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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

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
Received 12th March 2019, structures of full-length Src family kinases in an autoinhibitory state. Instead of the direct interaction and
Accepted 10th May 2019 desolvation that are primarily concerned in traditional studies, peptide flexibility and the entropy penalty
DOI: 10.1039/c9mo00041k 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
rsc.li/molomics disordered, and/or located at the middle region of their parent proteins.

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


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

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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
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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
Fig. 1 There is a doubt whether PMI is functionally equivalent or analogous domain to its target motifs involves a small interface that
(e.g. affinity, specificity, conformation and binding mode, etc.) to the PTI is unlikely to fully account for the PDZ–target interaction
involved in the PMI? (A) A peptide segment is protruded on the surface of its
behavior; sequence context surrounding the motifs has been
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 suggested to introduce structural diversity, to impact the
segment is split from its parent protein to obtain an isolated peptide ligand, stability and solubility of the constructs, and to influence the
which then binds to its partner protein to form a PTI. The PTI can be binding affinity and specificity of the interaction.18 The context
regarded as a reduced counterpart version of the PMI. 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
have been addressed on the PPI/PMI fuzziness phenomenon to, H. sapiens.19 More importantly, Stein and Aloy have system-
for example, explore their thermodynamics basis,9 compare atically dissected 3D structural data matching the known
with folding coupled binding mechanisms10 and develop bio- eukaryotic linear motifs (ELMs) and found that the protein
informatics tools and databases to annotate them.11 Never- structural context contributes to B20% PMI binding events
theless, due to the large flexibility and high independence of and may play an important role in determining the binding
the peptide segment, previous studies normally had an implicit specificity, by either improving affinity with the native partner
hypothesis that a PMI is functionally equivalent or analogous or impeding non-native interactions.20
to the protein–peptide interaction (PTI) involved in the PMI Recently, we have examined a special PMI phenomenon in
system, while ignoring the protein context contribution to the proto-oncogene tyrosine kinase c-Src, termed as self-binding
peptide binding. Surely, such a hypothesis can considerably peptide (SBP),21 which is an intramolecular interaction between
simplify the PMI system since only a short peptide is used to the SH3 domain and PPII peptide of the kinase. We found that
represent its large parent protein. For example, many existing the PPII lacks the standard PxxP motif that is normally required
crystallographic analyses and binding affinity assays of PMIs for SH3 recognition, and thus can only interact effectively with
only adopted the reduced PTI versions (but not the full-length SH3 domains when it is integrated into the full-length c-Src
PMI protein–protein interactions) to conduct the investiga- kinase protein; splitting of the PPII peptide segment from the
tions, but the obtained conclusions were directly applied to intact protein would considerably impair SH3 affinity by
explain the PMI events. However, one would doubt whether increasing the entropy penalty upon domain–peptide binding,
the hypothesis is reasonable and which conditions it can revealing that the protein context plays an essential role in the
(or cannot) work in (Fig. 1). SBP biological function.22 On the other hand, during a compu-
In fact, there were some studies that had shed light on the tational design of antiangiogenic peptibody PbHRH by fusing
importance of protein context in PMIs. For instance, Fuxreiter an HRH peptide agent to human IgG1 Fc fragment we found
and coworkers found that peptide linear motifs (LMs) are that the HRH is structurally and functionally independent of
usually constructed by grafting a few specificity-determining the Fc fragment in the designed peptibody; integration of HRH
residues favoring structural order on a highly flexible carrier peptide into the peptibody does not alter the peptide inter-
region. They also established a connection between LMs and action with its target protein substantially. Instead, the
molecular recognition elements of intrinsically disordered designed peptibody may indirectly help to improve the phar-
proteins (IDPs), which realize a non-conventional mode of macokinetic profile and bioavailability of HRH.23 This finding
partner binding mostly in regulatory functions.12 Later, the suggests that the protein context may not be crucial for
contribution of context-specific evolutionary conservation to every PMI event, and in some cases the peptide segment can
LM recognition and the context dependence of fuzzy interaction work independently of its parent context to interact with its
were revealed, implicating the context role in PMIs.13,14 Recently, partner protein.
Konrat et al. showed how structural unfolding compensates for Here, we performed a deep investigation of the molecular
entropic losses through ligand binding in IDPs and elucidated the mechanism of the protein context contribution to diverse PMIs,
interplay between structure and thermodynamics of rapid substrate- attempting to answer questions like whether the context is
binding and -release events in IDP interaction networks.15 responsible for PMIs, how the context works in PMIs, and what

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kinds of contexts contribute significantly or insignificantly to monomer or a multiprotein complex. Most residues in the
PMIs. First, we systematically compared the crystal conforma- peptide segment belong to the interfacial residues of M1–M2
tions of peptide ligands in complex with their protein receptors interaction, although a few non-interfacial residues are allowed
and the same peptide segments in their parent proteins. to discretely distribute over peptide sequence to keep the
Second, twenty-third representative PMI systems were curated peptide continuity. We considered two residues separately
and subjected to atomistic molecular dynamics simulations to coming from M1 and M2 to be ‘‘in contact’’ (and thus as
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characterize the structural dynamics of the peptide segments interfacial residues) if there was a hydrogen bond, a water-
with or without the support from their parent context. Thirdly, mediated hydrogen bond, a van der Waals interaction, or at
the PMI binding energy was also derived, analyzed and decom- least one pair of contacting nonhydrogen atoms (r4.5 Å)
posed in detail to examine the context contribution to different between them. This strategy was modified from our previous
energetic components involved in the PMIs. This work would work on protein–nucleic acid interactions.25 The hydrogen
establish a complete profile for the structural basis, energetic bonds, water-mediated hydrogen bonds and van der Waals
property and dynamic behavior of the context effect on PMI interactions were detected using HBPLUS26 and PROBE27
intermolecular recognition and interaction, and may help to programs, respectively.
correct the inappropriate applications of PTI succedaneums in Here, we only chose the PMI systems that have high-
future PMI studies. 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
Materials and methods peptide-mediated interaction whose interaction site only
Crystal structure survey presents a sole successive peptide segment and other inter-
facial residues, if present, out of the segment in the parent
The current deposition (Sept. 2018) of the RCSB Protein Data protein are very few (r1/5 of the peptide residues). According
Bank (PDB) database24 was systematically surveyed using the to the above rule, a total of 23 representative PMI systems
following criteria implemented with an advanced search tool were curated from a number of literature reports.20,28,29 The
provided by the database: collected PMIs are diverse in terms of their biological functions
(i) Macromolecule types: only protein entities, no nucleic as well as the size, location, structural type and amino acid
acids. composition of their core peptide segments (Table 1). Some
(ii) Experimental method: X-ray diffraction. examples of these PMI structural architectures are shown in
(iii) X-ray resolution: r3 Å. ESI,† Fig. S1. The crystal structures of the PMI complexes were
(iv) Number of chains in biological assembly: Z2. used to derive their respective PTI counterparts. As shown in
(v) Length of at least one chain: 5–30. Fig. 2, the PMI system of subtilisin–chymotrypsin inhibitor 2
(vi) Sequence identity: r30%. complex (PDB: 2SNI_I:E) is mediated by the intermolecular
The criteria were applied for filtering the entire PDB database interaction between subtilisin (partner protein) and a loop
to obtain the high-resolution, non-homologous crystal structure peptide segment of chymotrypsin inhibitor 2 (parent protein).
data of the PTI complexes. Consequently, in total 6497 records The peptide bonds separately at the N- and C-termini of the
meeting the criteria were retrieved from the database, which peptide segment are manually broken and then capped by
represents a distinct panel of PTI complex crystal structures acetyl (–Ac) and amide (–NH2) moieties to eliminate the formal
(listed in ESI,† Table S1). We manually excluded some invalid charges at the free N- and C-termini of the generated peptide
samples from the panel, such as those that were largely incom- ligand, respectively, while rest of the parent protein (protein
plete and highly modified. Next, the peptide sequences were context) is discarded.
extracted from these collected complexes, which were then one-
by-one searched back against the database to identify the crystal Molecular dynamics simulation
structures that contain the same peptides as a segment of their The 23 representative PMIs and their PTI counterparts (Table 1)
parent proteins. The crystal structures are solved in high resolu- were subjected to atomistic molecular dynamics (MD) simula-
tion (r3 Å) and their peptide segment regions should be intact tions to examine the context effect on the structural dynamics
(e.g. no backbone breaking and/or side-chain missing). of peptide segments involved in these PMIs. All simulations were
carried out using the AMBER ff03 force field30 implemented with
Definition, curation, and setup of PMI systems and their PTI the sander module of the Amber package.31 According to our
counterparts previous studies,32–34 dynamics may be sensitive to the combi-
A PMI system is defined as a binary or multi-PPI complex that nation of parameter settings, solvent conditions, forcefield
consists of two members M1 and M2. The M1 is termed as the versions and so on for different systems. Here, we have per-
parent protein that contains a functional peptide segment as the formed pre-simulations to optimize the combination, although
core sequence element to mediate M1–M2 interaction; the M2 the optimization procedure is empirical and intuitive, and no
is designated as the partner protein that recognizes and inter- general/objective rule is applicable for guiding it. Briefly, a
acts with the peptide segment of M1. The M1 is commonly a truncated octahedral box of TIP3P water molecules35 was added
monomeric protein, whereas the M2 can be either a protein with a 12 Å buffer around the simulated system. Counterions of

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

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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
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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


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

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

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

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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 moderately touch on the surface of their partner proteins.
regarded as an indirect indicator to reflect the context effect The two peptide segments can well keep around the native
on PMIs. As can be seen in Fig. 6A, numerous conformational crystal conformation in intact PMI systems during the whole
snapshots of the peptide segment in the PMI system and simulation course, but the conformation and even the binding
peptide ligand in the PTI counterpart were extracted from mode of the peptide ligands in PTI counterparts have a distinct
their respective 50 ns MD trajectories and then their RMSD story from that of corresponding PMI systems. The loop peptide
variations relative to the native crystal structures were calcu- ligand of 1AK4_D:A PTI displays a strong thermal motion and
lated. Here, the obtained RMSD variations for the 23 PMI/PTI its conformation varies largely during the simulations, while
systems are illustrated in Fig. 6B. Evidently, the variation the b-strand peptide ligand of 1KTZ_B:A PTI is totally unfolded
profiles are considerably different between PMI peptide into disorder and gradually unbound from its partner along the
segments and PTI peptide ligands; the former (red bar) is simulations, suggesting that the context factor should play an
generally smaller than the latter (grey bar), indicating that the essential role in the conformation and binding of functional
same peptides have a low flexibility and small disorder in peptide segments in the two PMI systems.
protein context as compared to that out of the context. The Next, the conformational snapshots of the PMI peptide
difference is very significant for a few PMI/PTI systems such as segments and PTI peptide ligands extracted from MD equili-
1AK4_D:A, 2HLE_B:A, 1D6R_I:A and 1KTZ_B:Z; structural brium trajectories were clustered into single representatives. In
examination found that these peptides are highly flexible this way, the peptide region of a PMI/PTI system can be
segments and/or bound weakly to their partners, and context represented by three conformations: (i) the native crystal struc-
is therefore essential for conformational constraint of the ture, (ii) the MD equilibrium conformation cluster of the
intermolecular recognition and interaction involved in these peptide segment in PMI, and (iii) the MD equilibrium con-
systems. formation cluster of the peptide ligand in PTI. Here, the
Here, two typical PMI systems 1AK4_D:A and 1KTZ_B:A that differences (RMSD values) between the three conformations
are influenced significantly by context are discussed visually. for the 23 PMI/PTI systems are shown in Fig. 7A. As might be
Conformational snapshots of the two PMIs and their PTI expected, after dynamics equilibrium all the 23 peptide con-
counterparts were separately extracted at 0, 10, 20, 30, 40 and formations were changed largely in PTIs as compared to those
50 ns of MD trajectories and compared in ESI,† Fig. S2. The in PMIs relative to their respective crystal structures, imparting
functional peptide segments of 1AK4_D:A and 1KTZ_B:A are, that protein context can, more or less, address a conforma-
respectively, a loop and a short b-strand that can only tional constraint on peptide binding to their partner proteins,

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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 The functional peptide segment of 7CEI_B:A is a long helix
systems. More importantly, by comparing MD equilibrium con- extending out of the active site; splitting and truncation of the
formations of the same peptides in PMIs and PTIs a significant helical peptide appear not to influence its core binding region,
difference is revealed for most systems (RMSD 4 2 Å), while only but would cause a significant effect on its two termini, that is,
a few samples have a moderate or modest conformational without context support the two termini become totally
difference at their dynamics equilibrium state (RMSD o 2 Å). disordered in PTI as compared to the structured helix in PMI.
Here, three typical examples that separately represent small, Consequently, the PMI peptide segments are very similar in
moderate and large context contribution to peptide conforma- both the native crystal structure and MD equilibrium confor-
tion in PMI/PTI systems 4JLR_S:HL, 7CEI_B:A, and 2MTA_C:A mation (RMSD = 0.87 Å), but the two conformations (in PMI)
are shown in Fig. 7B, in which the three distinct conformations differ moderately or considerably to the MD equilibrium con-
for a peptide are superposed in the active site of its partner formation of the peptide ligand in the PTI counterpart (RMSD =
protein. The helical hairpin peptide of 4JLR_S:HL is tightly 1.70 and 2.18 Å). In addition, the highly flexible loop peptide of
fixed by the intramolecular hydrogen bonds and hydrophobic 2MTA_C:A is also very similar in both the crystal structure and
contacts across the two helical arms of the hairpin, which MD conformation (red and blue) (RMSD = 1.21 Å), but different
define an independent, rigid structure for the peptide and thus largely relative to its PTI peptide counterpart (green) (RMSD =
is invulnerable to context impact, with small differences 3.07 and 3.27 Å), indicating a solid context contribution to the
between its three conformations (RMSD = 0.72, 0.84 and 0.65 Å). peptide binding in PMI.

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

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values. This is a common phenomenon that the absolute about 80% of the total interaction energy for PMIs using an
binding energy of protein–peptide interactions is generally empirical alanine scanning approach,20 which is considerably
overestimated by MM/PBSA calculations, and we also discussed higher than the F-values (30–60%) determined in this study.
this issue previously.33,39 In fact, the MM term overestimates This could be attributed to the facts that: (i) we only selected a
the favorable interaction energy between protein and peptide small set of typical PMI samples in which the peptide segments
due to its lack of electrostatic screening at the loosely packed were defined as those few residues of parent proteins that can
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protein–peptide interface, while the PBSA term underestimates directly contact partner proteins, (ii) rigorous dynamics simula-
the unfavorable solvent effect since the hydrophobic contribu- tions and post energetics analysis were employed to accurately
tion cannot be described accurately using the very empirical calculate the binding energy of these PMI samples, and (iii)
surface area (SA) model. However, the calculated energy values peptide flexibility and the entropy effect were added to the
are also useful for comparing the relative binding potencies of energetics analysis.
PMI and its PTI counterpart, since the bias can be largely offset Second, the binding energetic components of peptide seg-
between different versions of the same system. ments in PMI relative to the same peptide ligands in PTI were
First, we discussed the energetic contribution of peptide examined. In order to straightforwardly observe the context
segment section to the whole PMI protein–protein interaction. effect on energetic components, the average value and standard
The total binding energy of the peptide segment (and its parent error (a.v.  s.e.) of differences of interaction energy (DDEint),
protein) to its partner protein in a PMI system was calculated solvent effect (DDGslv), entropy penalty (TDDS), and total
with or without consideration of entropy penalty (DGttl = DEint + binding energy (DDGttl) between the PMI peptide segment
DGslv or DGttl = DEint + DGslv  TDS), and the fraction of PMI and PTI peptide ligand were computed over the 23 investigated
total binding energy contributed by the peptide segment can be PMI/PTI systems (see ESI,† Fig. S3). It is seen that the parent
derived as F = DGttl(segment–partner)/DGttl(parent–partner)  context can influence the interaction energy and solvent effect
100%. The resulting F-values are plotted in Fig. 8, which range of peptide–partner binding considerably, with the a.v.  s.e. of
between 30 and 60% over the 23 investigated PMI systems, 33.6  16.0 (DDEint) and 39.2  19.8 (DDGslv) kcal mol1,
suggesting that these functional peptide segments (within their while the context can also contribute moderately to the
parent context) can only contribute to about a half or less of the entropy penalty and total binding energy, with a.v.  s.e. of
total binding affinity between the parent and partner proteins 14.5  7.3 (TDDS) and 8.9  3.7 (DDGttl) kcal mol1. As might
of PMI systems, and other regions out of the peptide segments be expected, without context support the peptides generally
(parent context) should also exert potential interactions with exhibit larger conformational disorder and weaker packing
the partners through, for example, long-range electrostatic tightness against their partner proteins, thus impairing the
forces and marginal van der Waals contacts. In addition, interaction energy of peptide–partner binding (viz. DDEint 4 0,
consideration of conformational flexibility and the entropy unfavorable) but also reducing the desolvation effect associated
penalty (TDS) can further decrease F-values for most samples, with the binding (viz. DDGslv o 0, favorable). Moreover, stripping
albeit the decrease is not solid (DF o 10%). Previously, Stein of the context constraint can also enhance peptide flexibility in
and Aloy estimated that interfacial peptides can contribute an unbound state, thus incurring increased entropy penalty

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).

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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 Dynamics and energetics analysis of context effect on PMI
their parent context (viz. DDGttl 4 0, unfavorable). specificity and selectivity
Third, the total binding energies DGttl of intact PMI systems The Src non-receptor tyrosine kinase family members share a
and their PTI counterparts were compared. The ratio (R-value) conserved structural architecture that contains a catalytic kinase
of DGttl(PMI) to DGttl(PTI) ranges from 2 to 14-fold for the 23 (CK) domain as well as two regulatory peptide-recognition
investigated PMI/PTI systems (Fig. 9), denoting a large differ- domains SH2 and SH3; the latter can recognize and bind
ence between the binding behaviors of PMI protein–protein to a polyproline II (PPII) peptide segment between the CK and
and PTI protein–peptide ligand interactions. The difference is SH2 domains to lock the kinase in an autoinhibitory state.54
considerably enlarged from that between PMI protein–protein Previously, we found that the PPII sequence of the first Src
and PTI protein–peptide segment interactions, implying that kinase family member, Src, does not contain the standard SH3
the parent context does not only contribute to PMIs by directly recognition motif PxxP, which may need the context help of full-
interacting with their partner proteins, but also promote the length kinase to maintain in the PPII helix conformation, thus
binding of functional peptide segments to the partners through promoting the SH3–PPII interaction.21,22 Here, we further sup-
indirect conformational constraint and allosteric effect. The large posed that the kinase protein context not only enhances the
difference also suggests that the PTI counterparts are not func- domain–peptide binding affinity, but also improves the PPII
tionally equivalent for most PMI systems from an energetic peptide selectivity for its cognate SH3 domain.
point of view. Three systems, namely, 1GHQ_B:A, 1AVX_B:A The autoinhibitory crystal structures of Src family members
and 1KTZ_B:A have the highest R-values (48-fold); structural Src, Abl and Hck were examined, and their PPII peptide sequences
analysis revealed that they all have a small functional peptide as well as the peptide structural dynamics and binding energetics
segment (size o 10-mer) in disorder or strand conformation that (with or without context) were calculated and listed in Table 2. As
can only pack weakly at the interface (see subplots in Fig. 9), and expected, all three PPII peptides can bind more tightly to their
thus their context effect is very significant. In particular, the PTI cognate SH3 domains in a kinase context (SH3–peptide segment)
counterpart of 1KTZ_B:A PMI systems, as described above, than in a split state (SH3–peptide ligand) (11.3 vs. 6.7 kcal mol1
cannot maintain in a stable bound state and its peptide ligand for Src, 13.0 vs. 6.4 kcal mol1 for Abl and 10.8 vs.
(without the context help) was observed to spontaneously unbind 4.2 kcal mol1 for Hck). Interestingly, energetic decomposition
from the partner protein during the MD simulations, with a revealed a considerable difference (TDDS 4 15 kcal mol1)
DGttl value of only 1.3 kcal mol1 as compared to 7.4 between the entropy penalties of SH3–peptide segment and
and 19.2 kcal mol1 for the corresponding peptide segment SH3–peptide ligand interactions; the latter is significantly larger
(within parent context) and intact PMI system, respectively. than the former. This can be confirmed by conformational

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

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peptides have high specificity with their context support. In 19 S. Panni, L. Montecchi-Palazzi, L. Kiemer, A. Cabibbo,
contrast, the mean selectivity is largely impaired or even reversed S. Paoluzi, E. Santonico, C. Landgraf, R. Volkmer-Engert,
to 6.0, 0.74 and 1.2-fold (global selectivity = 1.7-fold close to A. Bachi, L. Castagnoli and G. Cesareni, Proteomics, 2011,
1-fold), respectively, when the peptides were split from the 11, 128–143.
context and bind independently to SH3 domains. 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.
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Model., 2017, 57, 835–845.


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

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Research Article Molecular Omics

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

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


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

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