Inversion of the Side-Chain Stereochemistry of Indvidual Thr or Ile Residues in a Protein Molecule: Impact on the Folding, Stability, and Structure of the ShK Toxin

Article abstract of DOI:10.1002/anie.201612398

ShK toxin is a cysteine-rich 35-residue protein ion-channel ligand isolated from the sea anemone Stichodactyla helianthus. In this work, we studied the effect of inverting the side chain stereochemistry of individual Thr or Ile residues on the properties of the ShK protein. Molecular dynamics simulations were used to calculate the free energy cost of inverting the side-chain stereochemistry of individual Thr or Ile residues. Guided by the computational results, we used chemical protein synthesis to prepare three ShK polypeptide chain analogues, each containing either an allo-Thr or an allo-Ile residue. The three allo-Thr or allo-Ile-containing ShK polypeptides were able to fold into defined protein products, but with different folding propensities. Their relative thermal stabilities were measured and were consistent with the MD simulation data. Structures of the three ShK analogue proteins were determined by quasi-racemic X-ray crystallography and were similar to wild-type ShK. All three ShK analogues retained ion-channel blocking activity.

Full text of DOI:10.1002/anie.201612398

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Communications
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International Edition: DOI: 10.1002/anie.201612398
German Edition: DOI: 10.1002/ange.201612398
Protein Chemistry
Inversion of the Side-Chain Stereochemistry of Indvidual Thr or Ile
Residues in a Protein Molecule: Impact on the Folding, Stability, and
Structure of the ShK Toxin
Bobo Dang,* Rong Shen, Tomoya Kubota, Kalyaneswar Mandal, Francisco Bezanilla,
Benoit Roux, and Stephen B. H. Kent
Abstract: ShK toxin is a cysteine-rich 35-residue protein ion-
channel ligand isolated from the sea anemone Stichodactyla
helianthus. In this work, we studied the effect of inverting the
side chain stereochemistry of individual Thr or Ile residues on
the properties of the ShK protein. Molecular dynamics
Scheme 1. Stereochemistry of l-Ile, l-allo-Ile and l-Thr, l-allo-Thr.[1].
simulations were used to calculate the free energy cost of
inverting the side-chain stereochemistry of individual Thr or Ile
residues. Guided by the computational results, we used
chemical protein synthesis to prepare three ShK polypeptide
chain analogues, each containing either an allo-Thr or an allo-
Ile residue. The three allo-Thr or allo-Ile-containing ShK
polypeptides were able to fold into defined protein products,
but with different folding propensities. Their relative thermal
stabilities were measured and were consistent with the MD
simulation data. Structures of the three ShK analogue proteins
were determined by quasi-racemic X-ray crystallography and
were similar to wild-type ShK. All three ShK analogues
retained ion-channel blocking activity.
very limited work on the effect of inversion of Thr and/or Ile
side-chain stereochemistry on the properties of globular
protein molecules.[2]
The small protein toxin ShK was originally isolated from
the sea anemone Stichodactyla helianthus.[3] It binds to the
Kv1.3 ion channel with very high affinity.[3] There are 4 Thr
and 2 Ile residues in the 35 amino acid ShK toxin polypeptide
chain (Scheme 2).
Scheme 2. Amino acid sequence of the ShK toxin protein. The native
chemical ligation site used in the synthesis is underlined.
P
roteins are chiral molecules. Natural proteins have the l-
configuration since they are made by ribosomal translation
and thus consist only of l-amino acids and the achiral amino
acid glycine. Of the 20 principal genetically-encoded protei-
nogenic amino acids, isoleucine and threonine uniquely have
a second chiral center in their side chains. Inversion of the
alpha-carbon chiral center of most natural l-amino acids will
generate the mirror-image d-amino acids. In the case of
isoleucine and threonine, both chiral centers must be inverted
to generate the mirror-image amino acids d-isoleucine and d-
threonine. Inversion of stereochemistry only at the alpha
carbon will generate the diastereomeric amino acids d-
alloisoleucine (d-allo-Ile) and d-allothreonine (d-allo-Thr).
Conversely, inversion of the alpha carbon stereochemistry in
d-Ile and d-Thr generates l-alloisoleucine (l-allo-Ile) and l-
allothreonine (l-allo-Thr;[1] Scheme 1). There has been only
Recently, we reported a convergent total synthesis of wild-
type ShK toxin and its mirror-image form (d-ShK) for
determination of the ShK protein structure by racemic
protein X-ray crystallography.[4] Among other things, we
showed that d-ShK protein does not bind to the Kv1.3 ion
channel. Surprisingly, it had been reported that a diastereomer
of the mirror-image ShK protein, “d-allo-ShK”, retained its
biological activity and could bind to the Kv1.3 ion channel.[5]
This “d-allo-ShK” was described as being made up of d-
amino acids (and Gly) but with the natural stereochemistry of
the chiral side chains of the Ile and Thr residues, that is, d-
allo-Ile and d-allo-Thr.
In this work, In the work reported here, as a prelude to
a systematic reinvestigation of the structure and properties of
the d-allo-ShK protein molecule, we set out to explore the
effects of inverting the side chain stereochemistry of individ-
ual Thr or Ile residues on the foldability/stability/structure/
biological activity of the ShK protein, and to correlate
biological activities with atomic-resolution structures of ShK
analogues containing allo-amino acids.
[*] Dr. B. Dang, Dr. K. Mandal, Prof. Dr. S. B. H. Kent
Department of Chemistry
Department of Biochemistry & Molecular Biology
Institute for Biophysical Dynamics, University of Chicago
Chicago, IL 60637 (USA)
E-mail: skent@uchicago.edu
Dr. R. Shen, Dr. T. Kubota, Prof. Dr. F. Bezanilla, Prof. Dr. B. Roux
Department of Biochemistry & Molecular Biology
Institute for Biophysical Dynamics, University of Chicago
Chicago, IL 60637 (USA)
First, we calculated the free-energy cost of substituting
individual Thr or Ile residues in ShK protein with an allo-Thr
or allo-Ile residue, respectively, using bidirectional alchemical
free energy perturbation molecular dynamics (FEP/MD)
simulations. The FEP/MD calculations allowed us to estimate
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201612398.
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the influence of inverting the side-chain stereochemistry of
each of the six Thr/Ile amino acid residues on the stability of
the ShK protein. The molecular systems were set up using the
VMD program[6] and simulated with the NAMD program.[7]
The results of the forward and the backward FEP/MD
simulations were then combined using the Bennett accept-
ance ratio method[8] to calculate the free energy change for
each of the Thr/allo-Thr or Ile/allo-Ile mutations within the
VMD plugin ParseFEP.[9] The results are shown in Figure 1.
Scheme 3. Convergent synthesis of ShK toxin by native chemical
ligation of two unprotected peptide segments, followed by folding and
formation of disulfides. The figure is adapted from Ref. [4].
Figure 1. Calculated free-energy differences between ShK protein and
allo-amino acid ShK analogues. Error bars represent standard devia-
tions of the means.
To be rigorous, it would be necessary to close the
thermodynamic cycle of FEP/MD by including the free-
energy cost of the chirality change in the unfolded protein.
However, because the aqueous solvent is not a chiral environ-
ment, the solvation free energy of the side-chain moiety is
invariant upon such a change. Thus, it can be reasonably
assumed that the free-energy difference is negligible in the
unfolded protein.
From these MD calculations, we found that replacing Ile7
with allo-Ile7 will stabilize the folded structure by a free
energy of 0.7 kcalmolÀ1; and replacing Thr31 with allo-Thr31
will destabilize the folded structure by a free energy of
3.2 kcalmolÀ1. Other Thr/Ile residues did not show significant
effects upon substitution with allo-Thr/allo-Ile. (Figure 1)
Therefore, we chose three amino acid sites (Ile7, Thr13, and
Thr31) for further investigation to determine the impact of
side-chain chirality inversion of individual Ile and Thr
residues on the stability of the ShK protein.
Figure 2. LCMS data for the three allo-amino acid containing ShK
analogues and the wild-type ShK polypeptide chain. a) [allo-Ile7]ShK
polypeptide. Mass: Obsd. 4061.0Æ0.2 Da, calcd 4060.8 Da (av. iso-
tope composition). b) [allo-Thr13]ShK polypeptide. Mass: Obsd.
4060.8Æ0.2 Da, calcd 4060.8 Da (av. isotope composition). c) [allo-
Thr31]ShK polypeptide. Mass: Obsd. 4060.9Æ0.2 Da, calcd 4060.8 Da
(av. isotope composition). d) ShK polypeptide. Mass: Obsd.
4060.9Æ0.2 Da, calcd 4060.8 Da (av. isotope composition). The MS
data shown [insets] were collected across the entire UV-absorbing
main peak in each chromatogram.
Authentication of the identity and stereochemistry of the
protected l-allo-Ile and l-allo-Thr amino acids used is
described in the Supporting Information. All of the ShK
polypeptide chains were synthesized from two synthetic
peptide segments by following the method that was used for
ShK toxin (Scheme 3).[4] First, we chemically synthesized
peptides [allo-Ile7]Arg1–Gln16-COSR, [allo-Thr13]Arg1–
Gln16-COSR, and [allo-Thr31]Cys17–Cys35 using highly
optimized Boc chemistry “in situ neutralization” solid phase
peptide synthesis (SPPS)[10] The two appropriate peptide
segments were then covalently condensed by native chemical
ligation[11] at the -Gln16–Cys17- site.
Once we had obtained the three allo-amino acid contain-
ing ShK analogues, we carried out separate folding reactions
for each synthetic polypeptide under the conditions that were
used to fold wild-type ShK toxin: 50 mm AcONH4, pH 8.0,
[polypeptide] 0.4 mgmLÀ1, air oxidation (without stirring).
Each of the four folding reactions was monitored at chosen
time points by HPLC analysis. Data for the folding reactions
at time points of 3 hours and 18 hours are shown in Figure 3.
Folded [allo-Ile7]ShK protein, which has mass 6 Da less
than the [allo-Ile7]ShK polypeptide, corresponding to the
formation of three disulfide bonds, was observed to form most
rapidly (ca. 30 minutes), followed by wild-type l-ShK (ca.
1 hour), [allo-Thr13]ShK (ca. 2 hours), and [allo-Thr31]ShK
(ca. 3 hours). For preparative-scale folding reactions, the yield
The resulting synthetic polypeptides were purified by
HPLC on a C4 reverse-phase semi-prep column; LCMS data
for the purified allo-amino acid containing polypeptide chains
are shown in Figure 2.
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we hypothesized that less stable analogues were partially
denatured and thus had later retention times, and with the
rates of formation of the folded protein molecules in our
analytical folding studies, where the most stable protein
folded first and gave the highest yields.
Next, we set out to determine the crystal structures of the
synthetic ShK protein diastereomers. We have reported the
use of quasi-racemic protein mixtures to facilitate protein
crystallization.[12,13] Since we already had the d-ShK protein
from previous studies,[4] we attempted to crystallize the three
new ShK analogues by using quasi-racemic protein crystal-
lization under the seven conditions that produced true
racemate ShK protein crystals. We performed crystallization
trials under identical conditions using both a quasi-racemic
protein mixture and conventional l-protein alone.
For [allo-Ile7]ShK, none of the conditions that we
screened produced any crystals after one week. At that
point, when we checked for crystals, we accidentally spilled
well solution into the quasi-racemic mixture hanging drop in
one condition, and this one condition produced crystals after
another week. Later, we tried to mimic this adventitious
condition by mixing 1.6 mL well solution and 0.8 mL protein
solution to form the hanging drop, and this method indeed
produced crystals after two weeks. In the case of [allo-
Thr13]ShK, two conditions produced crystals overnight from
the quasi-racemic mixture. For [allo-Thr31]ShK, six of the
seven conditions (both quasi-racemic mixture and l-protein
alone) produced needle-shaped crystals overnight. Condi-
tions used to produce diffraction-quality crystals for each
analogue are given in the Supporting Information. Diffraction
resolution and space group data are summarized in Table 1.
Figure 3. Folding the three allo-amino acid containing ShK analogues
and wild-type ShK. The folding reactions were monitored by LCMS
(MS data not shown). Analytical HPLC data are shown for each folding
reaction at time points 3 hours (left panel) and 18 hours (right panel).
Folding conditions: 50 mm AcONH4, pH 8.0, [polypeptide]
0.4 mgmLÀ1, air oxidation (without stirring). * indicates the folded
protein products.
of each folded protein product was calculated based on HPLC
analysis (peak integrations). The folded product yields were:
[allo-Ile7]ShK 67%, ShK 61%, [allo-Thr13]ShK 51% and
[allo-Thr31]ShK 40%.
In the HPLC analysis of these folded products (Figure 3),
we observed that under identical reverse-phase analytical
HPLC conditions, the folded protein molecules [allo-
Ile7]ShK and ShK eluted at approximately the same time,
while [allo-Thr13]ShK eluted later, and [allo-Thr31]ShK
eluted later still. Our structural studies (see below) indicated
that the crystal structures of these four protein molecules
were essentially the same. The reason for the difference in
retention times might be that the folded products have
different stability properties under the denaturing reverse-
phase HPLC conditions: if the less stable ShK analogues were
partially denatured so that more hydrophobic surfaces were
exposed, it would lead to the later retention times observed
on HPLC analysis.
To study the thermal stability of these ShK protein
analogues, we performed CD experiments to measure the
thermal melting temperature. Each purified ShK analogue
(see the Supporting Information) was dissolved in 10 mm PBS
buffer at pH 7.4, at a protein concentration of 0.3 mgmLÀ1.
Peak absorptions were measured at a wavelength of 220 nm.
After fitting the experiment data with a sigmoid function, the
approximate thermal melting temperatures were: wild type
ShK ca. 1208C; [allo-Ile7]ShK ca. 1208C; [allo-Thr13]ShK
ca. 1008C; and, [allo-Thr31]ShK ca. 808C (see the Supporting
Information).
Table 1: X-ray diffraction data for the allo-amino acid ShK analogues.
Resolution Space group Molecules in
[ꢀ]
asymmetric unit
[allo-Ile7]ShK/d-ShK
1.20
C2
P1
1 [allo-Ile7]ShK
and 1 d-ShK
1 [allo-Thr13]ShK
and 1 d-ShK
[allo-Thr13]ShK/d-ShK 0.90
[allo-Thr31]ShK/d-ShK 1.30
P21
P21
3 [allo-Thr31]ShK
3 [allo-Thr31]ShK
[allo-Thr31]ShK
1.56
The X-ray structure of quasi-racemic crystalline l-[allo-
Ile7]ShK/d-ShK was solved by molecular replacement[14]
using the true racemate l/d-ShK (PDB ID: 4LFS) as
a search model. The asymmetric unit contained one l-[allo-
Ile7]ShK protein and one d-ShK protein. The final model was
refined to a crystallographic R-factor of 0.14 (R-free 0.20)
using CCP4.[15] The X-ray structure of quasi-racemic crystal-
line l-[allo-Thr13]ShK/d-ShK was solved by molecular
replacement[14] using PDB ID: 4LFS as a search model. The
asymmetric unit contained one l-[allo-Thr13]ShK protein
and one d-ShK protein. The final model was refined to
a crystallographic R-factor of 0.11 (R-free 0.13) using
CCP4.[15]
For [allo-Thr31]ShK, the fact that all of the crystals have
three molecules in each asymmetric unit, and all of the
crystals (from both quasi-racemic crystallization and l-
This order of ShK protein thermal stabilities matches our
observations on the retention times in HPLC analysis, where
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protein crystallization) obtained have the same needle shape,
while racemic ShK protein and the other quasi-racemic ShK
proteins produced cube-shaped crystals, thus suggested that
the crystals from both the quasi-racemic mixture and the l-
protein alone might be the same and that they might only
contain [allo-Thr31]ShK. The protein structures in both
crystals were solved by molecular replacement[14] using l-
ShK (PDB ID: 4LFQ) as the search model. Indeed, neither of
the [allo-Thr31]ShK crystals contained the d-ShK molecule;
that is, the quasi-racemic mixture gave crystals of the l-[allo-
Thr31]ShK protein, not quasi-racemate crystals. The final
model of the l-[allo-Thr31]ShK protein was refined to
a crystallographic R-factor of 0.14 (R-free 0.20) using
CCP4.[15]
ing ShK polypeptide analogues and to then perform folding
and stability studies. The structures of the synthetic proteins
were determined by X-ray crystallography; each of the three
allo-amino acid ShK analogues proteins had essentially the
same folded structure as wild-type ShK protein. The three
allo-amino acid ShK analogues all retained Kv1.3 ion channel
blocking activity.
The three allo-Thr/allo-Ile-containing ShK polypeptides
have distinct folding properties, and the folded protein
products have different stabilities. Indeed, the experimental
folding and thermal stability data we obtained were in good
agreement with the computational data. These results illus-
trate the utility of combining computational calculations of
protein properties and total protein synthesis enabled by
modern chemical ligation methods for the systematic inves-
tigation of the molecular basis of protein structure and
function.
The structures of the three allo-amino acid containing
ShK protein analogues are all very similar to wild-type ShK
protein. The structure comparisons are shown in Figure 4.
Acknowledgements
This research was supported in part by funds from NIH
Grants U54 GM087519, and R01-GM030376 to F. Bezanilla.
Use of NE-CAT beamline 24-ID-C at the Advanced Photon
Source is supported by the National Institute of General
Medical Sciences of the National Institutes of Health (P41
GM103403). The Pilatus 6M detector on 24-ID-C beam line is
funded by a NIH-ORIP HEI grant (S10 RR029205). Use of
the Advanced Photon Source is supported by the U.S.
Department of Energy, Office of Basic Energy Sciences,
under Contract No. DE-AC02-06CH11357.
Figure 4. Comparison of the crystal structures of l-ShK protein and the
allo-amino acid containing analogues. Left: structure alignment of l-
ShK (green) and [allo-Ile7]ShK (cyan) with RMSD 0.38 ꢀ. Center:
structure alignment of l-ShK (green) and [allo-Thr13]ShK (magenta)
with RMSD 0.34 ꢀ. Right: structure alignment of l-ShK (green) and
[allo-Thr31]ShK (yellow) with RMSD 0.76 ꢀ.
Crystal structure data for [allo-Ile7]ShK, [allo-Thr13]ShK,
and [allo-Thr31]ShK have been deposited in the Protein Data
Bank with PDB IDs 5I5A, 5I5B, and 5I5C, respectively.
To examine the channel-blocking abilities of allo-ShK
analogues, we used the cut-open oocyte voltage clamp
method to measure potassium ionic currents from Xenopus
laevis oocytes expressing human Kv1.3 (hKv1.3) ion channels,
before and after addition of the toxin. All of the allo-ShK
analogues retain Kv1.3-blocking activity. However, their
activities are 4–6-times lower than wild type ShK toxin, with
no significant difference between the analogues (Table 2).
In conclusion, we have explored the impact of threonine
and isoleucine side-chain chirality on the folding and struc-
tural stability of the ShK protein. Free energy perturbation
molecule dynamics (FEP/MD) simulations helped us to
estimate the thermodynamic cost of inverting the stereo-
chemistry of individual Thr/Ile side chains. Chemical protein
synthesis enabled us to prepare the allo-Thr/allo-Ile-contain-
Conflict of interest
The authors declare no conflict of interest.
Keywords: allo-isoleucine · allo-threonine ·
protein crystallography · protein synthesis · stereochemistry
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Wild type
140Æ30
1.1Æ0.1
0.7Æ0.2
0.8Æ0.1
0.8Æ0.1
[Allo-Ile7]ShK
[Allo-Thr13]ShK
[Allo-Thr31]ShK
440Æ220
540Æ200
860Æ260
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Manuscript received: December 21, 2016
Final Article published: && &&, &&&&
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Communications
Protein Chemistry
B. Dang,* R. Shen, T. Kubota, K. Mandal,
F. Bezanilla, B. Roux,
S. B. H. Kent
&&&& — &&&&
Inversion of the Side-Chain
Stereochemistry of Indvidual Thr or Ile
Residues in a Protein Molecule: Impact
on the Folding, Stability, and Structure of
the ShK Toxin
Ch-ch-ch-changes: Guided by molecular
dynamics calculations, chemical protein
synthesis was used to explore the impact
of allo-Thr and allo-Ile substitutions for
individual Thr and Ile residues on the
folding, crystal structure, and stability of
the ShK protein. The experimental folding
and thermal stability data matched well
with the computational results.
6
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