞ ðiÞ ðiÞ
¼ DEelc þ DEvdW þ DGplr þ DGnplr  TDS ðiÞ
(1)
where h  i represents average over snapshots from a single
simulation trajectory and (i) corresponds to the ith snapshot of
the complex system. The DEint is the intermolecular interaction
energy between the complex members, which can be divided
into electrostatic (DEelc) and van der Waals (DEvdW) potentials
and was calculated with a molecular mechanics (MM) approach.
DGslv is the solvent effect associated with the complex formation,
which is contributed from polar (DGplr) and nonpolar (DGnplr)
desolvations. The polar aspect was computed by numerical
solution of the nonlinear Poisson–Boltzmann (PB) equation,
while the nonpolar facet was described using a surface model
(SA) as DGnplr = b + gSASA.43 The grid size for the PB calculations
was set to 0.5 Å, and the interior and exterior dielectric constants
were 2 and 78, respectively. TDS is the conformational free
energy due to the entropy penalty upon complex binding,
which was characterized by normal mode analysis (NMA).44
Frequencies of the vibrational modes were computed at 300 K
for conformational snapshots and using a harmonic approxi-
mation of the energies.34 Considering the high computational
demand, only 100 snapshots for each system were utilized to
estimate the entropy penalty.45 Here, the MM/PBSA and NMA
calculations were implemented using the mmpbsa and nmode
modules of the Amber package,31 respectively.
Mol. Omics
 View Article Online

Research Article Molecular Omics

Table 2 Binding energetic components of the PPII peptide (with or without context) to SH3 domain of Src family kinases Src, Abl and Hck

Binding energetics (kcal mol1)

Kinase PDB PPII peptidea Context RMSD variation (Å) DEint DGslv TDS DGttl
248 257
Src 1FMK SKPQTQGLAK Yes 0.21 113.6 80.6 21.7 11.3
No 0.89 102.8 58.7 37.4 6.7
Published on 13 May 2019. Downloaded by MTA Research Centre for Natural Science on 5/27/2019 2:40:11 PM.

240
Abl 1OPL NKPTVYGVSP249 Yes 0.19 127.0 89.8 24.2 13.0
No 0.83 115.4 69.5 39.5 6.4
223
Hck 2C0T SKPQKPWEKD232 Yes 0.25 109.4 71.0 27.6 10.8
No 0.91 96.3 49.3 42.8 4.2
a
The class II binding motif PxxPx+ is in bold. The three PPII peptides cannot meet the motif, although they bind to SH3 domains in the class II
orientation according to our previous analysis.11

PMI specificity across Src family kinases

The autoinhibitory crystal structures of the full-length proteins
of three Src kinase family members, namely, Src, Abl and Hck,
were retrieved from the PDB database13 and listed in Table 2, in
which the polyproline II (PPII) peptide segments are in a bound
state with their intramolecular targets (SH3 domains). Each
PPII peptide segment was computationally grafted into the
protein context of three kinases to derive noncognate inter-
actions of the peptide with three SH3 domains. For example,
the PPII peptide segments of Src and Abl kinases are two
10-mer sequences 248SKPQTQGLAK257 and 240NKPTVYGVSP249,
respectively. Grafting of the Src PPII peptide into the Abl kinase
context was manipulated simply as the virtual mutation of the
Abl PPII peptide segment to the Src PPII peptide segment
in an Abl crystal structure using the rotamer-based SCWRL4
program.46 In this way, the kinase structures of three cognate
(crystal) and six noncognate (modeled) domain–peptide inter-
actions across the three SH3 domains and three PPII peptides
were obtained and, based on the structures, their binding
energies DGttl can be derived using MD-based energetics
analysis.
Here, we described a method to straightforwardly quantify
the peptide selectivity from the calculated binding energies
DGttl. According to Gibbs formula the domain–peptide binding
free energy DGttl = RT ln(1/Kd) = RT ln Kd p ln Kd, therefore
Kd p exp(DGttl), where Kd is the dissociation constant of the
domain–peptide binding, which is a direct indicator of the
binding affinity. If a PPII peptide can bind to its one cognate
SH3 domain A and two noncognate SH3 domains B and C with
affinity values Kd(A), Kd(B) and Kd(C), respectively, the peptide
selectivity for A over B and for A over C can be expressed as:
S[AxB] = Kd[B]/Kd[A] = exp(DGttl)[B]/exp(DGttl)[A]
S[AxC] = exp(DGttl)[C]/exp(DGttl)[A]
Geometric mean of the two selectivity values can be
written as:
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
^ . B and A . C ¼
S½A S½A . B  S½A . B
which represents the mean selectivity of a peptide for its
cognate A over its noncognate B and C.
Results and discussion
Statistical survey and conformational analysis of crystal
structures
A total of 6497 PTI complex crystal structures were retrieved
from the PDB database,24 from which the primary sequences of
the peptide ligands were extracted and then one-by-one
searched back against the database to identify the crystal
structures that contain the same peptides as a segment of their
parent proteins. Consequently, the peptide ligands of 109 PTI
complex samples were found to match the sequences of
segments of protein crystal structures deposited in the data-
base. Next, these segments were split from their parent proteins
and superposed onto the conformations of matched peptide
ligands in PTI complexes using the least squares fitting
method. The difference between two superposed conforma-
tions of a peptide was measured using root-mean-squares
deviation (RMSD), which is a statistic representing the spatial
separation over all heavy atoms of the peptide in two different
conformations. Consequently, the RMSD values of the 109
samples were calculated, and they were plotted as three histo-
grams to investigate statistical information involved in the
RMSD distribution (Fig. 3).
Distribution of the 109 peptide samples in different RMSD
intervals (in 1 Å bin) is shown in Fig. 3A. As can be seen, the
conformational difference of these peptides within their parent
context and in complex with their partner proteins is moderate,
with a significant RMSD variation over different peptides.
Strictly, the conformational difference is originated from four
aspects: (i) protein context contribution, (ii) folding-upon-
binding effect, (iii) peptide disorder and (iv) experimental error
and bias, in which the (i) and (ii) are considered as the primary
(2)
factors. Therefore, the RMSD distribution profile can pre-
(3) liminarily shed light on the context contribution to the binding
conformation and mode of peptide ligands to their partner
proteins.
In addition, we examined the influence of peptide length
and structural type on the conformational difference. The
(4) average RMSD  SE values of the peptide samples at different
length intervals are shown in Fig. 3B. It is seen that both the
conformational difference and variation have an enlarging
tendency with increasing peptide length; this tendency is linear

Mol. Omics This journal is © The Royal Society of Chemistry 2019


Molecular Omics
Published on 13 May 2019. Downloaded by MTA Research Centre for Natural Science on 5/27/2019 2:40:11 PM.
Fig. 3 Histogram characterization of context effect on 109 peptide crystal conformations when binding to their partner proteins. (A) Frequency
distribution plot of peptide samples in different RMSD intervals (in 1 Å bin). (B) Average RMSD  SE values of peptide samples in different length intervals
(5 AAs bin). The distribution can be fitted using a linear function with Pearson’s correlation of Rp = 0.86. (C) Average RMSD  SE values of peptide samples
with different structural types.
and positively correlated, with Pearson’s correlation of Rp = 0.86.
The average RMSD  SE value of the 27 shortest peptides is only
3.1  0.8 Å, which is considerably smaller than that (7.2  3.2 Å)
of the 7 longest peptides, suggesting that the polypeptide seg-
ments can be impacted more significantly by their parent
context as compared to oligopeptide segments—this is not
unexpected if considering that, relative to short peptides, long
peptides generally have a large conformational degree of free-
dom and thus are more vulnerable to context influence. How-
ever, the long peptides are structurally diverse and should be
inconsistent in response to different protein contexts, thus
exhibiting a large RMSD variation (SE). On the other hand, the
peptide structural type (assigned using the DSSP dictionary47)
has also a substantial effect on conformational difference. As can
be seen in Fig. 3C, the 32 structured a-helical peptides are quite
rigid and can only be impacted modestly by protein context with
RMSD  SE of 1.8  0.9 Å, while the 17 b-stranded peptides are
partially flexible and can be influenced moderately by the context
with RMSD  SE of 4.4  2.4 Å. The folding-upon-binding effect
seems to be also responsible for the conformational change of
these b-stranded peptides upon splitting and binding. In con-
trast, the other 60 peptides, most of them coiled or loops, are
intrinsically disordered and have large flexibility, and are very
vulnerable to the context with RMSD  SE of 6.0  2.9 Å. For
these flexible systems fuzziness was also observed, which,
according to a previous study, may modulate the entropy and
enthalpy by adapting their bound-state structural ensemble
to achieve optimal binding, thus allowing PMIs to minimize
energetically unfavorable folding.9
Several peptide conformational difference cases are shown
in Fig. 4. The details can be found in the figure legends. As can
be seen, a short b-hairpin peptide segment of complement
protein C8a (Fig. 4A) and a long helical hairpin peptide
segment of RSV F glycoprotein (Fig. 4E-a) are rigid and highly
structured. The two peptide conformations are changed quite
modestly when they are split from their parent context and then
bind to their partner proteins C8g and motavizumab, with
superposed RMSD values of 0.87 and 1.45 Å, respectively.
Structural analysis revealed that the rigid structure of the two
hairpin peptides is tightly maintained by a number of hydrogen
This journal is © The Royal Society of Chemistry 2019
View Article Online
Research Article
bonds and hydrophobic contacts across the hairpin arms, that
is to say, the two peptides can keep in the well-structured
hairpin structures and are affected slightly by the splitting
and binding.
The long thrombin L peptide is highly flexible and locally
helicated within its protein context of prothrombin (Fig. 4B).
However, the peptide has a substantial conformational differ-
ence relative to its split counterpart in complex with its partner
protein prothrombin H, with a superposed RMSD value of
5.32 Å, although the peptide backbone trace seems to be
roughly consistent between the two conformations. The short
H4 peptide and epitope peptide 2 are located in the intrinsically
disordered regions of histone H4 (Fig. 4C) and glycoprotein
RSV F (Fig. 4E-b), respectively. As might be expected, their
conformations are also changed considerably with superposed
RMSD values of 5.19 and 4.17 Å, respectively. This is expected
due to the highly flexible nature of the two peptides and context
support should play an important role in their respective PMIs.
The midsized peptide segment of tumor-suppressor p53 is
partially structured within its protein context, which contains
a a-helix core capped by disordered N- and C-termini (Fig. 4D).
Splitting and binding of the p53 peptide would not have a
significant effect on the core helical conformation, but can
largely influence the two termini, thus inducing a moderate
conformational change for the global structure of the peptide,
with a superposed RMSD value of 3.18 Å.
Structural dynamics analysis and comparison of representative
PMIs and their PTI counterparts
A total of 23 representative PMI systems are compiled in
Table 1, and their PTI counterparts are set as shown in Fig. 2.
These PMI/PTI systems were one-by-one subjected to 50 ns MD
simulations and their root mean-square deviation (RMSD)
fluctuations (only regarding the regions of PMI peptide
segments or PTI peptide ligands) during the simulations were
analyzed and compared. Here, the RMSD fluctuations of
peptide segments/peptide ligands in six PMI/PTI systems are
shown in Fig. 5. At a first glance, an evident difference can be
observed between the RMSD fluctuations of these systems. The
dynamic conformation of highly flexible loop peptides such as
Mol. Omics
 View Article Online

Research Article Molecular Omics

Published on 13 May 2019. Downloaded by MTA Research Centre for Natural Science on 5/27/2019 2:40:11 PM.

Fig. 4 Conformational difference of the same peptides in complex with their partner proteins and within the context of their parent proteins. (A) The
short peptide segment 158LRYDSTAERLY168 of complement protein C8a is highly structured and folded into a two-stranded b-hairpin conformation
within its protein context (PDB: 3OJY). When the peptide is split from its parent protein C8a, it can also maintain in the hairpin conformation to interact
with its partner protein C8g (PDB: 2QOS), with superposed RMSD value of 0.87 Å. (B) The long peptide segment 276EADCGLRPLFEKKSLEDKTERELLE-
SYID304 of prothrombin is highly flexible and locally helicated within its protein context (PDB: 5EDM). When the peptide is split from its parent protein
prothrombin, it exhibits a substantial conformational change upon binding to its partner protein prothrombin H (PDB: 5A2M), with a superposed RMSD
value of 5.32 Å. (C) The short peptide segment 7GKGLGKGGA15 of histone H4 is intrinsically disordered within its protein context (PDB: 4PSX). When the
peptide is split from its parent protein H4, it exhibits a large conformational change upon binding to its partner protein Brd2 (PDB: 2E3K), with a
superposed RMSD value of 5.10 Å. (D) The midsize peptide segment 15SQETFSDLWKLLPEN20 of tumor-suppressor p53 is partially structured in a helical
conformation within its protein context (PDB: 2LY4). When the peptide is split from its parent protein p53, it exhibits a moderate conformational change
upon binding to its partner protein MDMX (PDB: 2MWY), with a superposed RMSD value of 4.18 Å. (E-a) The long peptide segment 254NSELLSLINDM-
PITNDQKKLMSNN277 of respiratory syncytial virus fusion RSV F glycoprotein is highly structured and folded into a two-stranded helical hairpin
conformation within its protein context (PDB: 3RRR). When the peptide is split from its parent protein RSV F, it can also maintain in the hairpin
conformation to interact with its partner protein motavizumab (PDB: 3IXT), with a superposed RMSD value of 1.45 Å. (E-b) The short peptide segment
427
KNRGIIKTFSN437 of RSV F glycoprotein is intrinsically disordered within its protein context (PDB: 3RRR). When the peptide is split from its parent
protein RSV F, it exhibits a moderate conformational change upon binding to its partner protein 101F (PDB: 3O41), with superposed RMSD value of 4.17 Å.

1AK4_D:A (A) and 2MTA_C:A (D) can be roughly constrained
around their native state and is changed moderately during MD
simulations. In contrast, the conformation exhibits a large
variation and alters dramatically when splitting these peptide
segments from their parent context in PMIs and re-performing
simulations of the resulting PTI counterparts. In addition,
the partially structured strand/loop peptide 1D6R_I:A (F) has
also a considerable difference in its conformational dynamics
between PMI and PTI. During MD simulations the peptide
segment can well maintain in the native strand/loop conforma-
tion in intact PMI, whereas the conformation becomes intrin-
sically disordered and highly flexible in the PTI counterpart.
The other three PMI systems, namely, 1AY7_B:A (B), 7CEI_B:A
(C) and 4JLR_S:HL (E), are fully or partially structured as an
a-helix in their functional peptide regions and thus possess a
larger rigidity that is relatively insensitive to the context effect.
As can be seen, splitting of these peptide segments from their
parent context can only moderately or modestly shift their
RMSD profiles. In particular, the peptide segment in 4JLR_S:HL
(E) is rather rigid, which shows a small structural fluctuation
and a very similar RMSD profile with and without the context
support. In fact, this peptide is an epitope region of respiratory
syncytial virus immunogen that can well fold into a tightly
bound helical hairpin structure. Crystallographic study also
confirmed that the peptide can spontaneously form the hairpin
and then binds to antibody motavizumab in an independent
manner.48
The above structural dynamics analysis suggested that
flexible peptide segments such as loops are vulnerable to their
parent context, whereas rigid peptide segments such a helix can
work independently to interact with their partners. This is
expected because a rigid peptide configuration is primarily
maintained by intramolecular nonbonded interactions within
the peptide molecule that can be kept when splitting from its

Mol. Omics This journal is © The Royal Society of Chemistry 2019


Molecular Omics
Published on 13 May 2019. Downloaded by MTA Research Centre for Natural Science on 5/27/2019 2:40:11 PM.
Fig. 5 Comparison of the RMSD fluctuations between PMI peptide segment and PTI peptide ligand during 50 ns MD simulations of the PMI and PTI
systems: (A) 1AK4_D:A, (B) 1AY7_B:A, (C) 7CEI_B:A, (D) 2MTA_C:A, (E) 4JLR_S:HL, and (F) 1D6R_I:A. All atoms of only peptide segment/peptide ligand are
used to derive the profile, while the partner protein and parent protein context are not considered.
parent context. In this respect, peptide flexibility can be
regarded as an indirect indicator to reflect the context effect
on PMIs. As can be seen in Fig. 6A, numerous conformational
snapshots of the peptide segment in the PMI system and
peptide ligand in the PTI counterpart were extracted from
their respective 50 ns MD trajectories and then their RMSD
variations relative to the native crystal structures were calcu-
lated. Here, the obtained RMSD variations for the 23 PMI/PTI
systems are illustrated in Fig. 6B. Evidently, the variation
profiles are considerably different between PMI peptide
segments and PTI peptide ligands; the former (red bar) is
generally smaller than the latter (grey bar), indicating that the
same peptides have a low flexibility and small disorder in
protein context as compared to that out of the context. The
difference is very significant for a few PMI/PTI systems such as
1AK4_D:A, 2HLE_B:A, 1D6R_I:A and 1KTZ_B:Z; structural
examination found that these peptides are highly flexible
segments and/or bound weakly to their partners, and context
is therefore essential for conformational constraint of the
intermolecular recognition and interaction involved in these
systems.
Here, two typical PMI systems 1AK4_D:A and 1KTZ_B:A that
are influenced significantly by context are discussed visually.
Conformational snapshots of the two PMIs and their PTI
counterparts were separately extracted at 0, 10, 20, 30, 40 and
50 ns of MD trajectories and compared in ESI,† Fig. S2. The
functional peptide segments of 1AK4_D:A and 1KTZ_B:A are,
respectively, a loop and a short b-strand that can only
This journal is © The Royal Society of Chemistry 2019
View Article Online
Research Article
moderately touch on the surface of their partner proteins.
The two peptide segments can well keep around the native
crystal conformation in intact PMI systems during the whole
simulation course, but the conformation and even the binding
mode of the peptide ligands in PTI counterparts have a distinct
story from that of corresponding PMI systems. The loop peptide
ligand of 1AK4_D:A PTI displays a strong thermal motion and
its conformation varies largely during the simulations, while
the b-strand peptide ligand of 1KTZ_B:A PTI is totally unfolded
into disorder and gradually unbound from its partner along the
simulations, suggesting that the context factor should play an
essential role in the conformation and binding of functional
peptide segments in the two PMI systems.
Next, the conformational snapshots of the PMI peptide
segments and PTI peptide ligands extracted from MD equili-
brium trajectories were clustered into single representatives. In
this way, the peptide region of a PMI/PTI system can be
represented by three conformations: (i) the native crystal struc-
ture, (ii) the MD equilibrium conformation cluster of the
peptide segment in PMI, and (iii) the MD equilibrium con-
formation cluster of the peptide ligand in PTI. Here, the
differences (RMSD values) between the three conformations
for the 23 PMI/PTI systems are shown in Fig. 7A. As might be
expected, after dynamics equilibrium all the 23 peptide con-
formations were changed largely in PTIs as compared to those
in PMIs relative to their respective crystal structures, imparting
that protein context can, more or less, address a conforma-
tional constraint on peptide binding to their partner proteins,
Mol. Omics

Research Article
Published on 13 May 2019. Downloaded by MTA Research Centre for Natural Science on 5/27/2019 2:40:11 PM.
Fig. 6 (A) Schematic representation of extracting conformational snapshots of PMI peptide segments and PTI peptide ligands from MD trajectories as
well as computing the RMSD variation over these snapshots. (B) Comparison of the RMSD variations of PMI peptide segments and PTI peptide ligands for
the 23 PMI/PTI systems.
although the constraint effect varies considerably over different
systems. More importantly, by comparing MD equilibrium con-
formations of the same peptides in PMIs and PTIs a significant
difference is revealed for most systems (RMSD 4 2 Å), while only
a few samples have a moderate or modest conformational
difference at their dynamics equilibrium state (RMSD o 2 Å).
Here, three typical examples that separately represent small,
moderate and large context contribution to peptide conforma-
tion in PMI/PTI systems 4JLR_S:HL, 7CEI_B:A, and 2MTA_C:A
are shown in Fig. 7B, in which the three distinct conformations
for a peptide are superposed in the active site of its partner
protein. The helical hairpin peptide of 4JLR_S:HL is tightly
fixed by the intramolecular hydrogen bonds and hydrophobic
contacts across the two helical arms of the hairpin, which
define an independent, rigid structure for the peptide and thus
is invulnerable to context impact, with small differences
between its three conformations (RMSD = 0.72, 0.84 and 0.65 Å).
Mol. Omics
View Article Online
Molecular Omics
The functional peptide segment of 7CEI_B:A is a long helix
extending out of the active site; splitting and truncation of the
helical peptide appear not to influence its core binding region,
but would cause a significant effect on its two termini, that is,
without context support the two termini become totally
disordered in PTI as compared to the structured helix in PMI.
Consequently, the PMI peptide segments are very similar in
both the native crystal structure and MD equilibrium confor-
mation (RMSD = 0.87 Å), but the two conformations (in PMI)
differ moderately or considerably to the MD equilibrium con-
formation of the peptide ligand in the PTI counterpart (RMSD =
1.70 and 2.18 Å). In addition, the highly flexible loop peptide of
2MTA_C:A is also very similar in both the crystal structure and
MD conformation (red and blue) (RMSD = 1.21 Å), but different
largely relative to its PTI peptide counterpart (green) (RMSD =
3.07 and 3.27 Å), indicating a solid context contribution to the
peptide binding in PMI.
This journal is © The Royal Society of Chemistry 2019
 View Article Online

Molecular Omics Research Article

Published on 13 May 2019. Downloaded by MTA Research Centre for Natural Science on 5/27/2019 2:40:11 PM.

Fig. 7 (A) Comparison of the differences (RMSD values) between the crystal structure and MD equilibrium conformation of PMI peptide segments,
between the crystal structure and MD equilibrium conformation of PTI peptide ligands, and between the MD equilibrium conformations of PMI peptide
segments and PTI peptide ligands for the 23 PMI/PTI systems. (B) Superposition of PMI peptide segments (crystal structure, red), PMI peptide segments
(MD equilibrium conformation, blue) and PTI peptide ligands (MD equilibrium conformation, green) in the active site of their partner proteins:
(B-a) 4JLR_S:HL, (B-b) 7CEI_B:A, and (B-c) 2MTA_C:A.

systems (DEint { 0), which, however, would be largely counter-

Binding energetics analysis and comparison of representative
PMIs and their PTI counterparts
For each PMI or PTI system, a total of 1000 conformational
snapshots (frames) were evenly extracted from the equilibrium
trajectory of MD production simulations, which were then
analyzed with MM/PBSA and NMA methods to derive binding
energetic components (interaction energy DEint, solvent effect
DGslv, entropy penalty TDS and total binding energy DGttl) for
the system. For a PMI, the trajectory of the peptide segment
section can be isolated from its parent context, and hence the
binding energetics of the segment to its partner protein can be
calculated based on MD simulations of the whole PMI system.
The obtained energetic components of PMI protein–protein,
PMI protein–peptide segment, and PTI protein–peptide ligand
interactions for each of the 23 investigated PMI/PTI systems are
tabulated in ESI,† Table S2. As can be seen, the direct inter-
molecular interaction energy is very favorable for all these
acted by unfavorable desolvation (DGslv 4 0) and entropy
penalty (TDS 4 0) incurred from the system binding.
Recently, the contribution of structures, thermodynamics,
and dynamics to the intrinsic disorder interactions of
amyloid-b peptide and other IDPs has been investigated and
discussed by Lohr et al., substantiating the important role of
entropic effects in the coupled binding and folding of disor-
dered proteins.49–53 Consequently, the PMIs and PTIs generally
possess a modestly or moderately favorable affinity with a total
binding energy DGttl range between 0 and 90 kcal mol1. In
addition, the affinity seems to increase in the order: PTI
protein–peptide ligand o PMI protein–peptide segment o
PMI protein–protein, suggesting that the intact PMI systems
as well the peptide segments within their parent context can
bind more effectively than that of the split peptide ligand in PTI
without context support. It is worth noting that the calculated
absolute binding energy values appear to be larger than the real

This journal is © The Royal Society of Chemistry 2019 Mol. Omics


Research Article
values. This is a common phenomenon that the absolute
binding energy of protein–peptide interactions is generally
overestimated by MM/PBSA calculations, and we also discussed
this issue previously.33,39 In fact, the MM term overestimates
the favorable interaction energy between protein and peptide
due to its lack of electrostatic screening at the loosely packed
Published on 13 May 2019. Downloaded by MTA Research Centre for Natural Science on 5/27/2019 2:40:11 PM.
protein–peptide interface, while the PBSA term underestimates
the unfavorable solvent effect since the hydrophobic contribu-
tion cannot be described accurately using the very empirical
surface area (SA) model. However, the calculated energy values
are also useful for comparing the relative binding potencies of
PMI and its PTI counterpart, since the bias can be largely offset
between different versions of the same system.
First, we discussed the energetic contribution of peptide
segment section to the whole PMI protein–protein interaction.
The total binding energy of the peptide segment (and its parent
protein) to its partner protein in a PMI system was calculated
with or without consideration of entropy penalty (DGttl = DEint +
DGslv or DGttl = DEint + DGslv  TDS), and the fraction of PMI
total binding energy contributed by the peptide segment can be
derived as F = DGttl(segment–partner)/DGttl(parent–partner) 
100%. The resulting F-values are plotted in Fig. 8, which range
between 30 and 60% over the 23 investigated PMI systems,
suggesting that these functional peptide segments (within their
parent context) can only contribute to about a half or less of the
total binding affinity between the parent and partner proteins
of PMI systems, and other regions out of the peptide segments
(parent context) should also exert potential interactions with
the partners through, for example, long-range electrostatic
forces and marginal van der Waals contacts. In addition,
consideration of conformational flexibility and the entropy
penalty (TDS) can further decrease F-values for most samples,
albeit the decrease is not solid (DF o 10%). Previously, Stein
and Aloy estimated that interfacial peptides can contribute
Fig. 8 Fraction (F-value) of the total binding energy of 23 PMI systems contributed by peptide segments. The total binding energy was calculated with or
without consideration of entropy penalty (DGttl = DEint + DGslv or DGttl = DEint + DGslv  TDS).
Mol. Omics
View Article Online
Molecular Omics
about 80% of the total interaction energy for PMIs using an
empirical alanine scanning approach,20 which is considerably
higher than the F-values (30–60%) determined in this study.
This could be attributed to the facts that: (i) we only selected a
small set of typical PMI samples in which the peptide segments
were defined as those few residues of parent proteins that can
directly contact partner proteins, (ii) rigorous dynamics simula-
tions and post energetics analysis were employed to accurately
calculate the binding energy of these PMI samples, and (iii)
peptide flexibility and the entropy effect were added to the
energetics analysis.
Second, the binding energetic components of peptide seg-
ments in PMI relative to the same peptide ligands in PTI were
examined. In order to straightforwardly observe the context
effect on energetic components, the average value and standard
error (a.v.  s.e.) of differences of interaction energy (DDEint),
solvent effect (DDGslv), entropy penalty (TDDS), and total
binding energy (DDGttl) between the PMI peptide segment
and PTI peptide ligand were computed over the 23 investigated
PMI/PTI systems (see ESI,† Fig. S3). It is seen that the parent
context can influence the interaction energy and solvent effect
of peptide–partner binding considerably, with the a.v.  s.e. of
33.6  16.0 (DDEint) and 39.2  19.8 (DDGslv) kcal mol1,
while the context can also contribute moderately to the
entropy penalty and total binding energy, with a.v.  s.e. of
14.5  7.3 (TDDS) and 8.9  3.7 (DDGttl) kcal mol1. As might
be expected, without context support the peptides generally
exhibit larger conformational disorder and weaker packing
tightness against their partner proteins, thus impairing the
interaction energy of peptide–partner binding (viz. DDEint 4 0,
unfavorable) but also reducing the desolvation effect associated
with the binding (viz. DDGslv o 0, favorable). Moreover, stripping
of the context constraint can also enhance peptide flexibility in
an unbound state, thus incurring increased entropy penalty
This journal is © The Royal Society of Chemistry 2019

Molecular Omics
Published on 13 May 2019. Downloaded by MTA Research Centre for Natural Science on 5/27/2019 2:40:11 PM.
Fig. 9 Ratio (R-value) of the total binding energies of 23 PMI systems to their PTI counterparts, i.e. DGttl(PMI)/DGttl(PTI). The complex structures of three
systems 1GHQ_B:A, 1AVX_B:A and 1KTZ_B:A with the highest R-values are shown on top.
upon binding to partners (viz. TDDS 4 0, unfavorable). Con-
sequently, the peptide affinity is decreased due to the removal of
their parent context (viz. DDGttl 4 0, unfavorable).
Third, the total binding energies DGttl of intact PMI systems
and their PTI counterparts were compared. The ratio (R-value)
of DGttl(PMI) to DGttl(PTI) ranges from 2 to 14-fold for the 23
investigated PMI/PTI systems (Fig. 9), denoting a large differ-
ence between the binding behaviors of PMI protein–protein
and PTI protein–peptide ligand interactions. The difference is
considerably enlarged from that between PMI protein–protein
and PTI protein–peptide segment interactions, implying that
the parent context does not only contribute to PMIs by directly
interacting with their partner proteins, but also promote the
binding of functional peptide segments to the partners through
indirect conformational constraint and allosteric effect. The large
difference also suggests that the PTI counterparts are not func-
tionally equivalent for most PMI systems from an energetic
point of view. Three systems, namely, 1GHQ_B:A, 1AVX_B:A
and 1KTZ_B:A have the highest R-values (48-fold); structural
analysis revealed that they all have a small functional peptide
segment (size o 10-mer) in disorder or strand conformation that
can only pack weakly at the interface (see subplots in Fig. 9), and
thus their context effect is very significant. In particular, the PTI
counterpart of 1KTZ_B:A PMI systems, as described above,
cannot maintain in a stable bound state and its peptide ligand
(without the context help) was observed to spontaneously unbind
from the partner protein during the MD simulations, with a
DGttl value of only 1.3 kcal mol1 as compared to 7.4
and 19.2 kcal mol1 for the corresponding peptide segment
(within parent context) and intact PMI system, respectively.
This journal is © The Royal Society of Chemistry 2019
View Article Online
Research Article
Dynamics and energetics analysis of context effect on PMI
specificity and selectivity
The Src non-receptor tyrosine kinase family members share a
conserved structural architecture that contains a catalytic kinase
(CK) domain as well as two regulatory peptide-recognition
domains SH2 and SH3; the latter can recognize and bind
to a polyproline II (PPII) peptide segment between the CK and
SH2 domains to lock the kinase in an autoinhibitory state.54
Previously, we found that the PPII sequence of the first Src
kinase family member, Src, does not contain the standard SH3
recognition motif PxxP, which may need the context help of full-
length kinase to maintain in the PPII helix conformation, thus
promoting the SH3–PPII interaction.21,22 Here, we further sup-
posed that the kinase protein context not only enhances the
domain–peptide binding affinity, but also improves the PPII
peptide selectivity for its cognate SH3 domain.
The autoinhibitory crystal structures of Src family members
Src, Abl and Hck were examined, and their PPII peptide sequences
as well as the peptide structural dynamics and binding energetics
(with or without context) were calculated and listed in Table 2. As
expected, all three PPII peptides can bind more tightly to their
cognate SH3 domains in a kinase context (SH3–peptide segment)
than in a split state (SH3–peptide ligand) (11.3 vs. 6.7 kcal mol1
for Src, 13.0 vs. 6.4 kcal mol1 for Abl and 10.8 vs.
4.2 kcal mol1 for Hck). Interestingly, energetic decomposition
revealed a considerable difference (TDDS 4 15 kcal mol1)
between the entropy penalties of SH3–peptide segment and
SH3–peptide ligand interactions; the latter is significantly larger
than the former. This can be confirmed by conformational
Mol. Omics
 View Article Online

Research Article Molecular Omics

Published on 13 May 2019. Downloaded by MTA Research Centre for Natural Science on 5/27/2019 2:40:11 PM.

Fig. 10 (A) Crystal structures of full-length Src (A-a), Abl (A-b) and Hck (A-c) kinases in an autoinhibitory state, in which the SH3 domain and PPII peptide
are highlighted. (B) Heatmap of the total binding free energies of three PPII peptides binding to three SH3 domains. The diagonal elements represent
cognate interactions, otherwise, noncognate: (B-a) the binding is within the intact kinase protein context (SH3–peptide segment), and (B-b) the binding is
out of the context (SH3–peptide ligand). (C) Mean selectivity diagram of three PPII peptides with (C-a) or without (C-b) context.

sampling during MD simulations. As can be seen in ESI,† Fig. S4,
the peptide segment in a kinase context has a low disorder
(RMSD variation = 0.21 Å over the simulations), which can be
well structured in the PPII helical configuration that is suited
for interacting with the SH3 domain. In contrast, the split
peptide ligand is highly flexible (RMSD variation = 0.89 Å
over the simulations) and cannot hold in the PPII configuration
and would incur a large entropy penalty upon binding to the
SH3 domain. This is in line with our previous investigation of
conventional protein–peptide interactions, which were found to
be co-dominated by direct readout of the intermolecular inter-
action between the protein receptor and peptide ligand, and by
indirect readout of the entropy penalty upon the interaction.55
The SH3 domains and PPII peptide segments are high-
lighted in the crystal structures of full-length Src, Abl and
Hck kinases in an autoinhibitory state (Fig. 10A). Based on
the kinase crystal structures six noncognate domain–peptide
interactions between the three SH3 domains and three PPII
peptides were modeled and their binding energies were calcu-
lated and visualized as two heatmaps in Fig. 10B, in which the
(B-a) and (B-b) profiles represent the interactions in the
presence and absence of kinase context, respectively. A clear
difference can be observed between the two heatmap profiles,
that is, the PPII peptides can interact with SH3 domains with a
generally high affinity in context as compared to that out of the
context, no matter whether the interactions are cognate or
noncognate. This is consistent with the above analysis that
context can effectively enhance the binding potency of peptide
segments to their partner proteins. However, the two profiles
impart a different story about peptide selectivity: in the former
the three cognate SH3–PPII interactions (diagonal elements)
generally have a high affinity relative to those noncognate
interactions (non-diagonal elements), whereas in the latter no
obvious difference between the cognate and noncognate inter-
actions can be appreciated, implying that the context not only
enhances peptide affinity to their cognate partners, but also
improves peptide selectivity for cognate over noncognate.
In addition, the mean selectivity Ŝ of three PPII peptides for
their one cognate and two noncognate SH3 domains was
derived from the systematic binding energy profiles, which
are separately plotted as two diagrams in Fig. 10(C-a) and
(C-b). The mean selectivity of Src, Abl and Hck PPII peptides is
18.2, 77.5 and 28.5-fold (global selectivity = 34.2-fold c 1-fold)
in the full-length kinase, respectively, suggesting that these

Mol. Omics This journal is © The Royal Society of Chemistry 2019


Molecular Omics
peptides have high specificity with their context support. In
contrast, the mean selectivity is largely impaired or even reversed
to 6.0, 0.74 and 1.2-fold (global selectivity = 1.7-fold close to
1-fold), respectively, when the peptides were split from the
context and bind independently to SH3 domains.
Published on 13 May 2019. Downloaded by MTA Research Centre for Natural Science on 5/27/2019 2:40:11 PM.
Conflicts of interest
The authors declare that they have no conflicts of interest with
the contents of this article.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (No. 31671361), the Fundamental Research
Funds for the Central Universities of China (No. ZYGX2016J186
and ZYGX2015Z006), and the Science Strength Promotion
Programme of UESTC.
References
1 P. Braun and A. C. Gingras, Proteomics, 2012, 12, 1478–1498.
2 E. Petsalaki, A. Stark, E. Garcı́a-Urdiales and R. B. Russell,
PLoS Comput. Biol., 2009, 5, e1000335.
3 E. Petsalaki and R. B. Russell, Curr. Opin. Biotechnol., 2008,
19, 344–350.
4 V. Neduva and R. B. Russell, Curr. Opin. Biotechnol., 2006,
17, 465–471.
5 P. E. Wright and H. J. Dyson, Curr. Opin. Struct. Biol., 2009,
19, 31–38.
6 D. Bonetti, F. Troilo, M. Brunori, S. Longhi and S. Gianni,
Biophys. J., 2018, 114, 1889–1894.
7 P. Tompa and M. Fuxreiter, Trends Biochem. Sci., 2008, 33,
2–8.
8 M. Fuxreiter, J. Mol. Biol., 2018, 430, 2278–2287.
9 S. Hadži, A. Mernik, C. Podlipnik, R. Loris and J. Lah, Angew.
Chem., Int. Ed., 2017, 56, 14494–14497.
10 M. Fuxreiter, Curr. Opin. Struct. Biol., 2018, 54, 19–25.
11 M. Miskei, C. Antal and M. Fuxreiter, Nucleic Acids Res.,
2017, 45, D228–D235.
12 M. Fuxreiter, P. Tompa and I. Simon, Bioinformatics, 2007,
23, 950–956.
13 N. E. Davey, D. C. Shields and R. J. Edwards, Bioinformatics,
2009, 25, 443–450.
14 M. Miskei, A. Gregus, R. Sharma, N. Duro, F. Zsolyomi and
M. Fuxreiter, FEBS Lett., 2017, 591, 2682–2695.
15 D. Kurzbach, T. C. Schwarz, G. Platzer, S. Höfler,
D. Hinderberger and R. Konrat, Angew. Chem., Int. Ed.,
2014, 53, 3840–3843.
16 A. Beier, T. C. Schwarz, D. Kurzbach, G. Platzer, F. Tribuzio
and R. Konrat, J. Mol. Biol., 2018, 430, 2439–2452.
17 A. Tóth-Petróczy, I. Simon, M. Fuxreiter and Y. Levy, J. Am.
Chem. Soc., 2009, 131, 15084–15085.
18 K. Luck, S. Charbonnier and G. Travé, FEBS Lett., 2012, 586,
2648–2661.
This journal is © The Royal Society of Chemistry 2019
View Article Online
Research Article
19 S. Panni, L. Montecchi-Palazzi, L. Kiemer, A. Cabibbo,
S. Paoluzi, E. Santonico, C. Landgraf, R. Volkmer-Engert,
A. Bachi, L. Castagnoli and G. Cesareni, Proteomics, 2011,
11, 128–143.
20 A. Stein and P. Aloy, PLoS One, 2008, 3, e2524.
21 Z. Bai, S. Hou, S. Zhang, Z. Li and P. Zhou, J. Chem. Inf.
Model., 2017, 57, 835–845.
22 P. Zhou, S. Hou, Z. Bai, Z. Li, H. Wang, Z. Chen and
Y. Meng, Artif. Cells, Nanomed., Biotechnol., 2018, 46,
1122–1131.
23 L. Ning, Z. Li, Z. Bai, S. Hou, B. He, J. Huang and P. Zhou,
Int. J. Biol. Sci., 2018, 14, 930–937.
24 H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland,
T. N. Bhat, H. Weissig, I. N. Shindyalov and P. E. Bourne,
Nucleic Acids Res., 2000, 28, 235–242.
25 P. Zhou, F. Tian, Y. Ren and Z. Shang, J. Chem. Inf. Model.,
2010, 50, 1476–1488.
26 I. K. McDonald and J. M. Thornton, J. Mol. Biol., 1994, 238,
777–793.
27 J. M. Word, S. C. Lovell, T. H. LaBean, H. C. Taylor, M. E.
Zalis, B. K. Presley, J. S. Richardson and D. C. Richardson,
J. Mol. Biol., 1999, 285, 1711–1733.
28 T. S. Chen, D. Petrey, J. I. Garzon and B. Honig, PLoS
Comput. Biol., 2015, 11, e1004248.
29 A. Stein and P. Aloy, PLoS Comput. Biol., 2010, 6, e1000789.
30 Y. Duan, C. Wu, S. Chowdhury, M. C. Lee, G. Xiong,
W. Zhang, R. Yang, P. Cieplak, R. Luo, T. Lee, J. Caldwell,
J. Wang and P. A. Kollman, J. Comput. Chem., 2003, 24,
1999–2012.
31 D. A. Case, T. E. Cheatham, T. Darden, H. Gohlke, R. Luo,
K. M. Merz, A. Onufriev, C. Simmerling, B. Wang and
R. J. Woods, J. Comput. Chem., 2005, 26, 1668–1688.
32 P. Zhou, S. Zhang, Y. Wang, C. Yang and J. Huang, J. Biomol.
Struct. Dyn., 2016, 34, 1806–1817.
33 C. Yang, S. Zhang, P. He, C. Wang, J. Huang and P. Zhou,
J. Chem. Inf. Model., 2015, 55, 329–342.
34 C. Yang, S. Zhang, Z. Bai, S. Hou, D. Wu, J. Huang and
P. Zhou, Mol. BioSyst., 2016, 12, 1201–1213.
35 W. L. Jorgensen, J. Chandrasekhar, J. D. Madura, R. W. Impey
and M. L. Klein, J. Chem. Phys., 1983, 79, 926–935.
36 T. Darden, D. York and L. Pedersen, J. Chem. Phys., 1993, 98,
10089–10092.
37 J. P. Ryckaert, G. Ciccotti and H. J. C. Berendsen, J. Comput.
Phys., 1977, 23, 327–341.
38 X. W. Wu and B. R. Brooks, Chem. Phys. Lett., 2003, 381,
512–518.
39 C. Yang, C. Wang, S. Zhang, J. Huang and P. Zhou, Mol.
Simul., 2015, 41, 741–751.
40 L. A. Kelley, S. P. Gardner and M. J. Sutcliffe, Protein Eng.,
1996, 9, 1063–1065.
41 L. Saı́z-Urra, M. A. Cabrera and M. Froeyen, J. Mol. Graphics
Modell., 2011, 29, 726–739.
42 T. Wu, P. He, W. Wu, Y. Chen and F. Lv, Bioorg. Chem., 2018,
80, 1–10.
43 P. A. Kollman, I. Massova, C. Reyes, B. Kuhn, S. Huo,
L. Chong, M. Lee, T. Lee, Y. Duan, W. Wang, O. Donini,
Mol. Omics
 View Article Online

Research Article Molecular Omics

P. Cieplak, J. Srinivasan, D. A. Case and T. E. Cheatham,
Acc. Chem. Res., 2000, 33, 889–897.
44 D. Case, Curr. Opin. Struct. Biol., 1994, 4, 285–290.
45 T. Hou, K. Chen, W. A. McLaughlin, B. Lu and W. Wang,
PLoS Comput. Biol., 2006, 2, e1.
46 G. G. Krivov, M. V. Shapovalov and R. L. Dunbrack, Proteins,
Published on 13 May 2019. Downloaded by MTA Research Centre for Natural Science on 5/27/2019 2:40:11 PM.
50 T. Löhr, A. Jussupow and C. Camilloni, J. Chem. Phys., 2017,
146, 165102.
51 G. T. Heller, F. A. Aprile, M. Bonomi, C. Camilloni, A. De
Simone and M. Vendruscolo, J. Mol. Biol., 2017, 429,
2772–2779.
52 E. Karlsson, E. Andersson, J. Dogan, S. Gianni, P. Jemth and

2009, 77, 778–795. C. Camilloni, J. Biol. Chem., 2019, 294, 1230–1239.

47 W. Kabsch and C. Sander, Biopolymers, 1983, 22, 2577–2637.
48 J. S. McLellan, M. Chen, A. Kim, Y. Yang, B. S. Graham and
P. D. Kwong, Nat. Struct. Mol. Biol., 2010, 17, 248–250.
49 T. Löhr, K. Kohlhoff, G. Heller and M. Vendruscolo, Biophys. J.,
2019, 116, 437a.
53 A. Cavalli, C. Camilloni and M. Vendruscolo, J. Chem. Phys.,
2013, 138, 094112.
54 S. R. Hubbard, Nat. Struct. Biol., 1999, 6, 711–714.
55 H. Yu, P. Zhou, M. Deng and Z. Shang, J. Chem. Inf. Model.,
2014, 54, 2022–2032.

Mol. Omics This journal is © The Royal Society of Chemistry 2019