3D Structure Determination of an Unstable Transient Enzyme Intermediate by Paramagnetic NMR Spectroscopy

Article abstract of DOI:10.1002/anie.201606223

Enzyme catalysis relies on conformational plasticity, but structural information on transient intermediates is difficult to obtain. We show that the three-dimensional (3D) structure of an unstable, low-abundance enzymatic intermediate can be determined by nuclear magnetic resonance (NMR) spectroscopy. The approach is demonstrated for Staphylococcus aureus sortase A (SrtA), which is an established drug target and biotechnological reagent. SrtA is a transpeptidase that converts an amide bond of a substrate peptide into a thioester. By measuring pseudocontact shifts (PCSs) generated by a site-specific cysteine-reactive paramagnetic tag that does not react with the active-site residue Cys184, a sufficient number of restraints were collected to determine the 3D structure of the unstable thioester intermediate of SrtA that is present only as a minor species under non-equilibrium conditions. The 3D structure reveals structural changes that protect the thioester intermediate against hydrolysis.

Full text of DOI:10.1002/anie.201606223

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International Edition: DOI: 10.1002/anie.201606223
German Edition: DOI: 10.1002/ange.201606223
Enzyme Catalysis
3D Structure Determination of an Unstable Transient Enzyme
Intermediate by Paramagnetic NMR Spectroscopy
Jia-Liang Chen, Xiao Wang, Feng Yang, Chan Cao, Gottfried Otting, and Xun-Cheng Su*
Abstract: Enzyme catalysis relies on conformational plasticity,
but structural information on transient intermediates is difficult
to obtain. We show that the three-dimensional (3D) structure of
an unstable, low-abundance enzymatic intermediate can be
determined by nuclear magnetic resonance (NMR) spectros-
copy. The approach is demonstrated for Staphylococcus aureus
sortase A (SrtA), which is an established drug target and
biotechnological reagent. SrtA is a transpeptidase that converts
an amide bond of a substrate peptide into a thioester. By
measuring pseudocontact shifts (PCSs) generated by a site-
specific cysteine-reactive paramagnetic tag that does not react
with the active-site residue Cys184, a sufficient number of
restraints were collected to determine the 3D structure of the
unstable thioester intermediate of SrtA that is present only as
a minor species under non-equilibrium conditions. The 3D
structure reveals structural changes that protect the thioester
intermediate against hydrolysis.
formed by Staphylococcus aureus Sortase A (SrtA) and
a peptide substrate under non-equilibrium conditions. SrtA
is an important enzyme in Gram-positive bacteria. It converts
a backbone amide of a substrate peptide (the peptide bond
between threonine and glycine in polypeptides containing the
LPXTG motif, where X can be any amino acid) into
a
thioester with the active-site cysteine residue
(Cys184).[8–11] SrtA is an established drug target[10] and is
increasingly being used as a biotechnological tool for protein
ligations.[12,13] 3D structures of SrtA have been determined by
X-ray crystallography and NMR spectroscopy.[14,15] The
structure of the disulfide-bond-linked intermediate analogue
SrtA-LPAT* showed significantly different structural features
to free SrtA or SrtA in a non-covalent complex with a peptide
substrate.[16] While the occurrence of a SrtA thioester
intermediate can be detected by mass spectrometry,[17,18] the
3D structure of this unstable intermediate has not been
determined. Thioester complexes are intermediates in many
enzyme reactions,[19] thus making techniques for their struc-
tural elucidation highly desirable.
D
elineating conformational changes in enzymes at atomic
resolution is required for a detailed understanding of their
functions.[1,2] Different methods for the structural character-
ization of enzymes during chemical reaction have recently
been developed.[1–3] Structure determination of unstable
enzyme intermediates in solution, however, is still a challenge
in view of the short lifetime and low abundance of these
intermediates. Recent advances in NMR spectroscopy have
made it possible to gain structural information on sparsely
populated species present in equilibrium by using experi-
ments such as relaxation dispersion, paramagnetic relaxation
enhancement (PRE), pseudocontact shift (PCS), chemical
exchange saturation transfer (CEST), and dark exchange
saturation transfer (DEST) measurements.[4–7] Among these
parameters, PCSs stand out for providing localization
restraints for nuclear spins relative to the paramagnetic
center that can be measured in minutes in sensitive hetero-
nuclear single quantum coherence (HSQC) spectra.
We identified conditions under which SrtA (without the
N-terminal 58 amino acids, which serve as a membrane
anchor in full-length SrtA)[14] transiently displays resolved
NMR resonances for the thioester intermediate formed
between Cys184 of SrtA and the threonine residue of the
substrate peptide QALPETG-NH2, where the C-terminal
amide serves as an uncharged protection group. The 3D
structure of the intermediate was determined by measuring
PCSs from new site-specifically attached paramagnetic lan-
thanide tags.
NMR signals for the thioester intermediate were identi-
fied by monitoring the reaction of SrtA with the substrate
peptide QALPETG-NH2 by 15N-HSQC spectroscopy (Figur-
es S1 and S2 in the Supporting Information). Many new weak
cross-peaks appeared (Figure 1a) in the spectrum of the
mixture of SrtA, calcium, and peptide, and they decayed with
a half-life of about 2.5 hours (Figure S2). A five-fold increase
in the concentration of SrtA or peptide did not increase the
population of the minor species, which was most highly
populated at pH 6.4 (Figure S1). Mass spectrometry indicated
that the new species was the thioester intermediate (Fig-
ure S1).[17,18] The intermediate could not be detected in the
absence of calcium or in the presence of 1.0 mm tri-glycine
peptide, which is known to act as a second substrate and
resolve the thioester by forming a peptide bond with the
bound first substrate (Figures S3–S5). The affinity of SrtA for
the substrate and product peptide is very weak, as indicated
by the conservation of chemical shifts of the main species
(Figure 1a), which corresponds to free SrtA, even in the
presence of a 20-fold excess of substrate peptide and after
Herein, we describe the use of PCSs to determine the 3D
structure of the unstable transient thioester intermediate
[*] Dr. J.-L. Chen, X. Wang, F. Yang, C. Cao, Prof. X.-C. Su
State Key Laboratory of Elemento-Organic Chemistry, Collaborative
Innovation Center of Chemical Science and Engineering (Tianjin)
Nankai University, Tianjin 300071 (China)
E-mail: xunchengsu@nankai.edu.cn
Prof. G. Otting
Research School of Chemistry, Australian National University
Canberra, ACT 2601 (Australia)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under http://dx.doi.org/10.
1002/anie.201606223.
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chemical shift differences, the structural difference is much
greater with respect to free SrtA (Figures S6 and S7).
Next we collected structural restraints for the thioester
intermediate. Most of the 15N-HSQC cross-peaks of the
thioester intermediate were sufficiently well resolved to
enable PCS measurements of samples tagged with a para-
magnetic lanthanide ion. SrtA contains a Ca2+ binding motif
and Ca2+ enhances the enzymatic activity.[14] Any paramag-
netic lanthanide tag thus needs a higher binding affinity for
lanthanide than calcium ions. Moreover, the thiol group of
Cys184 must not react with the tag. To address these
constraints, we produced the D82C mutant of SrtA and
synthesized the new paramagnetic tags T1 and T2 (Figure 2;
Figure 1. NMR detection of the SrtA thioester intermediate during
hydrolysis of the substrate peptide. All 15N-HSQC spectra were
recorded in 0.1 mm solutions of uniformly 15N-labeled SrtA in 20 mm
MES buffer, pH 6.4, at 298 K in the presence of 1.0 mm CaCl2.
a) Superimposition of 15N-HSQC spectra before (red) and after (black)
mixing with 1.0 mm peptide QALPETG-NH2. Corresponding cross-
peaks of the thioester intermediate (minor species) and free SrtA
(major species) from the same residues are connected by lines.
b) Cross-peak heights of selected residues of the thioester intermediate
relative to the sum of the cross-peak amplitudes of minor and major
species, which provide an estimate of the population and decay rate of
the thioester species. Cross-peak amplitudes varied between different
residues due to different line widths in the minor and major species.
c) Chemical structure of the thioester intermediate formed between
SrtA and QALPETG-NH2.
Figure 2. Site-specific labeling of SrtA D82C with a paramagnetic
lanthanide tag. a) Chemical structures of the tags T1 and T2 used in
the present study. Leaving groups are highlighted in red. b) Ribbon
representation of the SrtA structure (PDB ID: 1T2P[15]). The active site
and calcium binding site are highlighted by showing the side chains of
catalytically important residues (His120, Cys184, and Arg197) and
residues involved in calcium binding (Glu105, Glu108, Asp112,
Asn114, and Glu171).[21] The strands b7 and b8 are shown in red, the
preceding b6/b7 loop in magenta, and the b7/b8 loop in orange. The
arrow points at the side chain of Asp82 that was mutated to cysteine
for paramagnetic tagging.
prolonged reaction times (Figures S3 and S5). There was no
evidence for slow exchange between free SrtA and any non-
covalent complex with the substrate or product peptide
(Figure S5).
The lifetime of the thioester complex was too short and its
concentration too low for NMR resonance assignments by
standard 3D triple-resonance experiments (Figures S1 and
S2). Therefore, we assigned the resonances by comparison
with a stable disulfide-bonded analogue produced by ligating
the peptide QALPECG-NH2 to Cys184 of SrtA
(Scheme S1).[20] This adduct displayed 15N-HSQC cross-
peaks with very similar chemical shifts as the SrtA–
QALPET thioester complex with Ca2+. The backbone
resonances of the SrtA–QALPECG-NH2 adduct were
assigned based on a 3D NOESY-15N-HSQC spectrum.
Large differences in chemical shift between the SrtA–
QALPECG-NH2 adduct and the thioester intermediate
were limited to amides near Cys184, reflecting the chemical
difference between the disulfide and thioester bond linkages.
The calcium binding motif displayed almost identical chem-
ical shifts in the 15N-HSQC spectra of the SrtA–QALPECG-
NH2 adduct and thioester intermediate. Judging by the
see the Schemes S2–S5 in the Supporting Information for tag
synthesis and ligation with SrtA). T1 reacts with a solvent-
exposed thiol group in aqueous solution at pH 7.6, releasing
phenylsulfinate as the leaving group. Owing to the bromine
atom, it is more reactive than the previously published
4PhSO2-PyMTA tag.[22] T2 reacts with a protein thiol in
a similar manner to 4,4-dithiobisdipicolinic acid, with for-
mation of a disulfide bond.[23] Both tags showed high chemo-
selectivity towards the thiol group of the cysteine at position
82, and were not reactive towards the side chain of Cys184, as
confirmed by chemical shift mapping and MALDI-TOF mass
spectra (Figures S8 and S9). The tags show high affinity for
lanthanide ions, and this was not compromised by a large
excess of Ca2+ (up to 20 equivalents).
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To generate the paramagnetically tagged thioester inter-
Table S2). The Dc tensor parameters of the thioester inter-
mediate, solutions of the SrtA D82C-T1 or D82C-T2 adducts
were first titrated with lanthanides ions in the presence of
calcium, which maintained saturation of the calcium binding
site with Ca2+ (Figures S10 and S11). Following addition of
the QALPETG-NH2 peptide, paramagnetic cross-peaks were
observed for free SrtA D82C-T1 or D82C-T2 and the
corresponding thioester intermediates (Figure 3 and Figure
mediate were similar to those of free SrtA, as expected for
structural conservation of much of the protein. Small quality
factors indicate that the tags were sufficiently rigid to allow
the fitting of each Dc tensor by a single effective Dc tensor
(Figure S13).[25]
Next we determined the structure of the thioester
intermediate. A total of 407 PCSs (262 PCSs for D82C-T1–
QALPET, 145 for D82C-T2–QALPET) collected for para-
magnetic lanthanide ions (Dy3+, Tb3+, and Tm3+ for the T1
tag, and Tb3+ and Tm3+ for the T2 tag) were used to compute
the structure of loop regions in the thioester intermediate
using the program Xplor-NIH.[26,27] Figure 4a presents the 20
Figure 4. 3D structure of the thioester intermediate defined by PCSs.
a) Superimposition of the 20 lowest-energy structures. PCS isosurfaces
of the thioester SrtA D82C-T1·Tb3+ complex are shown together with
the protein structure. The isosurfaces plotted correspond to PCSs of
0.8 and 0.2 ppm (blue), and À0.8 and À0.2 ppm (red). b) Backbone
Ca displacement of the lowest-energy structure of the thioester
intermediate relative to the crystal structure of free SrtA (black
squares; PDB ID: 1T2P[15]) and to the first conformer of the NMR
structure of the disulfide linked thioester analogue SrtA-LPAT* (red
spheres, PDB ID: 2KID[16]). c) Structure comparison between the
thioester intermediate (gray) and SrtA-LPAT* (blue). The side chains of
residues 120, 184, and 197, which mark the active site, and of Trp194
are shown in stick representation. Loop regions with significant
structural differences are labeled. d) Superimposition of the thioester
intermediate (gray) and SrtA with inhibitor (PDB ID: 2MLM[28], red).
Figure 3. PCSs measured for SrtA D82C labeled with the lanthanide
binding tags T1 or T2. Superimpositions of 15N-HSQC spectra mea-
sured of 0.15 mm solutions of tagged SrtA D82C in complex with one
equivalent of Y3+ (black), Tm3+ (red), or Tb3+ (blue). Selected cross-
peaks are labeled with their resonance assignment using dark gray
labels and blue labels with a star for the cross-peaks of the free protein
and of the thioester intermediate, respectively. a) Spectra of 0.15 mm
SrtA D82C with the T1 tag in the presence of 1.0 mm Ca2+ and 1.0 mm
QALPETG-NH2. b) Zoom into selected regions of the 15N-HSQC
spectra of the thioester intermediate, highlighting differences in the
PCSs for the thioester intermediate and the free protein. Left panel:
15N-HSQC spectra of SrtA D82C with the T1 tag. Right panel: 15N-
HSQC spectra of SrtA D82C with the T2 tag.
S11). PCSs were determined from the 1H chemical shift
differences between the paramagnetic species, generated with
Tb3+, Dy3+, or Tm3+ ions, and the diamagnetic species,
generated with Y3+. Different PCSs were observed for
backbone amide protons of the thioester intermediate and
free SrtA (Figure 3 and Figures S10–S12).
lowest-energy conformations. The root mean squared devia-
tions of Ca and heavy atoms to the average structure were 0.7
and 1.1 ꢀ, respectively. Starting from the crystal structure of
free SrtA and the NMR structure of the disulfide-bond-linked
thioester analogue SrtA-LPAT* (PDB ID: 2KID[16]) yielded
very similar conformations (Figures S14 and S15, Tables S3
and S4). Compared with the crystal structure of SrtA, the
thioester intermediate revealed notable structural changes in
the b6/b7 and b7/b8 loops (Figure 4). The residues in these
regions are important for catalytic function and were found to
undergo conformational exchange in earlier NMR studies of
free SrtA.[15,16] In addition, residues in the first helix (a1) and
the Ca2+ binding motif, including the loop segments b3/b4, b4/
a2, and b5/b6, also showed significant structural changes.
Using the program Numbat,[24] the magnetic susceptibility
anisotropy (Dc) tensors of the lanthanide complexes were
determined based on PCSs and the crystal structure of free
SrtA (PDB ID: 1T2P[15]), using only protein segments with
regular secondary structure, for which neither chemical shifts
nor PCSs changed significantly between free SrtA and the
thioester intermediate. The resulting Dc tensor parameters
are listed in Table S1. Similar Dc tensors were obtained by
using the NMR structure of SrtA (PDB ID: 1IJA,[14]
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The structure of the thioester intermediate most closely
resembles the structure of the disulfide-bonded SrtA-LPAT*
analogue (Figure 4b,c) and differs significantly from the
crystal structure of SrtA with non-covalently bound LPETG
peptide (PDB ID: 1T2W[15]), which has almost the same
conformation as free SrtA. The b6/b7 and b7/b8 loops in the
thioester intermediate also differ significantly from the
structure determined for SrtA with an inhibitor linked to
Cys184 via a disulfide bond (PDB ID: 2MLM,[28] Figure 4d).
As in the SrtA-LPAT* analogue, Ca2+ restricts conforma-
tion exchange in the thioester intermediate, as evidenced by
the appearance of 15N-HSQC cross-peaks for residues from
the b6/b7 loop that are broadened by chemical exchange in
free SrtA (Figure 1 and Figure S16).[21] The altered confor-
mation of the b6/b7 loop is interesting, because it forms
a connection between the calcium binding site and Cys184 in
the active site. The thioester intermediate also displays a more
extended conformation of the b7/b8 loop, which facilitates
nucleophilic attack on the thioester by an oligo-glycine
peptide in the next step of the catalytic cycle.[15]
Keywords: enzyme catalysis · NMR spectroscopy ·
protein dynamics · protein structures · transient intermediates
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Published online: && &&, &&&&
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Communications
Enzyme Catalysis
J.-L. Chen, X. Wang, F. Yang, C. Cao,
G. Otting, X.-C. Su*
&&&& — &&&&
3D Structure Determination of an
Unstable Transient Enzyme Intermediate
by Paramagnetic NMR Spectroscopy
Now you see it: 3D structures of low-
abundance transient enzyme intermedi-
ates can be determined from pseudo-
contact shifts (PCSs) measured by NMR
spectroscopy. The method is demon-
strated with the unstable thioester inter-
mediate formed between Staphylococcus
aureus sortase A and the peptide sub-
strate.
6
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!