X-ray Crystallographic Structure of a Teixobactin Derivative Reveals Amyloid-like Assembly

Article abstract of DOI:10.1021/jacs.8b07709

This paper describes the X-ray crystallographic structure of a derivative of the antibiotic teixobactin and shows that its supramolecular assembly through the formation of antiparallel β-sheets creates binding sites for oxyanions. An active derivative of

Full text of DOI:10.1021/jacs.8b07709

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Communication
X-ray Crystallographic Structure of a Teixobactin
Derivative Reveals Amyloid-Like Assembly
Hyunjun Yang, Micha# Wierzbicki, Derek R Du Bois, and James S. Nowick
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b07709 • Publication Date (Web): 08 Oct 2018
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X-ray Crystallographic Structure of a Teixobactin Derivative
Reveals Amyloid-Like Assembly
Hyunjun Yang, Michał Wierzbicki, Derek R. Du Bois, and James S. Nowick*
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Department of Chemistry, University of California, Irvine,
Irvine, California 92697-2025, United States
Abstract
This paper describes the X-ray crystallographic structure of a derivative of the antibiotic
teixobactin and shows that its supramolecular assembly through the formation of antiparallel β-
sheets creates binding sites for oxyanions. An active derivative of teixobactin containing lysine
in place of allo-enduracididine assembles to form amyloid-like fibrils, which are observed
through a thioflavin T fluorescence assay and by transmission electron microscopy. A
homologue, bearing an N-methyl substituent, to attenuate fibril formation, and an iodine atom, to
facilitate X-ray crystallographic phase determination, crystallizes as double helices of β-sheets
that bind sulfate anions. β-Sheet dimers are key subunits of these assemblies, with the N-terminal
methylammonium group of one monomer and the C-terminal macrocycle of the other monomer
binding each anion. These observations suggest a working model for the mechanism of action of
teixobactin, in which the antibiotic assembles and the assemblies bind lipid II and related
bacterial cell wall precursors on the surface of Gram-positive bacteria.
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The peptide antibiotic teixobactin has been the subject of intensive research efforts for its
promise of addressing antibiotic-resistant Gram-positive pathogens such as MRSA and VRE
1
,2,3,4,5,6,7,8,9
(Figure 1).
Teixobactin is thought to bind highly conserved prenyl-pyrophosphate-
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saccharide regions of lipid II and related membrane-bound cell wall precursors. Here we
describe the first X-ray crystallographic structure of a full-length teixobactin analogue, which
reveals an amphipathic amyloid-like assembly that acts as a multivalent receptor for sulfate
anions. This crystallographic structure suggests a working model for the mechanism of action of
teixobactin in which teixobactin forms fibrils or smaller assemblies that bind to the
pyrophosphate groups of lipid II and related cell wall precursors on the bacterial cell membrane
and thus disrupt cell wall biosynthesis. These findings should be of value both in understanding
the mechanism of action of teixobactin and in rationally designing new antibiotics that target
lipid II and related cell wall precursors.
teixobactin (1)
Ile11
O
NH
2
O
O
O
O
O
O
H
N
H
N
H
H
N
H
N
O
HN
N
N
N
N
N
NH
H
H
H
H
O
O
4
O
NH HN
N
H
OH
OH
O
O
N-Me-
D
-Phe
1
Ile
2
Ser
3
D-Gln
D
-allo-Ile
5
Ile
6
Ser
7
D-Thr
8
Ala
9
allo-End10
Lys10-teixobactin (2)
O
NH
2
O
O
O
O
O
H
N
H
H
H
N
O
O
HN
N
N
N
H
N
H
N
N
H
NH
2
H
O
O
O
NH HN
O
OH
OH
O
N-Me-
D
-PheI
1
,N-Me-
D
4
-Gln ,Lys10-teixobactin (3)
O
NH
2
O
O
O
O
O
H
N
H
H
N
H
O
O
HN
N
N
N
N
N
N
H
NH
2
H
H
O
Me
O
O
NH HN
O
OH
OH
O
I
I
Figure 1. Teixobactin (1), Lys10-teixobactin (2), and N-Me-D-Phe
teixobactin (3).
1
,N-Me-D-Gln ,Lys10-
4
2
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While studying structure-activity relationships among teixobactin analogues, we have
observed that teixobactin and analogues with good antibiotic activity (low MIC values) form gels,
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while analogues with poor activity (high MIC values) do not. For example, Lys10-teixobactin
2), a homologue of teixobactin in which allo-enduracididine at position 10 is replaced with
lysine (Figure 1), has an MIC of 0.5-1.0 µg/mL against S. aureus and forms a gel in PBS buffer,
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(
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while D-Ala ,Lys10-teixobactin (MIC≥16 µg/mL) does not. This observation suggested that
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supramolecular assembly of teixobactin analogues could be involved in antibiotic activity.
We began exploring the supramolecular assembly of teixobactin and its analogues by
performing thioflavin T (ThT) fluorescence assays and transmission electron microscopy (TEM)
studies upon Lys10-teixobactin. When we incubated Lys10-teixobactin with PBS buffer and ThT
and monitored fluorescence, we observed a lag phase of ca. 1 day, followed by an increase in
1
1
fluorescence (Figure 2A). This behavior is a hallmark of amyloidogenic peptides and proteins.
To further explore the assemblies that formed, we performed TEM studies. TEM images of the
aggregated Lys10-teixobactin revealed amyloid-like fibrils (Figure 2B). The fibrils range from
individual or paired filaments, ca. 8 nm across, through bundles of filaments ca. 100-200 nm in
diameter.
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Figure 2. (A) ThT fluorescence assay of Lys10-teixobactin (2, four replicate runs with 120 µM
peptide in PBS buffer at pH 7.4). (B) TEM images of the fibrils formed by Lys10-teixobactin (2).
To further study teixobactin supramolecular assembly, we turned to X-ray
crystallography. Although we had successfully crystallized a truncated teixobactin analogue
containing only residues 6–11, all efforts to crystallize full-length teixobactin analogues failed,
1
2
giving only amorphous aggregates. We postulated that N-methylation of the peptide backbone
13,14
would attenuate the aggregation and permit the growth of crystals.
We discovered that N-
methylation of D-Gln
4
indeed facilitated crystallization. We also incorporated an iodine atom in
I
N-Me-D-Phe
1
to give N-methyl-p-iodo-D-phenylalanine (N-Me-D-Phe
1
), to permit determination
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15,16
of the X-ray crystallographic phases.
Figure 1 illustrates the structure of the resulting
teixobactin analogue 3, a homologue of Lys10-teixobactin (2). Teixobactin analogue 3 does not
form a gel and exhibits only modest activity against S. aureus (MIC=16 µg/mL).ꢀ
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We began our crystallization efforts by screening teixobactin analogue 3 in 864
conditions in a 96-well plate format using crystallization kits from Hampton Research (PEG/Ion,
Index, and Crystal Screen). Rectangular rod-shaped crystals grew in conditions containing
sulfate salts (Li
2
SO
4
, MgSO
4
, Na
2
SO
4
, K
2
SO
4
, (NH
4
)
2
SO
4
) and polyethylene glycol (PEG) 3,350.
SO and 15% PEG 3,350
With further optimization in a 24-well plate format, 0.19 M Na
2
4
afforded crystals suitable for X-ray diffraction. Four X-ray diffraction datasets were acquired at
the Stanford Synchrotron Radiation Lightsource (SSRL) at a wavelength of 2.07 Å. The datasets
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18
were processed using XDS and merged using BLEND . The structure was solved by single-
wavelength anomalous diffraction (SAD) phasing using the iodine anomalous signal from N-Me-
I
19
D-Phe
1
. The structure was refined with REFMAC5 in the P2
1
2
1
1
2 space group at 2.20 Å
resolution. The asymmetric unit contains 32 crystallographically independent teixobactin
analogue molecules, as well as 32 sulfate anions and 53 ordered water molecules.
The 32 molecules of teixobactin analogue 3 form a double helix of β-sheet fibrils in
which each fibril is composed of 16 peptide molecules. Each fibril may be thought of as
comprising hydrogen-bonded dimers. Figure 3 illustrates the structure of a representative
hydrogen-bonded dimer. In the dimer, two molecules of teixobactin analogue 3 come together to
form an antiparallel β-sheet in which Ile
2
hydrogen bonds with Ile
6
, N-Me-D-Gln pairs with N-
4
Me-D-Gln , and Ile hydrogen bonds with Ile
4
6
2
. The N-methyl groups of the two N-Me-D-Gln
4
residues tilt upward, allowing the β-sheet to form in spite of the disruption of the hydrogen-
bonding pattern. As a result, the β-sheet has four hydrogen bonds instead of six hydrogen bonds.
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Figure 3. X-ray crystallographic structure of a representative dimer of N-Me-D-Phe ,N-Me-D-
1
Gln ,Lys10-teixobactin (3). (A) Top view. (B) Side view.
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In the X-ray crystallographic structure, the dimer acts as a receptor for two sulfate anions.
The amide NH groups of the macrocyclic ring of each monomer subunit act in conjunction with
the N-terminus of the other monomer subunit to bind each sulfate anion. Each sulfate anion
hydrogen bonds to the amide NH groups of D-Thr
8
, Ala
9
, Lys10, and Ile11 of one monomer
I
subunit and the methylammonium group of the N-Me-D-Phe
1
of the other subunit. The β-sheet
, D-allo-Ile , and Ile create a
, and Ser , as well as the N-
I
dimer is amphipathic: the side chains of N-Me-D-Phe
hydrophobic surface, and the side chains of Ser
1
, Ile
2
5
6
3
, N-Me-D-Gln
4
7
terminal methylammonium group, create a hydrophilic surface. The macrocyclic rings and the
sulfate anions lie above the hydrophilic surface.
Sixteen molecules of teixobactin analogue 3 assemble to form each β-sheet fibril (Figure
4
). The molecules assemble in an antiparallel fashion to form an extended amphiphilic β-sheet,
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with the hydrophobic residues on one face and hydrophilic residues on the other face. At each β-
sheet interface between the dimers, Ser
3
hydrogen bonds with Ser
7
, D-allo-Ile hydrogen bonds
5
with D-allo-Ile , and Ser hydrogen bonds with Ser
5
7
3
. Each dimer interface is thus shifted by two
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residues, which results in an offset fibril structure. (In an aligned fibril structure, N-Me-D-Phe
1
would hydrogen bond with Ser
7
, Ser
3
would hydrogen bond with D-allo-Ile
5
, D-allo-Ile would
5
I
hydrogen bond with Ser , and Ser
3
7
would hydrogen bond with N-Me-D-Phe
1
.)
I
Figure 4. β-Sheet fibril formed by N-Me-D-Phe
1
,N-Me-D-Gln ,Lys10-teixobactin (3). (A) Top
4
view. (B) Bottom view with hydrophobic side chains shown as spheres.ꢀ
Two β-sheet fibrils wrap around each other to form a right-handed double helix of β-
sheets, with the hydrophobic surfaces in the interior and the hydrophilic surfaces on the exterior
(Figure 5). Each double helix contains 32 molecules of teixobactin analogue 3 and corresponds
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to the asymmetric unit. The double helices are discrete structures in the crystal lattice and are not
part of extended superstructures. The double helix is ca. 9 nm in length and ca. 4 nm in diameter
in the middle, tapering to ca. 2 nm at the two ends. The ends of the double helix are closed, but
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the middle has a central cavity of ca. 1 nm in diameter and ca. 5 nm in length that is surrounded
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by the hydrophobic side chains of N-Me-D-Phe
1
, Ile
2
, D-allo-Ile
5
, and Ile (Figure S5). The
6
ordered water molecules surround the hydrophilic exterior of the double helix.
I
Figure 5. Double helix of β-sheet fibrils formed by N-Me-D-Phe
teixobactin (3). Sulfate anions are shown as spheres.
1
,N-Me-D-Gln ,Lys10-
4
The X-ray crystallographic structure of the discrete double helix of β-sheets formed by
teixobactin analogue 3 suggests a molecular model for the assembly of teixobactin analogue 2
into the filaments and fibrils observed by TEM (Figure 2). In this model, teixobactin analogue 2
assembles to form extended networks of β-sheet fibrils, which wrap around each other to form
extended double helices of β-sheets. Figure 6 illustrates this model. Unlike the discrete structures
formed by N-methylated analogue 3, these double helices persist for many hundreds of
nanometers and contain thousands of molecules. These fibrils further wrap or bundle together to
form the fibrils and bundles observed by TEM. Although the N-methyl group in teixobactin
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analogue 3 does not prevent β-sheet formation, it impedes the formation of extended fibrils by
reducing the stability of the β-sheets that form.
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Figure 6. Crystallographically based molecular model of an extended double helix of β-sheet
fibrils formed by teixobactin analogue 2 and observed by TEM (Figure 2).
The amphipathic assembly formed by teixobactin analogue 3 explains many of the
10,21,22
previously reported structure-activity relationships in teixobactin analogues.
Our laboratory
has previously reported that substituting residues 1, 2, 5, 6, and 7 with L- or D-alanine
dramatically reduces or eliminates the antibiotic activity of Lys10-teixobactin, while substituting
1
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residues 3 and 4 with L- or D-alanine has much smaller effects upon activity. Similar effects
2
1
have been observed upon replacement of residues 2–7 with L- or D-lysine. The densely packed
hydrophobic surface formed by residues 1, 2, 5, and 6 on the interior of the double helix of β-
sheet fibrils (Figure 4B) explains why mutating any of these bulky hydrophobic residues to L- or
D- alanine or lysine disrupts supramolecular assembly and causes loss of activity. The
hydrophilic side chains of residues 3 and 4 are on the hydrophilic exterior of the double helix of
β-sheet fibrils (Figure 4A) and are substantially more tolerant of substitution. The hydrophilic
side chain of residue 7 is also on the hydrophilic exterior of the double helix of β-sheet fibrils,
however the X-ray crystallographic structure does not appear to explain the loss of activity upon
mutating this residue to Ala or Lys. Additional studies have reported that substituting L-amino
acids for D-amino acids at residues 1, 4, and 5 in Arg10-teixobactin also dramatically reduces or
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eliminates antibiotic activity. Each of these stereochemical mutations disrupts the amphipathic
β-sheet formed by residues 1–7 and causes loss of antibiotic activity.
The X-ray crystallographic structure of teixobactin analogue 3, in conjunction with the
observation that Lys10-teixobactin (2) forms amyloid-like fibrils, suggest that supramolecular
assembly may be involved in the antibiotic activity of teixobactin. We thus propose a working
model for the antibiotic activity of teixobactin in which teixobactin forms dimers, higher-order
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assemblies, or fibrils through antiparallel β-sheet interactions. The dimers or dimer subunits
create binding sites for the pyrophosphate groups of lipid II and related membrane-bound cell
wall precursors, perhaps adhering strongly to the surface through contacts with multiple lipid
2
4
molecules. In the binding site, the amide NH groups of residues 8–11 of one teixobactin
molecule in the dimer and the N-terminus of the other teixobactin molecule interact with each
bound pyrophosphate group. In teixobactin (1), the guanidinium group of allo-End10 may make
additional contacts to the pyrophosphate group.
This model shares a number of features in common with those observed for other
25,26
antibiotics that target lipid II and related cell wall precursors, including ramoplanin and nisin.
Ramoplanin forms fibrils with lipid II analogues, and supramolecular assembly through the
formation of antiparallel β-sheet dimers is thought to be important in its mechanism of
27,28,29,30,31
action.
Nisin binds the pyrophosphate group of lipid II by means of a pyrophosphate
3
2,33
cage formed by amide NH groups in and adjacent to the 16-membered lanthionine A ring.
The unique pattern of hydrophobicity and stereochemistry of residues 1–7 of teixobactin
makes fibril formation possible. By having evolved a D-L-L-D-D-L-L pattern of stereochemistry
with
a
hydrophobic-hydrophobic-hydrophillic-hydrophilic-hydrophobic-hydrophobic-
hydrophilic pattern of side chains, Eleftheria terrae has achieved an amyloidogenic non-
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ribosomal peptide that can assemble to form amphiphilic β-sheets and amyloid-like fibrils that
can bind oxyanions. On the basis of our crystal structure, we have proposed a working model for
the mechanism of action of teixobactin involving the formation of β-sheet dimers or higher-order
supramolecular assemblies. We further recognize that the crystallographic observation of
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34,35
36,37
supramolecular assembly
and its potential involvement in antibiotic activity
does not
3
8,39
assure its biological relevance.
We envision the model put forth here to be worthy of further
study and anticipate reporting these studies in due course.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at
DOI: .
I
Procedures for the synthesis of N-Me-D-Phe
1
,N-Me-D-Gln ,Lys10-teixobactin (3), MIC
4
assays, solubility assays, ThT fluorescence assays, and TEM imaging; characterization data
(HPLC, ESI-MS) for analogue 3; details of X-ray crystallographic data collection, processing,
and refinement.
Crystallographic coordinates of analogue 3 were deposited into the Protein Data Bank
with PDB code 6E00 (data collected on a synchrotron source at 2.07 Å wavelength).
AUTHOR INFORMATION
Corresponding Author
*
jsnowick@uci.edu
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGEMENTS
We thank Drs. Nicholas Chim and Huiying Li for helpful advice with X-ray
crystallography and Dr. Li Xing for helpful assistance with TEM imaging. This work was
supported by the National Institutes of Health (grants 1R21AI121548 and 1R56AI137258). H. Y.
acknowledges the Allegan for fellowship support. M. W. acknowledges the support from the
Ministry of Science and Higher Education, Republic of Poland (Mobility Plus grant no.
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647/MOB/V/2017/0). Use of the Stanford Synchrotron Radiation Lightsource, SLAC National
Accelerator Laboratory is supported by the US Department of Energy, Office of Science, Office
of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural
Molecular Biology Program is supported by the DOE Office of Biological and Environmental
Research,
and
by
the
NIH,
NIGMS
(including
P41GM103393).
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REFERENCES
1
Ling, L. L.; Schneider, T.; Peoples, A. J.; Spoering, A. L.; Engels, I.; Conlon, B. P.; Mueller,
A.; Schäberle, T. F.; Hughes, D. E.; Epstein, S.; Jones, M.; Lazarides, L.; Steadman, V.
A.; Cohen, D. R.; Felix, C. R.; Fetterman, K. A.; Millett, W. P.; Nitti, A. G.; Zullo, A.
M.; Chen, C.; Lewis, K. A new antibiotic kills pathogens without detectable resistance.
Nature 2015, 517, 455–459.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
4
Homma, T.; Nuxoll, A.; Gandt, A. B.; Ebner, P.; Engels, I.; Schneider, T.; Götz, F.; Lewis,
K.; Conlon, B. P. Dual Targeting of Cell Wall Precursors by Teixobactin Leads to Cell
Lysis. Antimicrob. Agents Chemother. 2016, 60, 6510–6517.
Zong, Y.; Sun, X.; Gao, H.; Meyer, K. J.; Lewis, K.; Rao, Y. Developing Equipotent
Teixobactin Analogues against Drug-Resistant Bacteria and Discovering a Hydrophobic
Interaction between Lipid II and Teixobactin. J. Med. Chem. 2018, 61, 3409–3421.
Jin, K.; Sam, I. H.; Po, K. H. L.; Lin, D.; Ghazvini Zadeh, E. H.; Chen, S.; Yuan, Y.; Li, X.
Total synthesis of teixobactin. Nat. Commun. 2016, 7, 12394.
5 Parmar, A.; Lakshminarayanan, R.; Iyer, A.; Mayandi, V.; Leng Goh, E. T.; Lloyd, D. G.;
Chalasani, M. L. S.; Verma, N. K.; Prior, S. H.; Beuerman, R. W.; Madder, A.; Taylor, E.
J.; Singh, I. Design and Syntheses of Highly Potent Teixobactin Analogues against
Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus (MRSA), and
Vancomycin-Resistant Enterococci (VRE) in Vitro and in Vivo. J. Med. Chem. 2018, 61,
2
009–2017.
6 Abdel Monaim, S. A. H.; Jad, Y. E.; El-Faham, A.; de la Torre, B. G.; Albericio, F.
Teixobactin as a scaffold for unlimited new antimicrobial peptides: SAR study.
ꢀB ioorganic Med. Chem. 2018, 26, 2788–2796.
7
Yang, H.; Chen, K. H.; Nowick, J. S. Elucidation of the Teixobactin Pharmacophore. ACS
Chem. Biol. 2016, 11, 1823–1826.
8 Kåhrström, C. T. Antimicrobials: A new drug for resistant bugs. Nat. Rev. Microbiol. 2015,
3, 126–127.
1
9
Wen, P.; Vanegas, J. M.; Rempe, S. B.; Tajkhorshid. E. Probing Key Elements of
Teixobactin-Lipid II Interactions in Membrane. Chem. Sci. 2018, 9, 6997–7008.
10 Chen, K. H.; Le, S. P.; Han, X.; Frais, J. M.; Nowick, J. S. Alanine scan reveals modifiable
residues in teixobactin. Chem. Commun. 2017, 53, 11357–11359.
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1
1 Upon further incubation, the fluorescence declines variably. This subsequent change in
fluorescence may reflect further reorganization of the amyloid-like fibrils that form, such
as assembly into the bundles of filaments that are observed by TEM.
12 Yang, H.; Du Bois, D. R.; Ziller, J. W.; Nowick, J. S. X-ray crystallographic structure of a
teixobactin analogue reveals key interactions of the teixobactin pharmacophore. Chem.
Commun. 2017, 53, 2772–2775.
0
1
2
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5
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7
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6
7
8
9
0
1
2
3
4
5
6
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0
1
3 Spencer, R.; Li, H.; Nowick, J. S. X-ray Crystallographic Structures of Trimers and Higher-
Order Oligomeric Assemblies of a Peptide Derived from Aβ17–36. J. Am. Chem. Soc.
2014, 136, 5595–5598.
1
4 Spencer, R. K.; Kreutzer, A. G.; Salveson, P. J.; Li, H.; Nowick, J. S. X-ray Crystallographic
Structures of Oligomers of Peptides Derived from β2-Microglobulin. J. Am. Chem. Soc.
2
015, 137, 6304–6311.
15 Richardson, M. B.; Brown, D. B.; Vasquez, C. A.; Ziller, J. W.; Johnston, K. M.; Weiss, G.
A. Synthesis and Explosion Hazards of 4-Azido-l-phenylalanine. J. Org. Chem. 2018, 83,
4
525–4536.
1
6 Malkov, A. V.; Stončius, S.; MacDougall, K, N.; Mariani, A.; McGeoch, G. D.; Kočovský,
P. Formamides derived from N-methyl amino acids serve as new chiral organocatalysts
in the enantioselective reduction of aromatic ketimines with trichlorosilane. Tetrahedron
2006, 62, 264–284.
1
7 Kabsch, W. XDS. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 125−132.
1
8 Foadi, J.; Aller, P.; Alguel, Y.; Cameron, A.; Axford, D.; Owen, R. L.; Armour, W.;
Waterman, D. G.; Iwata, S.; Evans, G. Clustering procedures for the optimal selection of
data sets from multiple crystals in macromolecular crystallography. Acta Crystallogr.,
Sect. D: Biol. Crystallogr. 2013, 69, 1617−1632.
1
9 Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of macromolecular structures by
the maximum-likelihood method. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53,
2
40−255.
20 Sangwan, S.; Zhao, A.; Adams, K. L.; Jayson, C. K.; Sawaya, M. R.; Guenther, E. L.; Pan,
A. C.; Ngo, J.; Moore, D. M.; Soriaga, A. B.; Do, T. D.; Goldschmidt, L.; Nelson, R.;
Bowers, M. T.; Koehler, C. M.; Shaw, D. E.; Novitch, B. G.; Eisenberg, D. S. Atomic
structure of a toxic, oligomeric segment of SOD1 linked to amyotrophic lateral sclerosis
(ALS). Proc. Natl. Acad. Sci. U.S.A. 2017, 114, 8770−8775.
2
1 Abdel Monaim, S. A. H.; Jad, Y. E.; Ramchuran, E. J.; El-Faham, A.; Govender, T.; Kruger,
H. G.; de la Torre, B. G.; Albericio, F. Lysine Scanning of Arg10−Teixobactin:
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Deciphering the Role of Hydrophobic and Hydrophilic Residues. ACS Omega 2016, 1,
1
262−1265.
22 Parmar, A.; Prior, S. H.; Iyer, A.; Vincent, C. S.; Van Lysebetten, D.; Breukink, E.; Madder,
A.; Taylor, E. J.; Singh, I. Defining the molecular structure of teixobactin analogues and
understanding their role in antibacterial activities. Chem. Commun. 2017, 53, 2016−2019.
1
1
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3 Lewandowski et al. have concurrently reported NMR-based structural studies of teixobactin
in aqueous and membranelike environments, both with and without lipid II and lipid II
analogues. [Öster, C.; Walkowiak, G. P.; Hughes, D. E.; Spoering, A. L.; Peoples, A. J.;
Catherwood, A. C.; Tod, J. A.; Lloyd, A. J.; Herrmann, T.; Lewis, K.; Dowson, C.;
Lewandowski, J. R. Structural studies suggest aggregation as one of the modes of action
for teixobactin. Chem. Sci. 2018, Accepted Manuscript (DOI: 10.1039/C8SC03655A)]
These studies indicate that teixobactin, in the presence of lipid II, rapidly aggregates and
the residues 2–6 rearrange from random coil like conformation to a more extended β-
strand like conformation.
24 A 2:1 teixobactin:lipid II binding stoichiometry was reported in the original 2015 Nature
paper on teixobactin (reference 1). The crystallographic observation of putative
pyrophosphate binding sites created by dimers could potentially support either a 2:1 or a
2
:2 teixobactin:lipid II stoichiometry, depending on how the binding of one molecule of
lipid II to the dimer affects the accessibility of the other site of the dimer.
25 Breukink, E.; de Kruijff, B. Lipid II as a target for antibiotics. Nat. Rev. Drug Discov. 2006,
5, 321–332.
2
6 de Kruijff, B.; van Dam, V.; Breukink, E. Lipid II: a central component in bacterial cell wall
synthesis and a target for antibiotics. Prostaglandins Leukot. Essent. Fatty Acids 2008, 79,
1
17–121.
27 Lo, M. C.; Men, H.; Branstrom, A.; Helm, J.; Yao, N.; Goldman, R.; Walker, S. A new
mechanism of action proposed for ramoplanin. J. Am. Chem. Soc. 2000, 122, 3540−3541.
2
8 Lo, M. C.; Helm, J. S.; Sarngadharan, G.; Pelczer, I.; Walker, S. A new structure for the
substrate-binding antibiotic ramoplanin. J. Am. Chem. Soc. 2001, 123, 8640−8641.
29 Hu, Y.; Helm, J. S.; Chen, L.; Ye, X.-Y.; Walker, S. Ramoplanin inhibits bacterial
transglycosylases by binding as a dimer to lipid II. J. Am. Chem. Soc. 2003, 125,
8
736−8737.
3
0 Walker, S.; Chen, L.; Hu, Y.; Rew, Y.; Shin, D.; Boger, D. L. Chemistry and biology of
ramoplanin: a lipoglycodepsipeptide with potent antibiotic activity. Chem. Rev. 2005, 105,
4
49−476.
ꢀ
ꢀ
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1
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5
5
6
ꢀ
3
1 Hamburger, J. B.; Hoertz, A. J.; Lee, A.; Senturia, R. J.; McCafferty, D. G.; Loll, P. J.; A
crystal structure of a dimer of the antibiotic ramoplanin illustrates membrane positioning
and a potential Lipid II docking interface. Proc. Natl. Acad. Sci. U.S.A. 2009, 106,
13759−13764.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
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2
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8
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0
1
2
3
4
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6
7
8
9
0
3
2 Hsu, S. T.; Breukink, E.; Tischenko, E.; Lutters, M. A.; de Kruijff, B.; Kaptein, R.; Bonvin,
A. M.; Nuland, N. A. The nisin-lipid II complex reveals a pyrophosphate cage that
provides a blueprint for novel antibiotics. Nat. Struct. Mol. Biol. 2004, 11, 963−967.
3
3 Watson, J. D.; Milner-White, E. J. A novel main-chain anion-binding site in proteins: The
nest. A particular combination of phi, psi values in successive residues gives rise to
anion-binding sites that occur commonly and are found often at functionally important
regions. J. Mol. Biol. 2002, 315, 171–182.
3
4 Sheldrick, G. M.; Jones, P. G.; Kennard, O.; Williams, D. H.; Smith, G. A. Structure of
vancomycin and its complex with acetyl-D-alanyl-D-alanine. Nature 1978, 271, 223–225.
35 Schäfer, M.; Schneider, T. R.; Sheldrick, G. M. Crystal structure of vancomycin. Structure
996, 4, 1509–1515.
1
3
6 Sharman, G. J.; Try, A. C.; Dancer, R. J.; Cho, Y. R.; Staroske, T.; Bardsley, B.; Maguire, A.
J.; Cooper, M. A.; O'Brie, D. P.; Williams. D. H. The Roles of Dimerization and
Membrane Anchoring in Activity of Glycopeptide Antibiotics against Vancomycin-
Resistant Bacteria. J. Am. Chem. Soc. 1997, 119, 12041–12047.
3
3
7 Cooper, M. A.; Williams, D. H. Binding of glycopeptide antibiotics to a model of a
vancomycin-resistant bacterium. Chem. Biol. 1999, 6, 891–899.
8 Ge, M.; Chen, Z.; Onishi, H. R.; Kohler, J.; Silver, L. L.; Kerns, R.; Fukuzawa, S.;
Thompson, C.; Kahne, D. Vancomycin Derivatives That Inhibit Peptidoglycan
Biosynthesis Without Binding d-Ala-d-Ala. Science 1999, 284, 507–511.
3
9 Kahne, D.; Leimkuhler, C.; Lu, W.; Walsh, C. Glycopeptide and Lipoglycopeptide
Antibiotics. Chem. Rev. 2005, 105, 425–448.
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