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org/biochemistry Article

Structural, Functional, and Mutational Studies of a Potent Subtilisin

Inhibitor from Budgett’s Frog, Lepidobatrachus laevis
Mihir Rami, Mohd Shafique,* and Siddhartha P. Sarma*
Cite This: https://doi.org/10.1021/acs.biochem.3c00252 Read Online

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ABSTRACT: Subtilases play a significant role in microbial

pathogen infections by degrading the host proteins. Subtilisin
Downloaded via INDIAN INST OF SCIENCE on October 11, 2023 at 13:59:32 (UTC).

inhibitors are crucial in fighting against these harmful micro-

organisms. LL-TIL, from skin secretions of Lepidobatrachus laevis,
is a cysteine-rich peptide belonging to the I8 family of inhibitors.
Protease inhibitory assays demonstrated that LL-TIL acts as a slow-
tight binding inhibitor of subtilisin Carlsberg and proteinase K with
inhibition constants of 91 pM and 2.4 nM, respectively. The
solution structures of LL-TIL and a mutant peptide reveal that they
adopt a typical TIL-type fold with a canonical conformation of a
reactive site loop (RSL). The structure of the LL-TIL-subtilisin
complex and molecular dynamics (MD) simulations provided an
in-depth view of the structural basis of inhibition. NMR relaxation
data and molecular dynamics simulations indicated a rigid conformation of RSL, which does not alter significantly upon subtilisin
binding. The energy calculation for subtilisin inhibition predicted Ile31 as the highest contributor to the binding energy, which was
confirmed experimentally by site-directed mutagenesis. A chimeric mutant of LL-TIL broadened the inhibitory profile and
attenuated subtilisin inhibition by 2 orders of magnitude. These results provide a template to engineer more specific and potent TIL-
type subtilisin inhibitors.

■ INTRODUCTION
Being the first vertebrates to conquer the land, amphibians
have evolved diverse strategies to fight against a multitude of
endogenous and exogenous factors. One of the key strategies is
the secretion of peptides by granular glands located in the
dermis when the amphibian is stressed or injured. These skin
peptides have unique chemical properties and extreme
biochemical diversity. Based on the function, amphibian skin
peptides can be categorized into myotropical peptides, opioid
peptides, corticotropin-releasing peptides, angiotensins, pro-
tease−inhibitor peptides, neuropeptides, antioxidant peptides,
lectins, insulin-releasing peptides, wound healing promoting
peptides, immunomodulatory peptides, antimicrobial peptides,
antiviral peptides, and antitumor peptides.1
Historically, extensive studies have been carried out on
amphibian skin peptides due to their potential clinical
applications and unique chemical properties.1,2 Skin secretions
of frogs and toads are used for prey capture as poison darts,3 in
traditional Chinese medicine to regulate internal bodily
functions and fertility, and as part of ancient Egyptian
pharmacopeia to treat pain and diarrhea.2
Protease inhibitors play an important role in the amphibian
skin. In the literature, it is speculated that protease inhibitors
either regulate endogenous proteases residing in the skin4 and/
or provide protection from exogenous proteases secreted by
pathogens.5 Protease inhibitors can be classified by the type of
protease they inhibit, viz., aspartic, cysteine, metallo, serine,
and threonine protease inhibitors.6 However, there are also
known examples of proteins that can inhibit two classes of
proteases using different, nonoverlapping binding sites.7 Serine
protease inhibitors are the most studied and found in almost
every living organism. Based on the mechanism of action, three
distinct types of serine protease inhibitors are known:
canonical inhibitors, noncanonical inhibitors, and serpins.7
Canonical serine protease inhibitors interact with proteases
through an exposed loop with a conserved backbone
conformation called the “canonical” conformation.6 This
conformation is found in at least 19 families of proteinaceous
serine protease inhibitors with different structural topologies
described in the MEROPS database (https://www.ebi.ac.uk/
merops/).8 Remarkably, the reactive site loop (RSL) for these
inhibitors adopts a similar canonical conformation regardless of
the different structural folds. To date, several canonical
protease inhibitors have been characterized in amphibians,
Received: May 14, 2023
Revised: September 21, 2023

© XXXX American Chemical Society https://doi.org/10.1021/acs.biochem.3c00252

A Biochemistry XXXX, XXX, XXX−XXX
Biochemistry pubs.acs.org/biochemistry
including Kunitz-type,5,9 Kazal-type,10 and Bowman−Birk-type
inhibitors.11−13 The trypsin inhibitor-like (TIL) disulfide-rich
peptides form a distinct class of protease inhibitors and are
found to be secreted by animals across phyla.4,14−17 These
peptides are known to inhibit the S1, S8, and M4 families of
proteases.
While the structures and specific protease targets of several
TIL inhibitors are known,18−25 information on TIL peptides
found in amphibian secretions is limited. To the best of our
knowledge, a trypsin inhibitor isolated from Bombina bombina
is the only TIL peptide from amphibians with a known
structure.24 TIL inhibitors of other protease families, such as
the S8 (subtilisin family), have not been reported for
amphibian secretions.
Subtilisin-like proteases play a pivotal role in the
colonization of the host by certain pathogenic microorganisms;
this is primarily achieved by the degradation of antimicrobial
peptides secreted by hosts.26 Subtilase inhibitors provide an
additional layer of defense for the host by neutralizing the
proteolytic activity of subtilases. Commercially, subtilisin
inhibitors are used extensively in laundry detergents and
food processing to regulate the protease activity. The TIL
family of inhibitors generally inhibits the S1 family
(chymotrypsin-like) of proteases: trypsin, chymotrypsin, and
elastase. There are a few reports of subtilisin inhibitors from
the TIL family: BmSI-7 found in tick Boophilus microplus and
multiple variants of silkworm inhibitors from Bombyx mori.27,28
Although inhibitory properties of these TIL-type subtilisin
inhibitors have been characterized, no structural information is
available for TIL-type subtilisin inhibitors.
Here we report the structure and protease inhibitory
properties of a TIL peptide identified from cDNA in the
skin secretions of the South American frog Lepidobatrachus
laevis,29 also known as Budgett’s frog.30 This peptide, called
LL-TIL, belongs to the I8 family of inhibitors (according to the
MEROPS database8), incorporating a Trypsin inhibitor-like
(TIL) domain. The I8 family of inhibitors generally possesses
10 cysteine residues that form 5 disulfide bonds. Interestingly,
the TIL domain also occurs in large glycoproteins such as the
von Willebrand factor (VWF),25 mucins, and SCO-spondin.
The disulfide connectivity for the I8 family is C1-C7, C2-C6,
C3-C5, C4-C10, and C8-C9.15 In the first report of the
protease inhibitory activity of this peptide, it was shown that
LL-TIL exhibited moderate trypsin inhibitory activity.
However, we demonstrate unequivocally that LL-TIL strongly
inhibits the microbial protease subtilisin Carlsberg in a slow-
tight binding manner with the Ki of 91 pM. High-resolution,
multinuclear solution NMR methods were used to determine
the solution structure of LL-TIL. Data-driven HADDOCK
models31 of the LL-TIL-subtilisin complex were calculated.
The structural stability of the LL-TIL-subtilisin complex was
examined by using molecular dynamics simulation. Further-
more, mutations were introduced to understand the role of the
RSL residues in the inhibition profile of LL-TIL. The results of
our findings and their implications are discussed below.
■ MATERIALS AND METHODS
Protein Expression and Purification. The plasmid
vector (pET21(a)+) containing the genes of interest (LL-
TIL and mutant variants) were synthesized as cytochrome b5
fusion proteins32 and purchased from GenScript. The plasmid
constructs were designed to include an N-terminal hexahisti-
dine tag and a thrombin cleavage site between cytochrome b5
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Article
and the LL-TIL peptides. The plasmids were transformed into
Escherichia coli BL21 (DE3) cells and grown at 37 °C. Protein
expression was induced by the addition of IPTG to a final
concentration of 0.5 mM when the cells reached an optical
density of 0.8 to 0.9 at 600 nm.
The cells were harvested by centrifugation (6000 rpm, 15
min, 4 °C) 5 h postinduction, resuspended in lysis buffer 50
mM Tris-HCl, 150 mM NaCl, 0.1% NaN3, pH 8.0 (at 25 °C),
and stored at −80 °C. The cOmplete protease inhibitory
EDTA-free tablets from Roche were added to the resuspended
buffer before lysis. Cell lysis was carried out using French Press
(1100 psi). Cell debris and other insoluble matter were
removed by centrifugation (13000 rpm, 60 min, 4 °C).
The lysate was passed through a Ni2+-NTA column pre-
equilibrated with lysis buffer. The recombinant protein bound
to the column was then eluted using 1 M Imidazole. The
elution fraction containing the recombinant protein was
dialyzed against thrombin cleavage buffer (50 mM Tris-HCl,
10 mM CaCl2, 150 mM NaCl, and 0.01% NaN3, pH 8.0)
before the proteolytic cleavage reaction. Thrombin cleavage
reaction was performed at 22 °C with the fusion protein to
thrombin ratio being 50:1. The purified peptide was obtained
by separating the LL-TIL from fusion tag cytochrome b5 using
size exclusion chromatography with a Superdex S-30 column.
The mutants LL-TILmut1 and LL-TILmut2 were purified in
the same manner as the native form.
The isotope precursors for protein production were
purchased from Sigma Chemicals or Cambridge Isotope
Laboratories. Carbon-13, nitrogen-15, and nitrogen-15-en-
riched protein samples were prepared by culturing cells
bearing the appropriate plasmids in M9 minimal medium
with 15N-enriched ammonium chloride (1 g/L) and 13C6-
enriched glucose (2 g/L) or 15N-enriched ammonium chloride
(1 g/L) as metabolic sources of carbon and nitrogen,
respectively.
Molecular Characterization. Analytical Size Exclusion
Chromatography. Size exclusion chromatography studies
were performed on a GE Healthcare Superdex S75 (10/300
GL) analytical column at 4 °C. 100 μL of a 1.0 mg/mL
solution of LL-TIL was injected into the column and eluted
using a flow rate of 0.5 mL/min. The elution volumes of LL-
TIL were compared with those of the protein molecular weight
standards.
CD Spectroscopy. Circular dichroism (CD) spectra were
acquired on a JASCO-715 spectro-polarimeter over a 190 to
260 nm wavelength range. The CD data were acquired with a
scan rate of 100 nm/min and a data pitch of 1/nm using a 0.1
cm path length cuvette. The final spectra were averaged over
three scans and baseline-corrected by subtracting the spectrum
of the appropriate blank solution. The data were smoothed
using a Savitzky−Golay filter33 available with the JASCO
spectra analysis software.
Mass Spectrometry. Matrix-assisted laser desorption
ionization (MALDI) mass spectra were acquired on an Ultraex
time of flight (TOF)/TOF or UltraeXtreme of BrukerDal-
tonics, Germany. 1 μL of (0.1 to 1 mg/mL) sample was mixed
with an equal volume of a saturated solution of either sinapinic
acid (SA) or α-cyano-4-hydroxycinnamic acid (CCA) (in 50%
acetonitrile in water with 0.1% TFA) before spotting on the
MALDI plate. The data were acquired in positive ion mode
with a linear or reflectron mode detector. 500 to 1000 spectra
were co-added. The data were processed and analyzed using
data analysis software Flex Analysis 3.1.
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Biochemistry pubs.acs.org/biochemistry
Protease Inhibition Assays. Bovine trypsin and chymo-
trypsin, elastase from porcine pancreas, subtilisin Carlsberg
from Bacillus licheniformis, and proteinase K from Tritirachium
album were purchased from SIGMA. According to the
manufacturer’s instructions, the protease−inhibitor activities
of trypsin, chymotrypsin, and elastase were determined using
EnzChek Protease assay kits (E6638, Molecular probes). 50
nM concentration of each protease (Trypsin, chymotrypsin,
elastase) was incubated in 100 mM Tris buffer (pH = 7.4) with
different concentrations of LL-TIL and LL-TIL mutants for 15
min. The reaction was initiated by adding 100 μL of BODIPY
FL casein to the protease−inhibitor mixture. Fluorescence
intensity (485 nm excitation and 510 nm emission wave-
lengths) was measured continuously for 10 min at 37 °C on a
96-well plate reader (Thermo Scientific Verioscan reader).
Subtilisin and proteinase K inhibition was characterized by
using the synthetic substrate N-suc-AAPF-pNA (S7388,
SIGMA). Subtilisin (2 nM) and proteinase K (10 nM) were
incubated with LL-TIL and LL-TIL mutants in 50 mM HEPES
buffer (100 mM NaCl, 5 mM CaCl2, pH = 7.4) for 15 min,
followed by the addition of N-suc-AAPF-pNA. Initial velocities
were measured at 405 nm continuously for 5 min in a 96-well
plate reader. Apparent inhibition constant (Kapp i ) and active
enzyme concentration (Eo) were determined by nonlinear
regression curve fitting using the Morrison equation for tight
binding.34,35
vs E Io K iapp + [(Eo + Io + K iapp)2 4EoIo]1/2
= o
vo 2Eo
The inhibition constants (Ki) were extracted using the
Cheng−Prusoff equation for competitive inhibition.36
ij [S ] yzz
K iapp = K ijjj1 z
j
k K m zz{
where Eo is the active enzyme concentration, Io is the total
inhibitor concentration, vs is the steady-state velocity, and vo is
the initial velocity.
Slow binding kinetics was analyzed using equations
described by Morrison and Walsh for subtilisin inhibition.34
Inhibition progress curves were generated by initiating the
reaction by adding 1 nM subtilisin to LL-TIL (1−7 nM) and
N-suc-AAPF-pNA (100 μM). The reaction was monitored at
405 nm for 45 min at 37 °C. Data curves for each inhibitor
concentration were fitted by nonlinear regression analysis to
the integrated rate equation for the slow-tight binding
inhibitors.37 The resultant Kobs values were plotted against
inhibitor concentration to determine the inhibition mecha-
nism: Schemes 1, 2, or 3.34,35,38
Scheme 1
Scheme 2
The equations for Scheme 1 predict that Kobs increases
linearly with inhibitor concentration, the equations for the
Article
Scheme 3
Scheme 2 mechanism predict that Kobs increases hyperbolically
with inhibitor concentration, and the equations for the Scheme
3 mechanism predict that Kobs decreases hyperbolically with
inhibitor concentration. For the Scheme 1 mechanism, eq 4
was used to extract kon, koff, and Kobs values.
(vi vs)(1 ) ij 1 exp( Kobst ) yz
A = vst + lnjjj zz
z
Kobs k 1 {
+ Ao
2
[E ] ij vs yzz
where = t jjj1 z
vi zz{
[I ] jk
(3)
k1[I ]
Kobs = k 1 +
[S ]
1+
Km (4)
where A is the absorbance, Kobs is the first-order rate
constant, t is time, Ao is the initial absorbance, k−1 is the
reverse rate constant, k1 is the forward rate constant, and Km is
the Michaelis constant. All of the data fitting was carried out
using ORIGIN software version 10.5.117.
(1) NMR Spectroscopy. Sample Preparation. Data Acquis-
ition. All NMR experiments for structure determination were
performed on an Agilent 600 MHz spectrometer equipped
with a cryogenically cooled, triple-resonance (z-axis) pulsed
field gradient probe. NMR titration studies were performed on
(2) a Bruker 700 MHz spectrometer equipped with a triple-
resonance (z-axis) pulsed field gradient probe. NMR data were
recorded at 25 °C. Backbone and side-chain resonance
assignments for 1H, 15N, and 13C nuclei were obtained from
double- and triple-resonance NMR experiments that transfer
magnetization via scalar couplings.39 1H−15N correlations were
determined from the 15N-HSQC experiment. Triple-resonance
experiments, viz., CBCA(CO)NH, HNCACB, HNCO, and
HN(CA)CO experiments, were carried out to obtain
sequence-specific assignments. Side-chain assignments were
obtained from HCCCONH, HCCH-TOCSY, CCCONH,
HBHACONH, 13C-HSQC, and TOCSY-HSQC spectra.
three-dimensional (3D)-HNHA experiment was carried out
to obtain scaler coupling constant 3JHN − Hα. HNHB40 and
HN(CO)HB41 spectra were acquired to determine the χ1
angles (Figure S8a). Long-range HNCO42 experiment was
performed to detect the presence of hydrogen bonds. NOE
correlations were established from simultaneous 13C and 15N
edited NOESY-HSQC spectra acquired with a mixing time
(τc) of 175 ms. 15N relaxation data was acquired to estimate R1
(logitudinal relaxation rates), R1ρ (transverse relaxation rates),
and 1H−15N heteronuclear NOE. NMR experiments and
acquisition parameters are listed in Table S1. Model-free
analysis43 of the relaxation data was carried out to probe the
internal dynamics of the individual amino acid residues using
Model-free4 program44 incorporated in FAST-MF.45
Data Analysis. NMR data were processed on an Intel PC
workstation running SuSe Linux version 42.1 using NMRPipe/
NMRDraw processing software.46 The time domain data in
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Biochemistry XXXX, XXX, XXX−XXX
Biochemistry pubs.acs.org/biochemistry Article

Figure 1. (a) Multiple sequence alignment of LL-TIL, LL-TIL mutants, and other protease inhibitors of the TIL family. The position of the RSL is
indicated by a red box. Disulfide-bonded cysteines are denoted by same numbers below in magenta. The sequence alignment was performed on
ESPript server.62 (b) Sequence specifically assigned 2D 1H,15NHSQC spectrum of LL-TIL acquired at 600 MHz. A total of 57 well-resolved
correlation peaks (49 from backbone, 6 from side chains of N29, N47, N48, and 2 from side chains of E56) were assigned. Side-chain correlations are
connected by horizontal dotted lines. (c) 15N backbone dynamics of LL-TIL (blue) and LL-TILmut1 (green). The RSL residues are highlighted in
gray strip.

both the direct and indirect dimensions were apodized with a
90° phase-shifted sine bell with a power of one or two. Zero
filling was carried out before the Fourier transformation in
each dimension. Data were analyzed using CcpNmr Analysis
version 2.4.2.47
in the HNHB experiment and 1Hβ-CO J coupling in the
HN(CO)HB experiment. Hydrogen-bond restraints between
donor−acceptor pairs were derived from the long-range
HNCO experiment. The restraints were included as the
distance of acceptor oxygen from the donor nitrogen (dNiC′i) d d

Structure Determination. Structural Restraints. Distance and proton (dNiC′i) with upper limits of 3.5 and 2.5 Å,
d d

restraints were generated from the integrated peak intensities
of unambiguously and ambiguously assigned interproton
correlations in 15N, and 13C edited NOESY-HSQC spectra
using the “Make Distance Restraints” routine in the CcpNmr
suite. Upper distance limits were set to an absolute maximum
of 6.0 Å, while the lower limits were set to 1.8 Å. Restraints for
ϕ angles were generated using the 3JHN − Hα coupling constants
derived from the intensities of the cross and diagonal peaks.
The ψ angle restraints were generated from the prediction
program TALOS+.48 Side-chain χ1 torsion angles were
predicted from the qualitative analysis of 15N−1Hβ J coupling
D https://doi.org/10.1021/acs.biochem.3c00252
respectively. Disulfide connectivity was established by
assigning unique NOEs between protons on disulfide-paired
cysteines. Two disulfide-bonded cysteines, viz., i and j, were
restrained by distances between Sγi −Sγj , Cβi −Sγj , and Sγi −Cβj
atoms with both the upper and lower limits at 2.05, 3.05, and
3.05 Å respectively.
Structure Calculation. Structures were calculated using
torsion angle dynamics protocol incorporated in CYANA
version 3.98.13.49 NOE-based distances, hydrogen-bond
distances, disulfide bond restraints, and dihedral angles were
used for the structure calculations. In total, 500 structures were
Biochemistry XXXX, XXX, XXX−XXX
Biochemistry pubs.acs.org/biochemistry Article

Figure 2. Characterization of the protease−inhibitor activities of LL-TIL and mutants. (a) Inhibition of elastase, chymotrypsin, and trypsin by LL-
TIL. The wild-type peptide has no or minimal inhibitory activity against these proteases. (b) Inhibition of elastase, chymotrypsin, and trypsin by
LL-TILmut1. The mutant peptide has nominal inhibitory activity against these proteases and does not reach 100% inhibition at the LL-TILmut1
concentrations used in these studies. (c) Inhibition of subtilisin by LL-TIL and LL-TILmut2. The wild-type peptide inhibits subtilisin with a Kapp i of
131 pm, while the LL-TILmut2 has an inhibition constant 5-fold lower. The solid line passing through data points (blue for LL-TIL, red for LL-
TILmut2) reflects the best fit obtained from fitting the quadratic Morrison equation. (d) Inhibition of subtilisin by LL-TILmut1. The LL-TILmut1
peptide exhibits an inhibition constant that is ≈100-fold lower compared to the wild type. The solid line passing through the data points reflects the
best fit obtained from fitting the quadratic Morrison equation. (e) Progress curve analysis of LL-TIL-subtilis in inhibition, solid lines passing via
data points for each inhibitor concentration reflect the best fit obtained by fitting eq 3. (f) Kobs values obtained from progress curve analysis plotted
against inhibitor concentration of 0 to 11 nM. The solid line represents the linear fitting of the data to eq 4. All experiments were carried out in
duplicates and repeated three times (n = 3).

E https://doi.org/10.1021/acs.biochem.3c00252
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calculated from CYANA. The top 50 structures were selected
for further refinement based on target function criteria. Explicit
water refinement was conducted using XPLOR-NIH.50 Finally,
20 structures were selected for further analysis based on the
least energy criteria. Restraints from CYANA to XPLOR-NIH
were converted using pdbstat.51 Structures were analyzed in
MOLMOL52 and PyMOL.53
LL-TIL Subtilisin Binding. The lyophilized subtilisin
powder from SIGMA was dissolved in 50 mM HEPES buffer
(100 mM NaCl, 5 mM CaCl2, pH 7.4) followed by purification
using size exclusion chromatography. The purified LL-TIL and
subtilisin were incubated in a 2:1 ratio, and the complex
formation was monitored by size exclusion chromatography.
The binding of subtilisin to LL-TIL and LL-TILmut1 was
further investigated by NMR titration. This was achieved by
acquiring 15N HSQC spectra of 15N-labeled, 100 μM LL-TIL
and LL-TILmut1 with increasing concentrations of UL-
subtilisin. The combined and weighted backbone amide
chemical shift perturbations (Δδ) were calculated using the
below equation39
= [( H)
2
+( 2 1/2
N /5) ] (5)
where ΔδH and ΔδN are the chemical shift changes (in ppm) at
the beginning and end of the titration, respectively. Two-
dimensional and one-dimensional line shape analysis of NMR
titration data were performed using TITAN software package54
and NmrLineGuru55 respectively. All of the 1H−15N-HSQC
spectra of LL-TIL and LL-TILmut1 from the titration series
were processed identically. For LL-TIL and LL-TILmut1, a
two-state binding model and a three-state induced fit binding
model were used, respectively, to track changes in chemical
shift and intensities.
Structure Calculation of LL-TIL-Subtilisin Complex.
Data-driven structural models of the subtilisin complex with
LL-TIL were generated by HADDOCK 2.4. The ensemble of
the 20 lowest-energy structures of LL-TIL was submitted for
docking with subtilisin. Subtilisin structure was extracted from
subtilisin-eglinC complex (1cse.pdb).56 From NMR titration of
15
N-LL-TIL with subtilisin, residues that exhibited CSP values
above “average CSP + Standard deviation” were selected as
active site residues for LL-TIL: Cys19, Thr27, Asn29, Cys30, Ile31,
and Ala32. For subtilisin, residues forming hydrogen bonds with
eglinC reactive site loop in 1CSE were selected as active
residues: Gly100, Gly102, Tyr104, Gly127, Ala129, Ser130, Asn155,
Asn218, Ser221. The active residues of LL-TIL were introduced
as semiflexible. The resulting clusters were manually analyzed,
and the top four structures of each cluster were submitted to
the PRODIGY server to predict the binding affinity. The final
four least energy structures were analyzed in PyMOL and
LigPlot+.57
Molecular Dynamics Simulation. Molecular dynamics
(MD) simulations58 were carried out for LL-TIL, LL-
TILmut1, and LL-TIL-subtilisin complex with GROMACS
version 2021 employing the AMBER99sb all-atom force
field.59 The protein molecules were solvated in a water box
extending 10 Å from the outermost protein atom. The ionic
strength of the solvating solution was kept at 100 mM. The
starting structures of LL-TIL and LL-TILmut1 were selected
based on the least energy criteria from the final ensemble of
NMR structures. The initial structure of the LL-TIL-subtilisin
complex was selected based on the lowest predicted ΔG from
cluster 1 of the HADDOCK run. The structures were energy-
F https://doi.org/10.1021/acs.biochem.3c00252
Article
minimized and equilibrated before production runs. The MD
simulations were conducted for 200 ns under a constant
temperature of 298 K and a constant pressure of 1 atm. The
simulation trajectory was displayed, animated, and analyzed
with the visualization program PyMOL.
The MM-PBSA method60 was used to calculate the binding
free energy of the LL-TIL-subtilisin complex for each frame of
MD simulation. In the MM-PBSA method, the binding free
energy is described as the sum of the potential energy in a
vacuum, the polar and nonpolar solvation-free energies, and
the entropy contribution. Here, as the binding interface
remains rigid throughout the simulation, the calculation does
not include the entropic contribution. MM-PBSA calculations
were carried out using g_mmpbsa tool61 implemented within
GROMACS. Solvent and solute dielectric constants of 80 and
2, respectively, and salt concentrations of 150 mM were set in
the PB calculations. The binding energy contribution for each
residue was also obtained from g_mmpbsa. Bootstrap analysis
with 2000 steps was carried out to estimate free energy
calculation errors.
■ RESULTS AND DISCUSSION
Biophysical Characterization. The sequences of the LL-
TIL peptide and two mutant variants used in our studies are
given in Figure 1a. Overexpression of fusion LL-TIL protein
was good, and the yields were high enough to prepare
isotopically enriched samples for NMR. Analytical size
exclusion chromatography shows LL-TIL elutes as a single
peak with the elution volume corresponding to the molecular
mass of 8 kDa (Figure S1a). This is indicative of the
monomeric state of LL-TIL in solution. The molecular mass
obtained from MALDI spectra matches the calculated LL-TIL
mass (Figure S1b). CD spectrum of LL-TIL (Figure S2a)
shows a minima at 210 nm and predicts only the antiparallel β-
sheets as the secondary structure in the peptide.63 The one-
dimensional proton NMR spectrum of LL-TIL at 25 °C
exhibits upfield-shifted resonances and large chemical shift
dispersion in the spectral region between 6.5 and 10 ppm,
indicative of a well-folded peptide (Figure S2b). The CD and
one-dimensional NMR spectra of LL-TILmut1 and LL-
TILmut2 peptides showed similar spectral properties.
Functional Characterization. Trypsin, Chymotrypsin,
and Elastase Inhibition. The majority of the protease
inhibitors of the TIL family inhibit proteases from the S1
family (chymotrypsin-like): trypsin, chymotrypsin, and elas-
tase. Consequently, the protease inhibitory activity of LL-TIL
was tested against trypsin, chymotrypsin, and elastase using
EnzChek Protease assay kits (E6638, Molecular probes).
Surprisingly, LL-TIL did not inhibit trypsin, chymotrypsin, or
elastase, even at a maximum LL-TIL to protease ratio of 200
(Figure 2a). The putative reactive site loop (RSL), which is
responsible for inhibiting the proteases, was identified from the
sequence alignment of LL-TIL and other TIL-type protease
inhibitors (Figure 1a). The reactive site loop comprises Cys30,
Ile31, Ala32, Val33, Cys34, and Lys35, occupying positions P3, P2,
P1, P1′, P2′, and P3′ respectively.64 Previously identified
trypsin inhibitors of the TIL family show a preference for
charged residues in RSL (Figure 1a). To study the implication
of polar residues in the RSL on the specificity of LL-TIL, we
mutated three residues at position P2, P1, and P1′ to residues
found in RSL of Ascaris trypsin inhibitor (ATI), viz., Thr31,
Arg32, and Glu33. This chimeric LL-TIL mutant will be referred
to as LL-TILmut1 from here on. LL-TILmut1 weakly inhibited
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Biochemistry pubs.acs.org/biochemistry Article

trypsin and chymotrypsin but did not inhibit elastase (Figure
2b).
Subtilisin Inhibition. Sequence alignment of the TIL-type
subtilisin inhibitors FPI-F and BmSPI7 from B. mori65 showed
similar hydrophobic residues as LL-TIL in their RSL (Figure
1a). This prompted us to investigate the inhibitory profile of
LL-TIL against subtilisin. Subtilisin inhibition was charac-
terized using the N-suc-AAPF-pNA substrate. LL-TIL strongly
inhibits subtilisin and exhibits prolonged onset of inhibition, a
defining feature of slow-tight binding inhibitors. The nonlinear
curve fitting of the Morrison equation for LL-TIL-subtilisin
inhibition data yielded Kappi of 131 pM and active enzyme
concentration ([E]o) of 1.98 nM (Figure 2c). Considering
substrate concentration, the Cheng−Prusoff equation gener-
ated a Ki of 91 pM, comparable to the well-recognized potent
inhibitors like Streptomyces subtilisin inhibitor (SSI), hiproly
barley chymotrypsin inhibitor 2 (CI-2), turkey ovomucoid
third domain (OMTKY3), and leech eglin c (Table 1).
Table 1. Potent Subtilisin Inhibitors
no. name specificity RSL (P3−P3′)
1 SSI subtilisin Cys-Pro-Met-Val-Tyr-Asp
2 CI-2 subtilisin, Val-Thr-Met-Glu-Tyr-Arg
chymotrypsin
3 OMTKY3 subtilisin, Cys-Thr-Leu-Glu-Tyr-Arg
chymotrypsin
4 LL-TILa subtilisin Cys-Ile-Ala-Val-Cys-Lys
5 eglin C subtilisin, Val-Thr-Leu-Asp-Leu-Arg
chymotrypsin
6 FPI-Fa subtilisin Cys-Ile-Thr-Val-Ile-Arg
7 BmSI-7a subtilisin, Cys-Thr-Ala-Asp-Cys-Ile
elastase
a
TIL peptides.
The kinetics of the slow onset of subtilisin inhibition was
analyzed to get insight into the mechanism of inhibition.
Fitting of the progress curve data to eqs 2 and 3 estimated
values for vs, vi, and Kobs for each curve with different LL-TIL
concentrations (Figure 2e). The Kobs values show a linear
increase when plotted against LL-TIL concentration, in
accordance with the inhibition mechanism of Scheme 1,
indicating a single-step binding. However, this linearity can
also account for Scheme 2 (induced fit), when Ki is much
greater than Ki*, which reduces the hyperbolic equation for
Scheme 2 to a linear relationship.35 Consequently, it has not
been possible to distinguish which scheme is operational in the
case of LL-TIL-subtilisin inhibition. However, the slow-tight
binding inhibitors almost always follow the Scheme 2
mechanism.35 Subsequent linear fitting with eq 4, yields kon
and koff of (1.08 ± 0.08) 106 M−1 s−1 and (7.53 ± 6.5) 10−5
s−1, respectively (Figure 2f). The Ki calculated (69 pM) from
the ratio of kon and koff agrees well with the value obtained
from the Morrison plot (91 pM).
The fitting of the Morrison equation for LL-TILmut1-
subtilisin inhibition generated a Kapp
i value of 9.75 nM (Figure
2d). Since the fitted Kapp
i value was higher than [E]T in the case
of the LL-TILmut1, the [E]T value was treated as constant as
suggested by Kuzmic and colleagues.66 Finally, the Cheng−
Prusoff equation yielded a Ki value of 6.93 nM. All inhibition
constants and kinetic parameters for subtilisin inhibition are
tabulated in Table 2.
Proteinase K Inhibition. LL-TIL also shows strong
inhibitory activity against fungal protease proteinase K.
Proteinase K from T. album belongs to the subtilisin-like
family (S8), and it possesses keratin hydrolyzing activity.67 The
nonlinear curve fitting of the Morrison equation for LL-TIL-
proteinase K inhibition data yielded Kapp
i of 3.45 nM and active
enzyme concentration ([E]o) of 9.55 nM (Figure S3).
Considering substrate concentration, the Cheng−Prusoff
equation generated Ki of 2.44 nM. The inhibition of proteinase
K by LL-TIL was not surprising as the active sites of proteinase
K and subtilisin are almost identical.68 Since LL-TIL inhibits
subtilisin more strongly than proteinase K, we chose to focus
on the LL-TIL-subtilisin interaction in this study.
Solution Structure of LL-TIL. Sequence-Specific Reso-
Ki nance Assignments. The sequence-specific resonance assign-
(nM) ments of LL-TIL were obtained by using a set of double- and
0.005 triple-resonance NMR experiments. Strip plots of HNCACB
0.012 and CBCA(CO)NH experiments show the sequential assign-
ments of residues ranging from His49 to Cys58 (Figure S4).
0.023
Except for Gly43, the backbone amide correlation for each
0.091 residue of LL-TIL was observed in the 15N-HSQC spectrum
0.15 (Figure 1b). The single set of peaks observed for the peptide
indicates an absence of chemical exchange on the NMR time
0.148 scale. The 13Cβ chemical shifts of cysteines fall between 35 and
1.4 45 ppm (Figure 3a), suggestive of their oxidized nature.69 The
values of Δδ 13Cβγ for all proline residues lie between 3 and 6
ppm (Figure S5), a characteristic feature of “trans” X-Pro
peptide bond conformation.70 Chemical shift assignments were
obtained for 95% HN, 92% 15N, 96.5% 13Cα, and 94.8% CO of
the backbone nuclei and 98.1% 13Cβ of the side-chain nuclei.
Near-complete chemical shift assignment of LL-TIL and LL-
TILmut1 is deposited in the BioMagResBank (http://www.
bmrb.wisc.edu/) under accession numbers 36536 and 36559,
respectively.
Secondary Structure and Disulfide Connectivity. Secon-
dary structure was determined based on short- and medium-
range NOEs, 3JHN − Hα coupling constants, and chemical shifts
of Cβ, Cα, and Hα atoms as shown in Figure S6. It consists of
four β strands and extensive loop regions. The disulfide
connectivity was deduced from the 15N and 13C edited NOESY
spectra. Based on the NOEs found across cysteine residues,
Cys6-Cys38, Cys15-Cys34, Cys19-Cys30, and Cys40-Cys52 were
established to be disulfide-paired (Figure 3b). Cys23-Cys58
disulfide pairing was performed. No NOE correlations were
found across unpaired cysteine residues. Furthermore, later
stages of structure calculations did not detect significant
proximity between the nonbonded cysteine pairs, confirming
the disulfide connectivity.

Table 2. Inhibition Constants and Kinetic Parameters for Subtilisin Inhibition

name Kapp
i (nM) Ki (nM) kon (M−1 s−1) koff (s−1)
LL-TIL 0.131 ± 0.03 0.091 (1.08 ± 0.08) 10 6
(7.53 ± 6.5) 10−5
LL-TILmut1 9.75 ± 0.42 6.93
LL-TILmut2 0.78 ± 0.12 0.54

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Biochemistry pubs.acs.org/biochemistry Article

Figure 3. (a) Strip plots from the 3D-HNCACB spectrum showing the chemical shifts of Cβ nuclei (in red) of the 10 cysteine residues in LL-TIL.
Dashed lines indicate the upper and lower limits of Cβ chemical shifts. The values of the chemical shifts indicate that all 10 cysteine residues are in
the oxidized state. (b) Strip plots of 13C edited NOESY spectrum depicting NOEs found across disulfide-paired cysteine residues. (c) Strip plots
from the long-range H-bond 3D-HNCO (blue) and reference 3D-HNCO (black) spectra showing the 3JNiC′j scalar coupling correlations across the
d d

hydrogen bonds. The long-range carbonyl correlations obtained from the H-bond HNCO spectrum are connected by horizontal lines to the
corresponding correlations in the reference HNCO experiment for the hydrogen-bond donor−acceptor pairs found across β strands of LL-TIL. (d)
Schematic diagram depicting the β strand topology of LL-TIL with experimentally observed interstrand hydrogen bonds.

NMR Restraints. The NOE-based distances were obtained
from 3D 15N and 13C edited NOESY-HSQC spectra.
Sequential NOE correlations (Hαi to HNi+1 and Hαi to Hδi+1 for
prolines) helped to corroborate the sequence-specific assign-
ments determined from three-dimensional through bond NMR
experiments. The hydrogen bonds deduced from the long-
range HNCO experiment were crucial to establish comple-
mentarity between β strands (Figure 3c). Long-range NOEs
observed across the β strands confirm the β strand registry
(Figure S7) with β-sheet 1 consisting of Lys10-Gly16 and Lys35-
Cys40, while the β-sheet 2 comprises Thr44-Asp46 and Cys52-
H https://doi.org/10.1021/acs.biochem.3c00252
Lys54. The schematic diagram depicting the topology of the β
strands, along with assigned hydrogen bonds, is presented in
Figure 3d. Strong Hiα to Hi+1δ NOE correlations observed
across the X-Pro7, Pro18, Pro20, and Pro28 peptide bonds
provided additional evidence for the trans peptide bond
conformation. 95 backbone dihedral angles were predicted
using TALOS+ and incorporated into the structure calculation.
Analysis of HNHB and HN(CO)HB experiments provided χ1
angles for 28 residues: 12 residues with t2g3 (−60°)
conformation, 9 residues with g2g3 (+60°) conformation and
7 residues with g2t3 (180°) conformation. Strip plots for
Biochemistry XXXX, XXX, XXX−XXX
Biochemistry pubs.acs.org/biochemistry Article

HNHB and HN(CO)HB for cysteine residues are shown in antiparallel to each other. The hydrogen bonds in the β-
Figure S8b. sheet 1 are disrupted to accommodate a wide type β-bulge.71
Tertiary Structure. In total, 1399 NOE-based distances, 24 Phe14 and Cys15 from strand-1 and Lys35 from strand-3 are
hydrogen bonds (two for each H-bond), 5 disulfide bonds, and involved in the formation of this β-bulge. The relatively shorter
122 dihedral angles were used as restraints for the structure β-sheet 2 consists of two strands: β3 (residues 44−46) and β4
calculations of LL-TIL. The NMR restraints and structural (residues 52−54), which are antiparallel. Loop2, connecting
statistics are listed in Table 3. The final ensemble of the 20 the first and second β strand, is the longest loop in LL-TIL
(residue Ser17 to Cys34), harboring the reactive site loop. The
Table 3. NMR Restraints and Structural Statistics for LL- RSL acquires a characteristic canonical shape that is observed
TIL in other canonical protease inhibitors. Disulfide bonds between
Cys19-Cys30, Cys23-Cys58, and Cys15-Cys34 play a substantial
NMR restraints LL-TIL LL-TILmut1
role in stabilizing loop2. Loop3 (residues 41−44) protrudes
distance constraints out from the plane of the disk-shaped peptide, resulting in a
total 1399 1103 highly solvent-exposed region. This observation explains the
intra-residue (|i−j| = 0) 685 489 weak intensity of backbone amide peaks for residues Arg41 and
inter-residue Asp42, as well as the absence of a Gly43 resonance in the
sequential (|i−j| = 1) 318 264 1
H−15N HSQC spectrum. β3 and β4 strands are connected by
medium-range (|i−j| ≤ 4) 140 114 loop 4 (type I turn), forming a β hairpin. Finally, the structure
long-range (|i−j| ≥ 5) 256 236
of LL-TIL ends with a 310-helix formed by residues Lys55-
hydrogen-bond restraintsa 24 24
Asn57.
total dihedral angle restraints 122 125
The structure comparison server DALI72 showed that LL-
backbone dihedral angles (ϕ and ψ) 95 97
TIL is structurally similar to other protease inhibitors of this
side-chain dihedral angles (χ1)b 27 28
family, namely, Apis mellifera chymotrypsin/cathepsin G
structure statistics
inhibitor-1 (AMCI-1, 1ccv.pdb),22 ATI (Ascaris trypsin
restraints violations (mean and s.d)
inhibitor, 1ata.pdb),21 and C/E-1 (Ascaris chymotrypsin/
distance violations > 0.1 Å 0 0
elastase inhibitor, 1eai.pdb),20 with z-scores of 6.1, 5.8, and
dihedral angles > 5° 0 0
5.1, respectively. Interestingly, LL-TIL also resembles the
Ramachandran map statistics
recently determined structure of TIL′ domain (6n29.pdb) in
PROCHECK
assembly with the Human Von Willebrand factor73 (z-score:
most favored regions (%) 90.9 87.4
6.0). Pairwise structural alignment of LL-TIL with AMCI-1,
additionally allowed regions (%) 9.2 12.6
ATI, C/E-1, and BSTI (Bombina skin trypsin/thrombin
generously allowed regions (%) 0 0
inhibitor) reveals that the differences primarily lie in the
disallowed regions (%) 0 0
conformation and length of the extended loop region situated
Richardson MolProbity
between the first and second β strand, as shown in Figure S15.
favored regions (%) 99.0 97.8
The notable difference between the loop of LL-TIL and BSTI
allowed regions (%) 1.0 2.2
arises due to the two extra residues in BSTI, which gives rise to
disallowed regions (%) 0 0
an additional turn. Also, this loop in the C/E-1-elastase
close contacts and deviations from ideal
geometryc structure forms a pore of 5 Å diameter near the RSL of C/E-1,
number of close contacts 0 1 where the elastase side-chain arginine inserts itself. LL-TIL
RMS deviation for bond angles 0.6 0.6 structure lacks this pore. The residues P1 and P′1 of RSL are
RMS deviation for bond lengths (Å) 0.008 0.008 solvent-exposed across all of the serine protease inhibitors,
r.m.s deviation from average structure (Å)d making them readily available to the enzyme. In the case of LL-
backbone 0.76 ± 0.12 0.70 ± 0.14 TIL also, the relative accessible surface area is 89 and 52% for
heavy 1.18 ± 0.11 1.13 ± 0.14 P1 and P′1, making them highly accessible to the enzyme.
a
Hydrogen-bond restraints were obtained from a long-range HNCO Solution Structure of LL-TILmut1 and Comparison
experiment. bχ1 angles were obtained from analysis of HNHB and with LL-TIL. In total, 1103 NOE-based distances, 24 hydrogen
HN(CO)HB experiments. cInformation on close contacts and bonds (two for each H-bond), 5 disulfide bonds, and 125
deviations from ideal geometry was acquired from PSVS server. dihedral angle restraints were used for the structure
d
Root mean square deviation was calculated in PSVS server. calculations of LL-TILmut1. The NMR restraints and
structural statistics are tabulated in Table 3. The final ensemble
lowest-energy structures is shown in Figure 4c,d. The ensemble of the 20 lowest-energy structures is shown in Figure S10. The
of 20 structures shows a backbone root-mean-square deviation structure is similar to that of LL-TIL, with identical disulfide
(RMSD) of 0.7 Å and heavy atom RMSD of 1.1 Å for residues connectivity and secondary structure. The RMSD between the
6−58. PROCHECK-NMR analysis found 90.6 and 9.4% of the lowest-energy structure of LL-TIL and LL-TILmut1 is 1.46 Å
backbone ϕ and ψ dihedral angles lying in the most favored when superposed on Cα atoms. The overlay of the lowest-
region and additionally allowed region of the Ramachandran energy structures of LL-TIL and LL-TILmut1 is shown in
map, respectively (Figure S9). The solution structure of LL- Figure S11. Significant structural differences are observed in
TIL acquires a flat disk shape and lacks a hydrophobic core the mutated RSL region of the peptide. In the LL-TILmut1
(Figure 4b). The overall structure of LL-TIL consists of two structure, the backbone conformation of the RSL changes as
well-defined antiparallel β sheets and five loops held together Arg32 faces inward with relative accessible surface area reducing
by five disulfide bonds (Figure 4a). β1 strand is preceded by a from 0.91 (for Ala32 in LL-TIL) to 0.5. This can be attributed
type I turn formed by residues Pro7-Lys10. β-sheet 1 consists of to the hydrogen-bond formation between the side chain of
β1 (residues 10−16) and β2 (residues 35−40) strands, Arg32 to the backbone carbonyl of Glu33, also resulting in
I https://doi.org/10.1021/acs.biochem.3c00252
Biochemistry XXXX, XXX, XXX−XXX
Biochemistry
Figure 4. Structure of LL-TIL. (a) Superposed cartoon and surface representations of the lowest energy structure of LL-TIL. Disulfide bonds are
colored pink and β strands are colored blue. The position of RSL and loop2 of LL-TIL are also indicated. (b) Side view of LL-TIL. (c, d) Ensemble
of 20 lowest energy structures of LL-TIL. The color scheme is the same as in (a).
distortion of the canonical shape of the binding loop (8a). As
we will see further, this alteration in the conformation may
have some relevance in subtilisin inhibition. The structural
coordinates for LL-TIL and LL-TILmut1 have been submitted
to the Protein Data Bank (PDB) with the accession numbers
8HWU and 8J3Q, respectively.
15
N Backbone Dynamics of LL-TIL and LL-TILmut1.
The backbone amide 15N T1, T2, and {1H}-15N steady-state
heteronuclear NOE values of LL-TIL and LL-TILmut1 are
shown in Figure 1c. The {1H}-15N steady-state heteronuclear
NOE values predominantly lie above 0.6, suggesting that the
peptides are rigid and ordered. The average order parameters
for LL-TIL and LL-TILmut1 were 0.69 and 0.67, respectively.
The extracted order parameters are plotted against the residue
numbers in Figure 1c. Notably, the reactive site loop residues
Cys30 and Val33 for LL-TIL and Cys30 and Glu33 for LL-
TILmut1 yield order parameters below 0.6, suggesting a
relatively more dynamic nature of both residues.
LL-TIL Subtilisin Binding. LL-TIL-subtilisin binding was
studied by size exclusion chromatography and NMR titration.
The overlay of gel filtration chromatograms for LL-TIL,
subtilisin, and LL-TIL-subtilisin is shown in Figure S1a. When
LL-TIL and subtilisin are injected into the column in a 2:1
ratio, a new peak appears at a lower retention volume,
corresponding to the complex.
NMR titration data was instrumental in identifying the
residues residing at the binding interface of the LL-TIL
subtilisin complex. The titration was performed by adding
unlabeled subtilisin to the 15N-labeled LL-TIL sample. With
pubs.acs.org/biochemistry Article
increasing subtilisin concentration, the free LL-TIL (F)
resonances disappeared, while the peaks corresponding to
subtilisin-bound LL-TIL started to appear. There was an
abrupt change in chemical shift between the free and bound
states with increasing subtilisin/LL-TIL molar ratio, character-
istic of a slow exchange. The fitting and extraction of the
kinetic and thermodynamic parameters were performed by
using TITAN and NmrLineGuru. Both programs calculated
very similar values of 2.122 s−1 (TITAN) and 1.086 s−1
(NmrLineGuru) for the koff. The Kd values were calculated
to be 4.56 and 0.030 μM. However, as the LL-TIL
concentration used in the titration study (100 μM) ≫ Kd
(extracted from NMR titration studies) and Ki (extracted from
inhibition studies), the Kd and koff values extracted from NMR
titration data should only be taken as the upper limits and not
as the absolute values. As seen in Figure 5a,b, the RSL region
and the residues structurally proximal to it exhibit significant
chemical shift perturbation, while the chemical shifts of other
peptide regions are largely unperturbed.
15
N-labeled LL-TILmut1 also indicated slow exchange but
exhibited a different pattern of chemical shift perturbation with
the addition of subtilisin (Figure S12a). The free LL-TILmut1
peak disappeared, and two resonances appeared at different
chemical shift positions, presumably corresponding to two
subtilisin-bound states (B1 and B2). All of these states are in
slow exchange with each other on the NMR time scale. The
CSPs between the free state and B1 state resonances are
negligible. Interestingly, the CSPs between the B2 state and
free state resonances are greater and agree well with the CSPs
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Biochemistry XXXX, XXX, XXX−XXX
Biochemistry pubs.acs.org/biochemistry Article

Figure 5. Mapping the interaction site of subtilisin on LL-TIL. (a) Overlay of 15N-HSQC spectra of free 15N-LL-TIL (blue) and 15N-LL-TIL with
UL-subtilisin (green) in a 1:1 ratio. Significantly shifted residues of LL-TIL (circled) are considered to participate in the interaction with subtilisin.
(b) Plot of cumulative CSP as a function of the primary structure of LL-TIL. Average CSP and average CSP plus one standard deviation are
indicated by dotted lines. (c) CSPs are mapped on the structure of LL-TIL with color gradient indicating no change in CSP (gray) to CSP plus one
standard deviation (green).

of LL-TIL when titrated against subtilisin, indicating a similar
arrangement of the binding interface (Figure S12b).
Interestingly, when the subtilisin to LL-TILmut1 ratio is
under unity during the NMR titration, the B1 state intensities
are higher compared to the B2 state. This begins to change at
the subtilisin to LL-TILmut1 ratio of 1, ultimately resulting in
higher intensities of the B2 state at the ratio of 1.4 (Figure
S13). Implications of these results are discussed below.
Mechanism of Inhibition. The canonical inhibitors
interact with the proteases according to the “Standard
mechanism” or “Laskowski mechanism”, in which the inhibitor
RSL inserts itself into the protease binding site in a substrate-
like manner. This mechanism can be written in the following
way6
E + I F L F C F X* F L* F E + I*
where E is the protease, I and I* are intact and cleaved
inhibitors, respectively, and L and L* are loose, noncovalent
complexes of protease and intact or modified inhibitor,
respectively. C and X* are the stable, noncovalent protease−
inhibitor complexes with intact and cleaved inhibitors,
respectively. The formation of the “C” complex is usually
followed by a very slow hydrolysis of the peptide bond
between P1 and P1′ residue initiated by a nucleophilic attack
from Oγ atom of catalytic serine residue residing in the
protease active site.
As the LL-TIL RSL is flanked by two disulfide bonds, the
hydrolysis of the peptide bond between P1 and P1′ would
result in an increase in mass by 18 Da. MALDI spectra of LL-
K https://doi.org/10.1021/acs.biochem.3c00252
TIL and LL-TILmut1 in the absence and the presence of
subtilisin are shown in Figure S16. The MALDI spectra were
acquired following incubation of the inhibitor with subtilisin
for 6 h. It is clear from the spectra that the masses of LL-TIL
and LL-TILmut1 do not alter in the presence of subtilisin,
suggesting that the cleavage does not occur under experimental
conditions.
The crystal structures of the standard mechanism protease−
inhibitor complexes are almost exclusively found in the
thermodynamically favored “C” state (tight, noncovalent
complex with intact inhibitor), also called a Michaelis complex.
NMR titration of 15N LL-TIL with subtilisin resulted in the
emergence of only a single-bound state. Moreover, the mass
spectrometry data show that an “intact” LL-TIL forms a
complex with subtilisin (Figure S16a,b). In light of this, we can
safely assign this state to the Michaelis complex. The absence
of “L” state from the LL-TIL-subtilisin titration spectra
suggests that either LL-TIL directly forms the Michaelis
complex (C) or that a loose complex is formed, which rapidly
rearranges to the Michaelis complex. This agrees with the
progress curve analysis for LL-TIL-subtilisin inhibition, which
indicated either a single-step binding (Scheme 1) or an
induced fit mechanism (Scheme 2) with Ki much greater than
that of K*i .
The NMR titration of 15N LL-TILmut1 with subtilisin leads
to the partition of LL-TILmut1 into two bound states (B1 and
B2). The mass spectrometry data show that 15N LL-TILmut1
stays intact during complex formation with subtilisin (Figure
S16c,d). Therefore, according to the standard mechanism, the
Biochemistry XXXX, XXX, XXX−XXX
Biochemistry
two bound states can only be the loose complex (L) and the
Michaelis complex. As the B2 state resembles the subtilisin
bound form of LL-TIL with regard to chemical shifts, we
speculate that this state corresponds to a Michaelis complex.
On the other hand, the B1 state peaks are proximal to the free
state, which suggests the possibility of a loose complex “L”.
The NMR titration also shows that the “L” state (B1) is
predominant at lower subtilisin:LL-TILmut1 ratios (≤1.2),
while the “C” state (B2) becomes the major species at a higher
subtilisin:LL-TILmut1 ratio (1.4) (Figure S13). In other
words, the equilibrium seems to shift toward the B2 state as the
subtilisin/LL-TILmut1 ratio increases (Figure S14). The
presence of the loose complex and it is comparable populations
to the Michaelis complex observed for LL-TILmut1-subtilisin
titration are in stark contrast to what is observed for the LL-
TIL-subtilisin titration. It suggests that the equilibrium is not
heavily biased toward the Michaelis complex as the loose
complex does not readily transition into the Michaelis complex
in LL-TILmut1-subtilisin titration. This may also account for
the weaker inhibition of LL-TILmut1 toward subtilisin.
HADDOCK Model of LL-TIL-Subtilisin Complex. NMR
perturbation data confirm that the reactive site loop
significantly contributes to the LL-TIL-subtilisin interaction.
To gain insight into the structural basis for the LL-TIL-
subtilisin interaction, data-driven docking protocols in
HADDOCK were applied. Residues of LL-TIL that showed
chemical shift perturbation (CSP) more than average plus one
standard deviation were found to be present near or within the
RSL region and were considered active residues (cf. Materials
and Methods Section). HADDOCK clustered 158 structures
of the LL-TIL-subtilisin complex into 13 clusters. Analysis of
clusters revealed that cluster 1, with a size of 47 structures,
showed the lowest Z-score (−2.0) and HADDOCK score
(−81.7 ± 0.7). They also displayed the maximum number of
hydrogen bonds across the interface and highest binding
affinities. Structural statistics of the LL-TIL subtilisin complex
are tabulated in Table 4.
Figure 6a,b shows the representative structure of the LL-TIL
subtilisin complex from cluster 1. The arrangement of the
Table 4. Restraints and Structural Statistics for Data-Driven
Docking of LL-TIL and Subtilisin
ambiguous interaction restraints HADDOCK run
LL-TIL 19,27,29,30,31,32
subtilisin 100,102,104,127,129,130,155,218,221
restraints for LL-TIL
NOE 1399
H-bond 24
dihedral angle 122
structural statistics for haddock-
derived structures of
LL-TIL-subtilisin complex
(cluster 1)
HADDOCK score −81.0 ± 0.7
cluster size 47
RMSD (Å) 0.7 ± 0.5
Evdw (kcal mol−1) −61.1 ± 1.9
EEE (kcal mol−1) −90.3 ± 6.2
EDE (kcal mol−1) −3.8 ± 2.6
ERVE (kcal mol−1) 13.0 ± 12.4
BSA (Å2) 1307.8 ± 79.9
Z-score −2.0
pubs.acs.org/biochemistry Article
binding interface in the LL-TIL-subtilisin complex is similar to
the known subtilisin bound complexes of Greglin, CI-2, Eglin
C, SSI, and OMTKY3.56,74−77 LL-TIL-subtilisin interaction is
predominantly driven by residues Thr27 (P6) to Cys34 (P2′) of
LL-TIL, inserting themselves into the subtilisin active site cleft
in a substrate-like manner (Figure 6c). The LL-TIL RSL is
held together in the active site cleft of subtilisin by hydrogen
bonds formed between pairs Asn29 (P4)-Gly102, Ile31 (P2)-
Gly100 above the cleft, and Cys30 (P3)-Gly127, Ala32 (P1)-Ser125
below the cleft (subtilisin residues are indicated in “italics”
typeface) (Figure 6d). These interactions allow the insertion of
Asn29 (P4) and Ala32 (P1) residues into the binding pockets S4
and S1 of subtilisin, respectively78 (Figure 6e,f). LL-TIL
residues Asn29 (P4)-Ile31 (P2) form a β-zipper79 type of
interaction with the subtilisin residues Leu126-Gly128 (Figure
6a), which is also observed for subtilisin complexes of CI-275
and greglin.74 This type of β-sheet addition, a commonly
observed motif in protein−protein interactions, has been
pointed out by Waksman and co-workers.79 Furthermore, Ala32
(P1) forms two hydrogen bonds with Asn155 and catalytic
Ser221, including the one between the Oγ atom of Ser221 and
the N atom of Ala32. On the prime side of the P1 residue, a
hydrogen bond is observed between Cys34 and Asn218. The
complex’s interface area and the buried surface area were found
to be 601.5 and 1229.4 Å2, respectively.
The contact map shown in Figure S17 reveals the
hydrophobic contacts formed between LL-TIL with subtilisin
(http://mapiya.lcbio.pl/).80 In particular, the RSL residues
Ile31, Ala32, and Val33 form in total 10 hydrophobic contacts of
less than 5 Å with subtilisin (Table S2). Besides, the nonpolar
buried surface area of the LL-TIL-subtilisin complex (753.9
Å2) accounts for 61.3% of the total buried surface area (1229.4
Å2). These lines of evidence point to the significance of
hydrophobic interactions to the binding process. The distance
between the catalytic Oγ atom of Ser221 and the Ala32 carbonyl
carbon is 3.55 Å. This distance is ∼0.7 Å shorter for other
subtilisin inhibitors (≈2.8 Å), essential for the nucleophilic
attack to occur. Interestingly, a similar long distance of 3.1 Å is
also seen in the crystal structure of TIL peptide C/E-1 bound
to elastase (1eai.pdb).20 Perhaps, this long distance precludes
the nucleophilic attack, implying that LL-TIL does not readily
get cleaved by subtilisin.
Molecular Dynamics Simulation. MD simulation of LL-
TIL and LL-TILmut1 illustrates that the RMSD remains
steady with respect to the initial structure over the time span of
200 ns (Figure 7a). The root-mean-square fluctuation (RMSF)
values indicate the mobility of each amino acid residue. The
relatively higher RMSF values for the RSL region of both LL-
TIL and LL-TILmut1 can be extrapolated to higher mobility,
which agrees with the NMR relaxation data (Figure 7b).
MD simulation analysis of the LL-TIL-subtilisin complex
was carried out to determine the stability of the complex and
to further refine the interface interactions unbiasedly. The low
RMSD values of LL-TIL-subtilisin complex trajectories (<0.3
nm) and negligible change in buried surface area (14,515 ±
894 Å2) for the complex during the 200 ns simulation suggest
that the LL-TIL-subtilisin complex is stable. The hydrogen-
bond distribution for the trajectories remains narrow with an
average of 12.4 ± 1.8 hydrogen bonds per frame (Figure 7c).
Hydrogen-bond occupancies across all of the frames in the MD
simulation are tabulated in Table 5. Close examination of the
interface between LL-TIL and subtilisin in MD simulation
trajectories revealed that most of the hydrogen bonds observed
L https://doi.org/10.1021/acs.biochem.3c00252
Biochemistry XXXX, XXX, XXX−XXX
Biochemistry pubs.acs.org/biochemistry Article

Figure 6. Structure of the LL-TIL-subtilisin complex. (a, b) Superposed cartoon and surface representative HADDOCK structure of the LL-TIL
(orange) subtilisin (green) complex. The inset shows the β zipper formed on the interface of the LL-TIL-subtilisin complex. (c) Dimplot57 of LL-
TIL-subtilisin complex depicting interactions across the interface. The hydrogen bonds (pink dotted line) and corresponding distances between
donor and acceptor atoms are indicated. Residues or atoms involved in hydrophobic interactions are bordered by eyelashes and connected by
dotted black lines. (d) Hydrogen-bond interactions between RSL of LL-TIL and residues located above and below the active site cleft of subtilisin.
(e) Insertion of Asn29 (P4) of LL-TIL into the S4 pocket of subtilisin. (f) Insertion of Ala32 (P1) of LL-TIL to S1 pocket of subtlilsin.

in the initial structure remain intact. The hydrogen bond
between the backbone O atom of Thr27 and the side-chain Oϵ
atom of Tyr104 is lost during the simulation. Interestingly, the
residue occupying the P1 site (Ala32) strengthens its
interaction with subtilisin by forming another hydrogen bond
with the backbone of Thr220 (Figure 7e).
The free energy of binding was computed using the
Molecular Mechanics Poisson−Boltzmann Surface Area
(MM-PBSA) approach as implemented in g_mmpbsa. A
total of 400 frames extracted every 0.5 ns were used for the
calculations. The average free energy of binding and its
M https://doi.org/10.1021/acs.biochem.3c00252
components are tabulated in Table 6. VdW and electrostatic
energy are the major contributors to the binding energy. A per-
residue decomposition was performed to examine the residue-
wise contribution of the binding energy for the LL-TIL (Figure
7d). As expected, the RSL residues contribute substantially to
the binding energy. Ile31 contributes the most, with a binding
energy of −34.8 ± 0.3 kJ/mol. Even though the backbone of
Ile31 forms only a single hydrogen bond with Gly100, its side
chain forms multiple hydrophobic interactions at the interface,
accounting for high binding energy (Figure 6c). We generated
an I31A mutant to validate this experimentally, and we indeed
Biochemistry XXXX, XXX, XXX−XXX
Biochemistry pubs.acs.org/biochemistry Article

Figure 7. MD simulations (a) root-mean-square deviation (RMSD) of LL-TIL (black), LL-TIL-subtilisin complex (blue), and LL-TILmut1 (red)
with respect to the initial structure. (b) Residue-wise root-mean-square fluctuation (RMSF) of LL-TIL (black), LL-TIL-subtilisin complex (blue),
and LL-TILmut1 (red). RSL residues are highlighted in light gray. (c) Distribution of hydrogen bonds across the LL-TIL-subtilisin interface during
200 ns simulation. RSL residues are highlighted in light gray. (d) Per-residue contribution of LL-TIL to the binding energy. Our calculations
indicate that residue I31 imparts the highest contribution to the binding energy. (e) Hydrogen bonds formed by Ala32 (P1) of LL-TIL with
subtilisin.

observed weaker inhibition of this mutant against subtilisin RSL is preserved across the inhibitor families. The
with a Ki of 0.54 nM (Figure 2b). conformation of RSL bound to LL-TIL is similar to the free
Conformation of RSL. Although a wide variety of scaffolds form of LL-TIL with a backbone RMSD of 0.8 Å (Figure 8b).
exist for serine protease inhibitors, the conformation of the This observation reveals that there is a minimal conformational
N https://doi.org/10.1021/acs.biochem.3c00252
Biochemistry XXXX, XXX, XXX−XXX
Biochemistry pubs.acs.org/biochemistry Article

Table 5. Hydrogen-Bond Occupancies of LL-TIL-Subtilisin the relatively lower stability of the Michaelis complex in LL-
Complex during 200 ns MD Simulation TILmut1-subtilisin interaction.
Specificity of LL-TIL. The original study by Wang et al.
no. LL-TIL subtilisin occ. (%)
had reported that LL-TIL acts as a trypsin inhibitor with a Ki of
1 T27 (O) A129 (N) 42.26 16 μM.29 This was surprising, considering trypsin prefers a
2 N29 (O) G102 (N) 73.63 positively charged residue at their P1 position. We observed
3 N29 (ND2) G102 (O) 86.81 here that LL-TIL does not inhibit trypsin even at a high LL-
4 C30 (O) G127 (N) 99.30
TIL-to-trypsin concentration ratio of 400. Moreover, LL-TIL
5 C30 (N) G127 (O) 94.91
showed no inhibition against chymotrypsin or elastase also.
6 I31 (N) G100 (O) 99.00
LL-TIL is a specific and potent inhibitor of subtilisin. Among
7 A32 (N) S125 (O) 97.40
other potent subtilisin inhibitors (Table 1), only a few show
8 A32 (O) S221 (N) 98.80
specificity for subtilisin. The specificity of canonical serine
9 A32 (O) T220 (N) 44.26
protease inhibitors is generally attributed to the P1 residue.
10 A32 (O) N155 (ND2) 89.31
Trypsin inhibitors prefer Arg or Lys at its P1 site, while
11 A32 (N) S221 (OG) 18.68
chymotrypsin inhibitors favor Leu, Met, Phe, Tyr, Trp, or Asn
12 C34 (N) N218 (O) 99.00
at its P1 site. In contrast to these mammalian proteases,
Table 6. Free Energy of Binding and Its Components for subtilisin inhibitors have a much broader preference for P1
LL-TIL-Subtilisin Complex residue,81 which also includes Ala as found in LL-TIL.
The hydrophobic RSL residues (Ile31, Ala32, and Val33) form
no. energy parameter energy (kJ mol−1) multiple hydrophobic contacts with subtilisin at the binding
1 Van der Waals energy −360.93 ± 2.19 interface, make a large contribution to the nonpolar buried
2 electrostatic energy −321.04 ± 4.42 surface area (276.6 Å2), and also contribute significantly to the
3 polar solvation energy 409.41 ± 8.50 binding energy as revealed in the MM-PBSA analysis. All of
4 SASA energy −32.42 ± 0.28 this evidence suggests that the hydrophobic nature of these
5 binding energy −303.74 ± 8.29 amino acids plays an important role in governing the specificity
of LL-TIL. Replacing Ile31 with alanine (LL-TILmut2)
change in the RSL of LL-TIL upon binding to subtilisin. The weakens the subtilisin inhibitory activity. Moreover, mutating
rigidity of RSL is a consequence of two disulfide bonds all three hydrophobic RSL residues to hydrophilic residues
flanking the RSL and the hydrophobic interactions of Val33 (LL-TILmut1) not only weakens the subtilisin inhibition but
(P1′) to Pro18 and Ile31. The bound conformation of RSL also imparts trypsin inhibitory activity, resulting in losing its
closely mimics the RSL of other canonical subtilisin inhibitors, specificity. The specificity of canonical protease inhibitors is
as shown in Figure 8b. also ascribed to the flexibility of the RSL of the inhibitor in
The solution structure of Ascaris trypsin inhibitor (ATI)21 their free state.82,83 Here, the NMR relaxation data and
solved at two different pHs (2.4 and 4.75) shows that the molecular dynamics simulation showed that the RSL region of
canonical shape of RSL gets slightly distorted at higher pH as LL-TIL is not too flexible, which could further contribute to its
the side chain of P1 residue faces inward to the main body of high specificity.
peptide. We observed that in the structure of LL-TILmut1, Conclusions. In this study, we show that LL-TIL acts as a
RSL indeed acquires a conformation similar to ATI RSL with a potent inhibitor of subtilisin and proteinase K, with no effect
backbone RMSD of 0.802 Å and differs significantly from RSL on trypsin, chymotrypsin, or elastase. Most of the NMR
of LL-TIL with a backbone RMSD of 1.678 Å, as shown in structures of TIL peptides have been determined using 1H
Figure 8a. This structural difference could be responsible for NMR spectroscopy. Here, we have determined the structures

Figure 8. (a) Superposition of RSL of LL-TIL, LL-TILmut1, and ATI using backbone atoms N, Cα, and C. LL-TIL differs from LL-TILmut1 and
ATI in the conformation of the P1 loop, which is responsible for the binding and inhibition. (b) Superposition of RSL of subtilisin bound LL-TIL,
CI-2, OMTKY3, eglin C, and SSI and free LL-TIL using backbone atoms N, Cα, and C. The RMSD values of RSL of LL-TIL and subtilisin-bound
LL-TIL with RSL of other subtilisin inhibitors are indicated.

O https://doi.org/10.1021/acs.biochem.3c00252
Biochemistry XXXX, XXX, XXX−XXX
Biochemistry pubs.acs.org/biochemistry
of LL-TIL and LL-TILmut1 using high-resolution triple-
resonance NMR experiments. These experiments allowed a
deeper understanding of the structural and dynamic nature of
the peptide. Mutagenesis and isotope-filtered NMR experi-
ments have provided important mechanistic insights into the
subtilisin−inhibitor interaction. The high similarity between
subtilisin-like proteases secreted by pathogens and the ability
of LL-TIL to strongly inhibit subtilisin and proteinase K
suggests that LL-TIL might be helpful in providing protection
against various harmful microorganisms. The involvement of
subtilisin-like proteases as a virulence factor in the entry of
several microbial pathogens makes it an important target for
treating several infectious diseases.
■ DATA DEPOSITION
Chemical shift assignments for LL-TIL and LL-TILmut1 have
been deposited in the Biological Magnetic Resonance Bank
under accession numbers 36536 and 36559, respectively. 3D
structural data for LL-TIL and LL-TILmut1 have been
deposited in the RCSB PDB as entries 8HWU and 8J3Q,
respectively.
■ ASSOCIATED CONTENT
* Supporting Information
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.biochem.3c00252.
Gel filtration spectra of LL-TIL, subtilisin, and LL-TIL-
subtilisin complex; CD and one-dimensional (1D) NMR
spectra of LL-TIL; determination of Kapp i for LL-TIL-
proteinase K inhibition; sequence-specific assignments
of LL-TIL; difference in Cβ and Cγ chemical shifts for
each proline residue of LL-TIL; secondary structure
assignment for LL-TIL; long-distance NOE correlations
found across β strands of LL-TIL; determination of the
χ1 angles for LL-TIL; Ramachandran plot analysis of
LL-TIL using PROCHECK; structure of LL-TILmut1;
structural superposition of LL-TIL and LL-TILmut1;
NMR titration of LL-TILmut1 with subtilisin; modu-
lation in peak volumes during NMR titration of
subtilisin:LL-TILmut1; B2:B1 peak volume ratios during
NMR titration of subtilisin/LL-TILmut1; structural
superposition of LL-TIL with TIL inhibitors; MALDI
spectra of 15N LL-TIL and LL-TILmut1 in the presence
and absence of subtilisin; contact map of LL-TIL-
subtilisin interaction; NMR data acquisition parameters
for LL-TIL, and hydrophobic contacts between RSL
residues of LL-TIL and subtilisin (PDF)
■ AUTHOR INFORMATION
Corresponding Authors
Mohd Shafique − Molecular Biophysics Unit, Indian Institute
of Science, Bangalore, Karnataka 560012, India;
Email: msnmbb@gmail.com
Siddhartha P. Sarma − Molecular Biophysics Unit, Indian
Institute of Science, Bangalore, Karnataka 560012, India;
orcid.org/0000-0001-7619-8904; Phone: +91 80 2293
3454; Email: sidd@iisc.ac.in
Author
Mihir Rami − Molecular Biophysics Unit, Indian Institute of
Science, Bangalore, Karnataka 560012, India
Complete contact information is available at:
P https://doi.org/10.1021/acs.biochem.3c00252
Article
https://pubs.acs.org/10.1021/acs.biochem.3c00252
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
The authors thank the DST FIST programme for the 600 MHz
NMR and other instrumental facilities in the Molecular
Biophysics Unit, Indian Institute of Science (IISc.). The
authors also thank the DBT-IISc Partnership program for the
Mass Spectrometric Facilities in the Division of Biological
Sciences (DBS), Indian Institute of Science. M.S. is thankful to
SERB, India, for NPDF grant file number PDF/2016/003833.
The authors thank Sunita, Mass Spectrometry Facility, DBS,
IISc., for data acquisition. They also thank Sri Teja for useful
discussions. The image of the frog used in the TOC image was
purchased from iStock by Getty Images.
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