Characterization of the Fast and Promiscuous Macrocyclase from Plant PCY1 Enables the Use of Simple Substrates

Article abstract of DOI:10.1021/acschembio.8b00050

Cyclic ribosomally derived peptides possess diverse bioactivities and are currently of major interest in drug development. However, it can be chemically challenging to synthesize these molecules, hindering the diversification and testing of cyclic peptide leads. Enzymes used in vitro offer a solution to this; however peptide macrocyclization remains the bottleneck. PCY1, involved in the biosynthesis of plant orbitides, belongs to the class of prolyl oligopeptidases and natively displays substrate promiscuity. PCY1 is a promising candidate for in vitro utilization, but its substrates require an 11 to 16 residue C-terminal recognition tail. We have characterized PCY1 both kinetically and structurally with multiple substrate complexes revealing the molecular basis of recognition and catalysis. Using these insights, we have identified a three residue C-terminal extension that replaces the natural recognition tail permitting PCY1 to operate on synthetic substrates. We demonstrate that PCY1 can macrocyclize a variety of substrates with this short tail, including unnatural amino acids and nonamino acids, highlighting PCY1's potential in biocatalysis.

Full text of DOI:10.1021/acschembio.8b00050

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Article
Characterization of the fast and promiscuous macrocyclase
from plant PCY1 enables the use of simple substrates
Hannes Ludewig, Clarissa M. Czekster, Emilia Oueis, Elizabeth S Munday,
Mohammed Arshad, Silvia A. Synowsky, Andrew F. Bent, and James H. Naismith
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00050 • Publication Date (Web): 29 Jan 2018
Downloaded from http://pubs.acs.org on January 30, 2018
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Characterization of the fast and promiscuous macrocyclase from plant PCY1
enables the use of simple substrates
1
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Hannes Ludewig , Clarissa M. Czekster , Emilia Oueis , Elizabeth S. Munday ,
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Mohammed Arshad , Silvia A. Synowsky , Andrew F. Bent and James H.
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*1,4,5,6
Naismith
Funding information
H.L. is funded by the George & Stella Lee Scholarship and EPSRC. This project was
funded by the European Research Council project 339367 NCBꢀTNT and by the
BBSRC (J.H.N.). E.S.M. and M.A. are funded by EPSRC. S.A.S. is funded by BSRC
mass spec facility.
1
Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St
Andrews KY16 9ST UK
2
EaStChem, School of Chemistry, University of St Andrews, North Haugh, St
Andrews, KY16 9ST UK
3
Institute of Mechanical, Process and Energy Engineering, HeriotꢀWatt University,
Edinburgh, EH14 4AS, UK
4
Biotherapy Centre, Sichuan University, Chengdu, China.
5
Research Complex at Harwell, Didcot, OX11 0FA, UK
6
Division of Structural Biology, University of Oxford, Roosevelt Drive, Headington,
Oxford OX3 7BN, UK
*
To whom correspondence should be addressed
naismith@strubi.ox.ac.uk
%
ANDREW BENT IS DECEASED
Abbreviations: POP, prolyl oligopeptidase; PCY1, prolyl oligopeptidase from plant
Saponaria vaccaria; PatGmac, macrocyclase from patellamide biosynthesis; RiPPs,
ribosomally synthesized and postꢀtranslationally modified peptides; LCꢀMS, liquid
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chromatographyꢀmass spectrometry; ITC, isothermal titration calorimetry; Preseg,
presegetalin. Amino acids from PCY1 are referred to with one letter amino acid code,
while amino acids from the peptide substrates are referred to with three letter amino
acid code.
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Abstract
Cyclic ribosomally derived peptides possess diverse bioactivities and are currently of
major interest in drug development. However, it can be chemically challenging to
synthesize these molecules, hindering the diversification and testing of cyclic peptide
leads. Enzymes used in vitro offer a solution to this, however peptide
macrocyclization remains the bottleneck. PCY1, involved in the biosynthesis of plant
orbitides, belongs to the class of prolyl oligopeptidases and natively displays substrate
promiscuity. PCY1 is a promising candidate for in vitro utilization but its substrates
require an 11 to 16 residue Cꢀterminal recognition tail. We have characterized PCY1
both kinetically and structurally with multiple substrate complexes revealing for the
molecular basis of recognition and catalysis. Using these insights, we have identified
a three residue Cꢀterminal extension that replaces the natural recognition tail
permitting PCY1 to operate on synthetic substrates. We demonstrate that PCY1 can
macrocyclize a variety of substrates with this short tail, including unnatural amino
acids and nonꢀamino acids, highlighting PCY1’s potential in biocatalysis.
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Introduction
The class of natural products represented by ribosomally synthesized and postꢀ
translationally modified peptides (RiPPs) are of particular interest as novel
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therapeutics. Several macrocyclic compounds including cyclic RiPPs are orally
2
active despite the fact that they are in disagreement with Lipinskis’ Rule of Five.
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This behavior has lead to the term ‘beyond rule of five’ to describe peptide
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macrocycles. The activity and utility of macrocyclic peptides arises from their
increased stability (both chemically and to protease degradation), rigidity and
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hydrophobicity when compared to their linear peptide counterparts. Yet, analogously
to linear peptides, macrocyclic peptides can encode complex structural and chemical
information and thus may be particularly suitable to tackle difficult targets such as
proteinꢀprotein interactions, which are traditionally challenging to target using small
1
molecules. The de novo chemical synthesis of macrocyclic peptides is well known
but has some disadvantages such as the requirement of multiple steps and the
optimization of conditions necessary for the ring closing reaction for each peptide
5
,6
variant. Moreover, when the active macrocycle is highly modified, as many
5
cyanobactins are, their synthesis is not considered practical. Biotechnology provides
an alternative approach utilizing enzymes entirely in vivo or in vitro. The enzymes
involved in RiPPs biosynthesis are particularly attractive as these natural products are
genetically encoded, and the enzymes involved are able to carry out highly specific
modifications while operating with a very broad substrate range. This allows enzyme
7
activities from different pathways and organisms to be combined.
Cyanobactins possess a wide range of desirable and valuable bioactivities including
antiꢀcancer (cytostatic and cytotoxic), antifouling, immunomodulating and
8
antineoplastic. An understanding of the cyanobactin biosynthetic machinery has
9
,10
underpinned the production of a wide range of novel cyanobactins in vivo
and in
1
1–14
vitro.
Whilst in vivo approaches ꢀ sometimes termed ‘cell factory’ ꢀ hold the
advantage of simplicity, in vitro technologies have the unique capacity of harnessing
the power and diversity of chemical synthesis. This means that not only multiple
different nonꢀnatural amino acids can easily be accommodated, but hybrid molecules
(peptide and nonꢀpeptide) that are impossible to be produced in vivo can be
13
generated. To date, the majority of reports of in vitro macrocyclization have used
the macrocyclase domain from the patellamide pathway (PatGmac). Although
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extremely promiscuous, the enzyme has extremely slow catalytic rates in vitro, a
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major drawback that limits its application at large scale. A fast and promiscuous
macrocyclase enzyme is thus highly desirable. Of the macrocyclases known to date,
PatGmac (and other members of its family), the prolyl oligopeptidases (POPs) from
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Basidiomycete mushrooms (AbPOPB and GmPOPB), and PCY1 from the plant
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Saponaria vaccaria (syn. Saponaria hispanica) are known to catalyze the formation
of small macrocycles (containing between 5 and 9 amino acids). Although butelase 1
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19
from Clitoria ternatea is extremely fast and highly versatile, it does not produce
2
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small ring macrocycles efficiently, and a similar situation occurs with the MdnB
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from Microcystis aeruginosa (ATPꢀgrasp superfamily). Further limiting the
18,19
widespread use of butelase 1 is the difficulty of its overexpression in E. coli
limited solubility (partially overcome by engineering but with decreased activity).
and
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The POP class of macrocyclases has been underexplored in terms of biocatalysis.
GmPOPB has been kinetically and structurally characterized, and although it
16,23,24
possesses fast reaction rates, it displays narrow substrate scope.
PCY1, the
macrocyclase in the biosynthesis of a range of plant cyclic peptides known as
segetalins, has been shown to be a naturally promiscuous enzyme capable to generate
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5
to 9 residue segetalins, although the reported turnover rates are slow. A twoꢀstep
biosynthetic pathway for segetalins has been proposed, starting from a precursor
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peptide in which the core peptide is flanked by both a leader and tail peptide. Firstly
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a 10 amino acid Nꢀterminal sequence (termed the leader in RiPPs biosynthesis) is
removed by an uncharacterized peptidase to give the presegetalin, then PCY1
catalyzes peptide bond formation to yield the corresponding macrocyclic peptide and
cleaved Cꢀterminal peptide (Figure 1a). Two PCY1 complex structures have been
published, the substrate’s core sequence organization within PCY1’s active site were
not resolved, leaving unanswered questions about peptide recognition and
26
macrocyclization.
Here we report the first apo structure of PCY1 as well as complex structures with
natural substrates resolving substrate core within the active site for the first time.
Combining structure, kinetics and thermodynamics, we have discerned the rules
governing substrate recognition. Based on these rules we demonstrate the enzyme’s
will macrocyclize efficiently with a three residue Cꢀterminal recognition tail. Using
this tail we show PCY1 operate on peptides including a peptide alkyl chain hybrid,
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peptides containing Nꢀmethylated peptide bond as well as thiazoline containing
peptides. PCY1 is an attractive tool for in vitro biosynthesis of diverse cyclic
peptides.
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Results and Discussion
Mechanism of PCY1
PCY1 operates via an acyl enzyme intermediate in a classical serine protease
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mechanism, with the Nꢀterminus of the peptide rather than water acting as the
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nucleophile.
The PatGmac macrocyclase also operates by this mechanism,
although possessing an entirely different fold. The putative catalytic triad (D653,
H695 and S562) is located in the peptideꢀbinding site of PCY1. The catalytic serine
and histidine are further apart than is observed for the triad in other POPs, although a
24
similar ‘nonꢀoptimal’ arrangement is present in GmPOPB. Activity tests using
PresegB1 as a substrate for macrocyclization confirmed that PCY1 mutants S562A
and H695A were inactive (Figure S1).
Kinetic characterization of PCY1
ꢀ1
17
Barber et al. described PCY1 to have a turnover number of ~ 1 h for PresegA1.
We evaluated the enzyme produced heterologously in E. coli by LC/MSꢀbased time
course experiments macrocyclizing 200 µM of PresegA1, PresegB1 or PresegF1 with
3.6 µM PCY1. These substrates were chosen as they produce 6, 5 and 9 residue
macrocycles, respectively, show variability in the length of the Cꢀterminal tail
sequence, and have a different Cꢀterminal core residue (proline in PresegF1, alanine
in the other two) (Figure 1b, Figure S2). PresegB1 was completely macrocyclized in ~
30 min, PresegA1 in ~ 50 min and PresegF1 in > 3 h (Figure 2a). We chose to
investigate the slowest and fastest substrate by steadyꢀstate kinetics using an LC/MS
ꢀ1 ꢀ1
assay and UV280 nm quantification. This resulted in a k /K value of 830,000 M s
cat
m
for PresegB1 (Figure 2c and Table 1), which is not only considered above ‘average’
2
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for a typical enzyme but is also larger than the efficiency using the secondꢀorder rate
ꢀ1
ꢀ1
constant of butelase 1 from Clitoria ternatea (542,000 M s ), the fastest
18
macrocyclase described to date. The UV280 nm detection at low substrate
concentrations and thus accurate determination of K was problematic, especially for
m
PresegF1, which lacks a tryptophan in its sequence. We explored steadyꢀstate kinetics
with single injection kinetic ITC for PresegB1 (Figure 2d and Table 1) and this
allowed more accurate determination of Km (0.25 ± 0.01 µM). Under the ITC
ꢀ1
conditions, the measured kcat (0.14 ± 0.01 s ) was a factor of three lower than the
LC/MS derived value. However, to obtain kinetic parameters for PresegF1, a different
approach was required since single injection kinetic ITC only showed a small heat
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change. Therefore, we employed an LC/MS assay followed by quantification using
mass ion counts, relying on a calibration curve employing the cyclic peptide product
m
as a standard (Figure 2b). This approach gave a K of 2.40 ± 0.51 µM and kcat of 0.12
ꢀ1
±
0.01 s for PresegF1. These kinetic parameters line up with steadyꢀstate kinetics
26
performed using PresegA1 , and show that the difference in efficiency that we
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observed in the time course experiments is due to large differences in K rather than
in the turnover rate.
Crystallization of PCY1
PCY1 has been predicted (through homology modelling using a porcine muscle prolyl
17
oligopeptidase (POP) structure) and shown via Xꢀray crystallography studies to
26
belong to the S9 protease family , displaying the overall architecture of the POP
family comprising an α/βꢀ hydrolase domain and a seven bladed βꢀpropeller (Figure
S3). The structures of PCY1 bound to PresegA1 and to the covalent small molecule
inhibitor zꢀprolineꢀprolinal (ZPP) have been published but disorder meant only the
17
recognition of the Cꢀterminal NASAPV motif (conserved in PCY1 substrates Figure
26
S2) was visualised. Our 2.55 Å PCY1 apo structure with a cacodylate molecule at
the active site is similar (rmsd: 0.88 Å over 4,853 atoms; Table S1) to the previously
reported PCY1 complex adjacent to the active site comprising S562, H696 and D563
(Figure S4). Cacodylate is coordinated through hydrogen bonds by the side chain
hydroxyl group of Y481 and the amide of N563; both residues have been proposed to
30,26
form the oxyanion hole in the POPꢀfamily of enzymes (Figure S5).
The PCY1
structures all have a closed conformation in contrast to the open apo structure of
GmPOPB (Figure S3), a structural homologue in the POP family with low sequence
24
identity. Although coꢀcomplexes were described for GmPOPB the core peptide was
24
not visualized. To obtain more structural insight into substrate recognition for the
POP macrocyclase family we explored other substrates for coꢀcrystallisation with
26
PCY1.
Substrate recognition
We determined complex crystal structures of PCY1:S562A bound to PresegA1 (2.0
Å), PresegB1 (2.17 Å) and PresegF1 (1.86 Å) (Table S2). We did not observe
continuous electron density spanning the entire length of the peptide substrate in any
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of the structures solved (Figure 3a). In all three complexes however, the Cꢀterminal
peptide (NASA(S)PV) is clearly visible and adopts the same structure (Figure 3b,c
and Figure S6), seen in in previously published PCY1 complex structures with
26
PresegA1 (Figure S6). In the PresegF1 complex, the highest resolution structure, the
carboxylic acid group of the substrate’s Cꢀterminus V25 makes hydrogen bonds to the
backbone amide and side chain of S495 as well as the side chain of S493. The
carboxyl group is positioned at the Nꢀterminal end of the helix thus interacting with
the helical dipole (Figure 3b,c). In the substrate, the side chains of Val25, Pro24,
Ala23, and Ala21 make only a few van der Waal contacts with the protein, while the
main chain of Ala21 makes hydrogen bonds with the protein. Both the main and side
chain of Ser22 and Asn20 make either direct or water bridged hydrogen bonds.
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In the PresegA1 complex, no other residues could be modeled, as previously observed
26
by Chekan et al. However, in PresegF1 and PresegB1 complex structures, it was
possible to model the core peptide in at least one of the subunits within the
asymmetric unit, although the intervening residues that connect core to the tail were
not ordered. In PresegF1, we visualized Phe1 to Gln11 (Figure 3a) but not Thr12 to
Met19. In the other three subunits, electron density for the Nꢀterminal region of core
peptide has been less well defined, suggesting there could be multiple binding
conformations. In the bestꢀordered subunit, one face of the proline (Pro9) ring in the
substrate stacks against W603 whilst the edge of the ring interacts with F484. The
Leu8ꢀPro9 peptide bond (P2ꢀP1) adopts a trans configuration. The remainder of the
core peptide makes a small number of hydrogen bonds and van der Waal interactions
(Figure S7). The scissile bond P1ꢀP1′ (Pro9ꢀIle10, Figure S8) is positioned such that
the modeling of the hydroxyl at S562A results in the distance and orientation
expected for the formation of the acyl enzyme intermediate (Figure 3d), with the
Y481 and N563 acting to stabilize the negative charge (Figure S9). The chemically
sensible positioning of the scissile bond at the active site validates the relevance of the
structure. The P1′ side chain (Ile10) sits in a hydrophobic cleft making contacts with
the side chains of Y481 and I466 (Figure S8). The amino terminus of the substrate is
over 20 Å from the scissile bond, requiring extensive conformational flexibility from
the substrate for macrocyclization to occur.
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A comparison between complex structures of PCY1ꢀS562A bound to PresegF1 and
GmPOPBꢀS577A bound to a 35 amino acid substrate (GmAMA1) reveal that
although the overall protein architecture is remarkably similar, significant differences
exist between the location of the peptide ligand, and positioning of specific regions of
each peptide (Figure S10). In GmPOPB, the entire peptide tail or recognition
sequence (which is 17 amino acids long) can be seen in the structure, and spans
through the  ꢀpropeller domain. In contrast, in PCY1 the recognition sequence is not
inserted into the propeller domain, and only the last Cꢀterminal residues can be
observed, demonstrating greater flexibility in the peptide region that follows the core
peptide, different to GmPOPB. The core sequence of the macrocyclization substrate
was not experimentally observed in GmPOPB complexes (pdb codes 5N4B and
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5N4D).
In the PresegB1 PCY1 complex, the P2ꢀP1ꢀP1′ residues (Trp4, Ala5 and Phe6, Figure
S8 and S11) adopt an identical main chain conformation, make similar interactions as
PresegF1, and are thus positioned for attack by S562. However, the remaining three
residues of the core peptide have a different and more ringꢀlike conformation than
PresegF1 and as a result the Nꢀterminus is only 11 Å away from the scissile bond
(Figure S12). Thus, PresegB1 needs less reꢀorganization than PresegF1 and this may
underlie the difference in their macrocyclization turnover rates. Comparing the PCY1
structures in the PresegB1 and PresegF1 complexes reveals the structures are very
similar (root mean square deviation 0.8 Å over 707 Cα atoms). The most notable
change occurs in the main chain at Y696 which, in the PresegF1 complex, has flipped
so that the side chain points into the structure core, and as consequence the loop at
E154 has moved to accommodate it (Figure S13). In PresegB1, the residue adopts a
different rotamer but the position of the main chain is unchanged. The change at Y696
appears necessary to avoid a clash between this residue and the core of the substrate.
In both complexes, R655 makes hydrogen bonds to the substrate in the P2ꢀP1ꢀP1′
region although the precise interactions are different. Informed by the crystal
structures, we constructed mutants S493A, S495A, Y696G, R655A and W603A using
the reaction with PresgB1 as a test substrate. The relative activities of S495A, S493A,
R696G, and R655A were decreased by half, while W603A and H695Q showed
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around 10% relative activity, and H695A was inactive (Figure S1). No variant
resulted in an increase in proteolytic (as opposed to macrocyclase) activity.
To further investigate substrate binding, we performed ITC experiments using
PresegA1, PresegB1, PresegD1 and PresegF1 with PCY1:S562A. All four peptides
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d
showed tight binding with K values around 200 nM (Figure 4a, Table 2 and Figure
S14). The major contribution to binding of all tested peptides is enthalpic rather than
entropic (Figure 4b), pointing towards interactions between protein and substrate
rather than exclusion of water. The Cꢀterminal tail of PresegA1 was previously
17
reported to be essential for activity. The only conserved amino acids in all natural
substrates are the Cꢀterminal six residues (NASA(S)PV) (Figure S2). The peptide
NASAPV binds with a K
conserved does not dominate binding (Table 2, Figure S14). We measured the binding
of PresegA1ꢀNH (an amide rather than carboxylic acid at Cꢀterminus) to
d
of only 25 µM suggesting that this region although
2
d
PCY1:S562A and observed a ~70ꢀfold decrease of affinity (K : 12.1 µM) (Table 2,
Figure 4).
Structural data point to a key role for W603 in positioning the substrate with the
27
correct geometry for attack by S562. Unlike the PatGmac class of macrocyclase,
PCY1 does not require a proline residue or cisꢀlike geometry for activity. Instead, we
propose the site of macroyclization is controlled by the nature of the residues at P1
and P1′ of the substrate. The structure shows that PCY1 residues W603, F484, Y607,
and V558 would clash with the amino acids that have two nonꢀhydrogen substituents
at Cβ (Val, Ile, Thr) or larger. Although serine cannot be ruled out on purely steric
grounds, the hydrophobic nature of the environment favors proline or alanine. The
binding site at P1′ is hydrophobic but requires larger side chains such Ile, Phe, or Leu
to make interactions with the protein (Figure S8). This sequence pattern is found at
d
the point of scission for all known substrates for PCY1 (Figure S2c). The K for
DNASAPV (9.6 µM) and NASAPV (25.2 µM) with PCY1:S562A were larger than
observed for full length substrate (Table 2 and Figure S14). A peptide substrate
without the Cꢀterminus (PresegF1ꢀtruncated, FSASYSSKPIQT) was evaluated
d
although its K could not be accurately measured but was estimated to ~ 0.6 mM
(poor peptide solubility and weak binding prevent more accurate measurement)
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(Table 2 and Figure S14). The peptide that links P2ꢀP1ꢀP1′ of the core and the Cꢀ
terminus is highly variable in sequence and in length (Figures 1b and S2) consistent
with this region acting as a flexible linker rather than a recognition element. Whilst
the intact peptide binds very tightly, these two regions on their own bind more
weakly, suggesting a model where affinity is generated by linking binding sites
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(conceptually analogous to the chelate effect).
Exploring substrate promiscuity
It is known that PCY1 is tolerant of significant changes in sequence within the core
peptide and competent to make macrocycles as short as five and as large as nine
17
residues. We tested the ability of PCY1 to make larger macrocycles by extending
the core sequence of PresegF1 by one amino acid and PCY1 was able to macrocyclize
this substrate (Figure S15 and S16). This extends the full range of relevant peptide
macrocycles that PCY1 makes to include the upper limit (1000 Da) for ‘beyond rule
32
of five’ macrocycles. However, the requirement for a long disposable Cꢀterminus is
a major drawback of the use of PCY1. The molecular recognition of substrate peptide
by PCY1 prompted us to devise substrates with a short Cꢀterminal extension replacing
the native recognition sequence. We analyzed reactions of a peptide containing
FSASYSSKP (F1 core) with different truncated recognition sequences derived from
PresegB1 or PresegF1 added Cꢀterminal to the core. In both PresegB1 and PresegF1 a
three residue tail gave optimal results (Figure S17) but a diverse set of extensions
(sequence and length) supported catalysis. PresegB1 derived recognition sequences in
general yielded more SegF overnight in comparison to the PresegF1 derived
recognition sequences. Steadyꢀstate kinetic analysis of the variants revealed FQA is
the superior truncated recognition sequence over IQT with a ~ 17x larger secondꢀ
order rate constant, but both show synthetically useful kcat and K
m
values (Table 1 and
24
Figure S17). Although GmPOPB can also process short tailed substrates, it is slower
ꢀ1
(kcat at 20 °C using a shorter substrate is 0.010 s ), intolerant of changes in core
length and restricted in sequence acceptance. We tested the core sequence
VTACITWP (related to the natural substrate of PatGmac and unrelated to PCY1
natural substrates) with both short recognition sequences: FQA, and IQT. The
peptides (250 µM) were processed with 0.5 µM PCY1 and compared to a 25 µM
PatGmac reaction of VTACITWPAYDGE (AYDGE contains the PatGmac
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recognition sequence). After 90 minutes, PCY1 had turned over ~29 x and ~11 x
more substrate for the FQA and IQT variants as analyzed by LC/MS (Figure S18),
respectively than the PatGmac reaction (PatGmac was at 50ꢀfold higher
concentration). PCY1 therefore outperforms PatGmac on an eightꢀresidue core
sequence similar to PatGmac’s own substrate. Although known to process a wide
range of amino acids within the core peptide, the tolerance of PCY1 for nonꢀnatural
amino acids is unknown. Capitalizing on our discovery of truncated substrates, we
explored the scope of PCY1 with such shortened substrates containing unnatural and
modified amino acids. PresegF1 core peptide modified with an amino octanoic acid
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(
FSAꢀ8AocꢀFP) and VGAGꢀ8AocꢀFPIQT (derived from a known PatGmac substrate)
Figure S19ꢀS21), MALDI spectrum shows an approximate 50% conversion rate
(
overnight for the first peptide FSAꢀ8AOcꢀSKPIQT with only traces of the linearꢀ
cleavage peptide alongside the cyclic peptide (Figure S19), while the other precursor
peptide VGAGꢀ8AocꢀFPIQT is almost entirely converted overnight to a mixture of
the corresponding cyclic peptide and linearꢀcleavage peptide (Figure S20). The same
reaction with the highly promiscuous PatGmac would have required 60 times the
amount of enzyme and an average of 20 days for completion. Nonetheless, PatGmac
usually gives rise to either exclusively linearꢀcleavage peptide (especially for shorter
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peptides) or none at all, as was the case for the VGAGꢀ8AocꢀFP peptide.
m
Additionally, we showed that PCY1 can macrocyclize FS ASYSSKPIQT and
m
m
FS ASYSSKPFQA (where A is Nꢀmethylated alanine) and produce cyclic
m
FS ASYSSKP (Figure S22 and S23). Nꢀmethylated peptidic drugs are known to cross
cell membranes more efficiently than compounds lacking this chemical feature. Taken
together, our data show that the processing activity of PCY1 is not simply limited to
the canonical amino acids within the core. PCY1 failed to process a prolineꢀrich
PresegF1 variant yielding only a hydrolysis product (Figure S24). Such prolineꢀrich
peptides are also extremely poor substrates for PatGmac, an observation attributed to
28
the conformational rigidity imposed by multiple proline residues. Using LynDfus., an
33
engineered heterocyclase enzyme,
we converted VTACITWPIQT into
VTAThnITWPIQT (Thn: thiazoline) in situ to explore sensitivity to heterocycles
which will impose conformational restrictions. Incubation with 1 µM PCY1 produced
both macrocyclic and hydrolyzed linear peptides (Figure S25ꢀS26), yielding 10.9 µM
Thn containing macrocycle after ~ 20 h incubation, representing a 4.4 % conversion.
Although a conversion this low is not synthetically useful, based on well established
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methods of optimization of enzyme activity including directed evolution this turnover
34
represents a starting point for future improvement. Further investigations are being
conducted to study variants of the mutated enzyme and the reaction conditions in
order to improve the synthesis of such hybrid cyclic peptides.
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Summary
The importance of peptidic and amino acidꢀcontaining macrocycles in medicine is
now clear. Robust synthetic approaches for these molecules, which can combine
synthetic diversity with the ability of enzymes to catalyze challenging chemical steps,
are highly desirable. Here we have reported the characterization of PCY1, a
macrocyclase from plant. We show substrate recognition occurs in two distinct
patches of the substrate, joined by a flexible linker, which underpins the promiscuity
of PCY1. Based on understanding substrate recognition, we have demonstrated that
the enzyme can utilize simpler synthetically accessible substrates with a short Cꢀ
terminal extension at a synthetically useful rate. PCY1 can process hybrid molecules
than are not solely composed of canonical amino acids, suggesting PCY1 could
represent a valuable synthetic tool.
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Materials and Methods
Materials
All reagents, but peptides, were purchased from SigmaꢀAldrich, unless stated
otherwise. All peptides were commercially obtained from Biosynthesis, unless
otherwise stated. HPLC grade acetonitrile (MeCN) was purchased from Fisher.
Aqueous buffers and aqueous mobileꢀphases for HPLC were prepared using water
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®
®
purified with an Elga Purelab MilliꢀQ water purification system (purified to 18.2
Mꢁ.cm) and filtered over 0.45 ꢂm filters.
Cloning, expression and protein purification
Codon optimized fullꢀlength PCY1 (Saponaria vaccaria) including an Nꢀterminal
6
His ꢀtag and a cleavable Tobacco etch virus (TEV) protease site (sequence
ENLYFQG, cleaving between Q and G) encoded in pJ414 plasmid was purchased
from DNA2.0. Overꢀexpression of PCY1 was performed in E. coli BL21 (DE3)
grown in LB media. Cultures were inoculated and grown at 37 °C until OD600 nm: ~
0.6, after which the temperature was lowered to 16 °C and protein expression was
induced with 0.5 mM IPTG (final concentration, Generon). Bacteria were grown for
20 h at 16 °C, and cells were harvested by centrifugation at 8,983g for 10 min at 4 °C
(Avanti Jꢀ26S, rotor JLAꢀ8.1000, New Brunswick Scientific). Wet cell pellets were
stored at ꢀ80 °C until protein purification. Cell pellets were resuspended in buffer A
(50 mM HEPES, 300 mM NaCl, 10 % (v/v) glycerol, 3 mM βME (Fischer Chemical),
pH 8.0) supplemented with EDTAꢀfree protease inhibitor tablets (Roche) and 0.4 mg
DNase I from bovine pancreas per g wet cells, at 4 °C. Cells were disrupted by double
passage through a cell disruptor (Constant Systems Ltd) at 30 kPSI. The lysate was
subsequently cleared via centrifugation at 43,667g at 4 °C for 20 min (rotor JA 25.50,
New Brunswick Scientific). The cleared lysate was loaded onto a 5 mL Co HiTrap
TALON (GE Healthcare), preꢀequilibrated in buffer A. Protein was eluted in buffer B
(50 mM HEPES, 300 mM NaCl, 10 % (v/v) glycerol, 250 mM imidazole, 3 mM
βME, pH 8.0). TEV protease was added to the eluted sample (1 mg per 30 mg of
eluted protein) and this mixture was dialyzed into 1 L of buffer A at 4 °C overnight.
The dialyzed sample was applied onto a 5 mL Co HiTrap TALON, equilibrated in
buffer C (50 mM HEPES, 300 mM NaCl, 10 % (v/v) glycerol, 3 mM βME, pH 8.0).
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The flowꢀthrough of this reverse Niꢀaffinity chromatography step was pooled and
dialyzed into 1 L of buffer D (50 mM HEPES, 5 mM NaCl, 10 (v/v) glycerol, 3 mM
βME, pH 8.0) at 4 °C for 1 h and subsequently subjected to anion exchange
chromatography using a 5 mL HiTrap QꢀFF column (GE Healthcare) and a gradient
of buffer D and E (50 mM HEPES, 500 mM NaCl, 10 % (v/v) glycerol, 3 mM βME,
pH 8.0). Eluted samples containing PCY1 (verified by SDSꢀPAGE) were pooled,
concentrated and further purified using sizeꢀexclusion chromatography (HiLoad
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16/600 Superdex 200 pg, GE Healthcare) preꢀequilibrated in buffer F (50 mM
HEPES, 150 mM NaCl, 10 % (v/v) glycerol, 1 mM TCEP (tris(2ꢀ
carboxyethyl)phosphate), pH 8.0). Purity and identity of final protein sample were
confirmed by SDSꢀPAGE and mass spectrometry.
Site directed mutagenesis
Site directed mutagenesis was performed using an overlaying mutation carrying
primer pair (IDT) of ~30 nucleotides employing Pfu polymerase (Thermo Scientific)
for amplification (95 °C 3 min initial denaturation; 15 x 95 °C 30s denaturation
m
followed by T ꢀ5 °C 45 s annealing and 72 °C 2min/kb extension, 68 °C 20 min final
extension), according to manufacturer instructions. The amplification product was
DpnI digested (Thermo Scientific, according to manufacturer’s instructions), and used
for transformation of E. coli DH5α. Sequencing was performed by Eurofins.
Crystallization, data collection and crystallographic analysis
PCY1 and PCY1:S562A crystals (apo and as complexes) were obtained from hanging
drop vapor diffusion crystallization experiments with 500 µL reservoir solution and 2
µL drops (1:1 protein/precipitant ratio). When additive screens were performed, 0.2
µL of additive screen (Hampton Research) was added to the 2 µL crystallization drop.
Crystals were harvested, cryo protected and subsequently flash frozen in liquid
nitrogen. All data sets were collected at Diamond Light Source (UK).
Apo PCY1 was crystallized at 12.2 mg/mL in 33% (w/v) PEG 2000, 113.75 mM Mg
formate and 0.1 M Na cacodylate pH 7.0. For cryo protection 10% (v/v) glycerol were
added to reservoir solution. Data was collected at 100 K at beamline i04. Data was
3
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processed using the processing pipeline xia2 3d.
PCY1:S562A complex with
PresegA1 was crystallized at 13.3 mg/mL with 163 µM PresegA1 in 28% (w/v) PEG
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5000 MME, 140 mM Mg acetate tetrahydrate and 0.1 M Tris pH 7.5. For cryo
protection 7% (v/v) glycerol were added to reservoir solution. Data was collected at
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00 K at beamline i04ꢀ1. Data was processed using the processing pipeline xia2
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dii.
PCY1:S562A complex with PresegB1 was crystallized at 13.3 mg/mL with
63 µM PresegB1 in 34.5% (w/v) PEG 3350 and 70 mM Mg sulfate. For cryo
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protection 5 % (v/v) glycerol were added to reservoir solution. Data was collected at
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d.
PCY1:S562A complex with PresegF1 was crystallized at 13.0 mg/mL with
161.3 µM PresegB1 in 27% (w/v) PEG 3350, 100 mM Calcium chloride and 0.1 M
BisꢀTris at pH 6.5. For cryo protection 10% (v/v) glycerol were added to reservoir
solution. Data was collected at 100 K at beamline i24. Data processing was performed
3
5,37
with xia2 3dii.
Data processing, structure solution and structure refinement were
38
performed using the CCP4 software suite. Apo PCY1 structure was solved using
molecular replacement using PHASER and 1QFS as search model (α/βꢀhydrolase and
3
9
βꢀpropeller domain as separated search models).
The initial solution was then
completed with buccaneer and manual model building and refinement of the model
were performed using Coot and refmac5, respectively, including TLS refinement and
38,40–46
model validation using MolProbity.
For all coꢀcomplex crystal structures, apo
PCY1 structure was used as search model.
ITC studies with PCY1 variants and peptide ligands
Equilibrium binding experiments with PCY1:S562A and different substrates were
performed using a MicroCal PEAQꢀITC (Malvern) in buffer F (see ‘Protein
purification’). All samples were bufferꢀexchanged or solubilized into buffer F and
further diluted in the same batch of buffer F, in order to minimize buffer mismatches.
ITC binding experiments were performed using a cell solution containing
PCY1:S562A and a titrant solution with each peptide. All peptides were used at 200
2
µM except for presegetalin A1ꢀNH (700 µM), DNASAPV and NASAPV (1.5 mM)
as well as FSASYSSKPIQT (2 mM) . The cell contained 20 µM, 18.3 µM, 17.1 µM
15.5 µM, 100 µM, 52.6 µM, 52.5 µM and 59.8 µM PCY1:S562A for Presegetalin A1
,
B1 , D1 , F1 and A1ꢀNH
2
, DNASAPV, NASAPV and FSASYSSKPIQT,
respectively. The measurements were performed at a stir speed of 750 rpm at 25°C
using a reference power of 10 µcal/s, except for A1 ꢀNH , DNASAPVY, NASAPV
2
and FSASYSSKPIQT (5 µcal/s). A buffer titration control in which peptide was
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titrated into buffer was performed for each experiment. Data processing, fitting to a
nonꢀlinear oneꢀsite binding model, and data plotting was performed using MicroCal
PEAQ ITC analysis software (Malvern).
Single injection kinetic ITC measurements were performed using MicroCal PEAQꢀ
ITC with 60 nM PCY1 in the syringe and 193 µM presegetalin B1 in the cell. To start
the reaction, 2 µL of 9.06 µM PCY1 was injected into the cell (4 s injection time)
containing 300 µL of 193.5 µM Presegetalin B1. The measurement was performed in
buffer at 30 °C, at 750 rpm using a reference power of 5 µcal/s. Data processing for
kinetic ITC measurements was performed using MicroCal PEAQ ITC analysis
software. Data fitting and plotting were performed using Prism 6 (GraphPad
Software). Steadyꢀstate kinetics data were fitted according to Michaelis Menten
equation (corrected by enzyme concentration; equation (1)).
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ꢇꢈꢉꢊ∙ꢂꢋꢃ
∙ ꢂEꢃ ꢅꢄ ꢆ
ꢀꢁ
=
(1)
ꢌꢍꢎꢂꢋꢃ
LC/MS assays: time courses and steadyꢀstate kinetics
All kinetic measurements (activity tests, progress curves and initialꢀrate experiments)
were performed as discontinuous assays analyzing samples via LC/MS or MALDI
(10ꢀmer activity test). Reactions were performed in 20 mM Tris pH 8.5, 100 mM
NaCl, 5 mM DTT (for comparative time courses of PresegA1, PresegB1 and
ꢀ1
PresegF1 0.2 mg mL BSA was included). All reactions (comparative time courses,
steady state kinetics, PCY1 and PatGmac comparison and recognition sequence
analysis) were performed at 30 °C and at various time points 50 µL samples were
quenched with 20 µL of 6 % (v/v) TFA. Steadyꢀstate kinetics were performed with 30
nM PCY1 for PresegB1, 0.1 µM PCY1 for PresegF1, 0.25 µM for FSASYSSKPIQT
and 50 nM for FSASYSSKPIQT. Recognition sequence experiments were performed
at 250 µM [substrate] and 0.5 µM PCY1. Assays comparing PCY1 and PatGmac
were performed with 250 µM substrates and 0.5 µM PCY1 or 25 µM PatGmac.
Reactions were quenched at different time points with 1.7 % TFA (final
concentration), samples were spun down at 20,817g and 50 µL were loaded onto a
ZORBAX SBꢀC18, 5 µm, 9.4 mm × 50 mm (Agilent) column connected to an Agilent
LC−MS instrument (G6130B Single Quad, Agilent Technologies). Reactants were
2
separated from products using a gradient from A (H O with 0.1% formic acid) to 80%
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B (acetonitrile), at a flow rate of 1.5 mL/min for 8.75 min. For data analysis, ion
count peaks and UV absorbance peaks were integrated using Agilent ChemStation
software. For high resolution mass spectrometry and mass fragmentation of analysis
of requirement of recognition sequence in presegetalins, and comparison between
PCY1 and PatGmac, 10 µL of quenched reaction mixture was injected onto a
ZORBAX SBꢀC18, 5 µm, 9.4 mm × 50 mm (Agilent) column connected to an
UltiMate 3000 LC system (Thermo Scientific) connected to an Orbitrap Velos Pro
MS system (Thermo Scientific). Reactants were separated from products using a
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gradient from A (H O with 0.1% formic acid) to 95% B (acetonitrile), at a rate of 1.5
mL/min for 12 min. For data analysis, ion count peaks were integrated using Thermo
Xcalibur Qual browser software. For data analysis involving quantitative mass spec
integration, calibration curves with cyclic peptide (SegF) standards (treated as
reaction products) were performed in triplicate in order to linearly correlate ion counts
to cyclic product concentration. SegF1 was biosynthesized using 0.5 µM PCY1 for 50
µM PresegF1, overnight incubation at 30 °C in reaction buffer. Reaction as quenched
with 1.7 % TFA (final concentration). Semiꢀpreparative RPꢀHPLC was performed on
an Agilent Infinity 1260 series equipped with an MWD detector using a Machereyꢀ
Nagel Nucleodur C18 column (10 ꢂm x 21 x 250 mm at 21 mL/min), fractions were
collected automatically by peak detection at the specified wavelength using an
Agilent 1260 Infinity preparativeꢀscale fraction collector using the following
chromatographic system: MeCN and 0.1 % aqueous TFA [95% TFA (5 min), linear
gradient from 5 to 95% of MeCN (35 min), 95% MeCN (40 min)] and UV detection
at 280 nm for peak collection, while monitoring at the additional two wavelengths
220 and 254 nm. Fractions of pure SegF were combined and freeze dried before being
used for the assays.
Further data fitting and plotting was performed with Prism 6. Comparative time
course measurements of PresegA1, PresegB1 and PreseF1 were fitted nonꢀ
exponentially (equation (2)). Data for steadyꢀstate kinetics were fitted to a Michaelis
Menten equation (corrected by enzyme concentration; equation (1)). Reactions
investigating heterocycleꢀcontaining macrocyclic VTACITWP formation have been
performed under different conditions: 100 mM NaCl, 100 mM TRIS pH 8.5, 5 mM
DTT, 10 mM MgCl
2
and 5 mM ATP with 250 µM VTACITWPFQA or
VTACITWPIQT and 10 µM LynDfus. and 1 µM PCY1 (added after 19 h). Reactions
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were stopped via filtration through 30 kDa cut off VIVASPIN 500 (Sartorius)
concentrators.
ꢏ = ꢏ + ꢑ(1 − ꢒꢅꢇ∙ꢓ)
(2)
ꢐ
0
y : offset; k: apparent rate; a: amplitude
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Peptide analysis on the TripleTOF 5600
Peptides were injected on an Acclaim PepMap 100 C18 100 µm × 2 cm trap and
washed for 5 min to waste after which the trap was turned inꢀline with the analytical
column (Acclaim Pepmap RSLC 75 µm × 15 cm, both ThermoFisher Scientific)
using a nanoLC Ultra 2D plus loading pump and nanoLC asꢀ2 autosampler
(Eksigent).
The analytical solvent system consisted of buffer A (2% acetonitrile, 0.1% formic
acid in water) and buffer B (2% water, 0.1% formic acid in acetonitrile) at a flow rate
of 0.3 ꢂl/min with the following gradient: linear 5ꢀ40% of buffer B for 6 min, linear
40ꢀ95% of buffer B for 3 min, isocratic 95% of buffer B for 3 min, linear 95ꢀ1% of
buffer B for 2 min and isocratic 1% solvent buffer B for 6 min for reꢀequilibration.
The eluate was sprayed into a TripleTOF 5600 electrospray tandem mass
spectrometer (ABSciex, Foster City, CA) and analyzed in Information Dependent
Acquisition (IDA) positive ion mode. For each MS scan (m/z 150ꢀ1250) of 150ms, 20
MSMS (m/z 100ꢀ1800) of 80 ms with 10 s dynamic exclusion were acquired. Rolling
collision energy with a collision energy spread of 5 eV was used for fragmentation.
To obtain better MSMS spectra of the peptides of interest, a product ion scan (PIS) of
the relevant masses were analyzed. The LC condition were kept constant with an
adjusted gradient: linear 5ꢀ40% of buffer B for 16 min, linear 40ꢀ95% of buffer B for
3 min, isocratic 95% of buffer B for 3 min, linear 95ꢀ1% of buffer B for 2 min and
isocratic 1% solvent buffer B for 6 min for reꢀequilibration. The mass spectrometer
was operated in product ion mode with four selected masses of interest. Each MS and
MSMS scan (m/z 100ꢀ2400) was acquired for 100ms respectively.
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In house peptide synthesis
®
Automated solidꢀphase peptide synthesis (SPPS) was carried out on a Biotage Syro
Wave™ system in polypropylene (PP) syringe with a PTFE frit. Final cleavage and
deprotection were completed manually.
The different precursor peptides were synthesized by standard automated solidꢀphase
(SPPS) on a ChemꢀMatrix Rink amide resin (~ 0.5mmol/g) or a Trityl chloride resin
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(
~ 0.4 mmol/g) using the Fmoc strategy and Fmocꢀprotected amino acids. A double
coupling strategy using a 5ꢀfold excess with 2ꢀ(1Hꢀbenzotriazolꢀ1ꢀyl)ꢀ1,1,3,3,ꢀ
tetramethyluronium hexafluorophosphate (HBTU)/N,N-Diisopropylethylamine
DIPEA) (HBTU, 0.5M in DMF and DIPEA, 2M in NMP) and DIC/oxyma pure
DIC, 0.5M in DMF, Oxyma, 1M in DMF) was used for all amino acids for 30
(
(
minutes at 75 °C, except for the loading of the first amino acid on the trityl resin. In
order to achieve that, to the resin (swelled in CH Cl ) was added the amino acid (2
2
2
2 2
eq.) and DIPEA (4 eq.) in CH Cl and the reaction agitated at room temperature for 2
hours. The reaction mixture was discarded, the resin was washed and capped by the
addition of MeOH and DIPEA in CH Cl (2:1:18) for one hour at room temperature.
2
2
After washing the resin, the peptide synthesis is completed as described. The Fmoc
deprotection was done in 20% piperidine/DMF for 12 min at rt. For the final cleavage
and side chain deprotection, the beads (washed with CH Cl and dried) were
2
2
transferred into a flacon tube and the cleavage cocktail was added and left shaking for
h: 96% TFA, 2.5% H O, 1.5% triisopropyl silane. The resin was filtered, washed
with CH Cl , and the filtrate concentrated under reduced pressure. The peptides were
2
2
2
2
2
then precipitated in cold Et O and the precipitate purified by prepꢀHPLC.
Semiꢀpreparative RPꢀHPLC was performed on an Agilent Infinity 1260 series
equipped with an MWD detector using a MachereyꢀNagel Nucleodur C18 column (10
ꢂm x 21 x 250 mm at 21 mL/min) and fractions were collected automatically by peak
detection at the specified wavelength using an Agilent 1260 Infinity preparativeꢀscale
fraction collector using the following chromatographic system: MeCN and 0.1 %
aqueous TFA [95% TFA (5 min), linear gradient from 5 to 95% of MeCN (35 min),
95% MeCN (40 min)] and UV detection at 280 nm for peak collection, while
monitoring at the additional two wavelengths 220 and 254 nm. Fractions of the pure
peptides were combined and freeze dried before being used for the assays.
Analytical RPꢀHPLCꢀMS was performed on an Agilent infinity 1260 series equipped
with a MWD detector using a MachereyꢀNagel Nucleodur C18 column (10 ꢂm x 4.6
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x 250 mm) and connected to an Agilent 6130 single quad apparatus equipped with an
electrospray ionization source using the following chromatographic system. 1 mL/min
flow rate with MeCN and 0.1 % aqueous TFA [95% TFA (5 min), linear gradient
from 5 to 95% of MeCN (35 min), 95% MeCN (40 min)] and UV detection at 220
nm. Peptides with truncated recognition sequences were purified by semiꢀpreparative
RPꢀHPLC (Agilent infinity 1260 series, Agilent Technologies) using a Jupiter 300 C4
column (Phenomenex, 10 ꢂM x 16 x 250 mm at 10 mL/min) and fractions were
collected automatically by peak detection at 220nm using an Agilent 1260 Infinity
preparativeꢀscale fraction collector. Solvents were 0.1 % aqueous TFA (solvent A)
and acetonitrile (solvent B). Gradients used for purification of different peptides were
as follows: Peptides GVAWAI, GVAWAIQ and GVAWAIQT, 5% B (5 minutes),
linear gradient from 5 to 40% B (10 minutes), followed by linear gradient from 40ꢀ
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4% acetonitrile (20 minutes); Peptides GVAWAF, GVAWAFQ and GVAWAFQA,
5% B (5 minutes), linear gradient from 5 to 41% B (10 minutes), followed by linear
gradient from 41ꢀ50% acetonitrile (20 minutes); Peptides FSASYSSKPI,
FSASYSSKPIQ, FSASYSSKPIQT, FSASYSSKPF, FSASYSSKPFQ and
FSASYSSKPFQA, 5% B (5 minutes), linear gradient from 5 to 15% B (10 minutes),
followed by linear gradient from 15ꢀ20% acetonitrile (20 minutes). Fractions
containing each peptide of interest were pooled, their identity and purity verified by
LCꢀMS and lyophilized for later use.
PDB accession codes: Coordinates have been deposited in the Protein Data Bank,
with accession codes 5O3X (apo PCY1), 5O3W (PCY1ꢀS562A mutant bound to
PresegA1 peptide), 5O3V (PCY1ꢀS562A mutant bound to PresegB1 peptide), and
5O3U (PCY1ꢀS562A mutant bound to PresegF1 peptide).
ACKNOWLEDGEMENTS
We thank O. K. Miller for crystal handling, and C. H. Botting and S. L. Shirran, for
mass spectrometry services. We acknowledge DLS beamtime, R. J. M. Goss for usage
of an UltiMate3000 Orbitrap Velos Pro LC/MS & C. Cartmell and C. S. Bailey for
assistance with the instrument. H.L. is funded by the George & Stella Lee Scholarship
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and EPSRC. E.S.M. and M.A. are also funded by EPSRC. This project was funded by
the European Research Council project 339367 NCBꢀTNT (J.H.N.). We dedicate this
paper to the memory of our friend and coꢀauthor A. F. Bent, who died aged 32 from
cancer.
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SUPPLEMENTARY INFORMATION
The Supporting Information containing substrates used, LigPlot, additional ITC and
xꢀray crystallography figures is available free of charge on the ACS Publications
website.
COMPETING FINANCIAL INTERESTS
HL, CMC and JHN are named on a patent that utilizes PCY1.
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Tables
Table 1. Kinetic parameters for PCY1.
ꢀ
1
ꢀ1
Substrate
K (µM)
kcat (s )
k
cat/K
m
(s Method
m
ꢀ
1
M )
1
1
1
1
1
1
1
1
1
1
2
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*
PresegB1
PresegB1
PresegF1
0.45 ± 0.17
0.25 ± 0.01
2.4 ± 0.5
0.37 ± 0.02
0.14 ± 0.01
0.12 ± 0.01
0.1 ± 0.01
820,000
560,000
50,000
1,200
LC/MS (UV280 nm)
ITC
LC/MS (ion count)
LC/MS (ion count)
FSASYSSKP 83 ± 18
FQA
FSASYSSKP 227 ± 166
0.016 ± 0.001 70
LC/MS (ion count)
IQT
Table 2: Binding parameters of PCY1:s562A and various peptides obtained by ITC.
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Peptide
N
K
d
Δ
H Δ
G ꢀT Δ S
(sites) (µM)
(kcal/mol) (kcal/mol) (kcal/mol)
1
PresegA1
0.173 ꢀ10.0
0.1
± ꢀ9.23
± ꢀ9.04
0.81
3.25
ꢀ2.19
2.03
±
0
1
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0
0.027
1
1
1
PresegB1
PresegD1
PresegF1
0.238 ꢀ12.3
±
0.1
0.022
0.250 ꢀ6.8 ± 0.1 ꢀ9.01
±
0.039
0.136 ꢀ11.4
0.1
± ꢀ9.37
±
0.019
1
1
PresegA1ꢀNH2
12.1 ꢀ5.3 ± 0.2 ꢀ6.71
2.0
ꢀ1.41
ꢀ3.14
±
FSASYSSKPIQT
56.0 ꢀ2.8 ± 3.1 ꢀ5.90
±
76.5
1
1
DNASAPV
NASAPV
9.58 ꢀ14.5
0.2
± ꢀ6.85
± ꢀ6.27
7.64
7.70
±
0.58
25.2 ꢀ14.0
1.2 0.1
±
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Figure legends
Figure 1: Reaction scheme of macrocyclization catalyzed by PCY1 and sequence
alignment of presegetalins. a) amino acid one letter code is used to abbreviate amino
acids, not depicted with the chemical formula. Transparent red circle is highlighting
the peptide bond formed via macrocyclization. b) Red dots highlight end of core
sequence. Sequence identity is highlighted with a gradient from white to blue. Deep
blue showing highest sequence identity.
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Figure 2: Kinetic analysis of macrocyclization catalyzed by PCY1.
a) Comparative time course of PresegA1 (green), PresegB1 (blue) and PresegF1 (red)
formation with PCY1. Data were fitted mono exponentially. b) LC/MS based steady
state kinetics of PresegF1 with PCY1 using ion count quantification and product
calibration curve, fitted non exponentially. c) LC/MS based steady state kinetics of
PresegB1 with PCY1 using quantification with UV at 280 nm, fitted non
exponentially. d) Single injection ITC steadyꢀstate kinetics of PresegB1, fitted non
exponentially. Reaction temperature for all experiments: 30 °C
Figure 3: Binding studies of PCY1:S562A and presegetalins.
Structure of PCY1:S562A bound to PresegF1. PCY1:S562A is drawn in cartoon style,
αꢀhelices are colored orange, βꢀsheets are colored metallic blue and loops are colored
in light grey. PresegF1 is shown as ballꢀandꢀstick model in dark red. Nitrogen atoms
o c
are colored in blue and oxygen atoms are colored in light red. 2F -F electron density
map is shown in mesh style (in blue) and rendered at s: 1.0. a) Overview of the
structure with peptide shown in spheres showing details into peptide binding site. b)
Interactions between PresegF1 Cꢀterminus and PCY1:S562A, depicting interactions
between the peptide carboxylic acid and the enzyme αꢀhelix. c) Overlay between
complex structures, peptides are shown as ballꢀandꢀstick model and colored green
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(PresegA1), blue (PresegB1) and red (PresegF1). Figure was adapted from Ligplot ,
and additional figures depicting interactions with the peptide ligand are available in
the supporting information.
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Figure 4: Structural analysis of PCY1:S562A complex structures.
a) ITC curves showing equilibrium binding measurements for PresegA1 and
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PresegA1ꢀNH with PCY1:S562A. b) Thermodynamic parameters derived from
equilibrium binding experiments for a selection of presegetalins. c) Chemical
structure of PresegA1 variants.
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Figure 5: Expanding the substrate scope of PCY1. Native cyclic peptides (blue),
cyclic peptides containing nonꢀamino acids and nonꢀnative sequences produced by
PCY1 via macrocyclization.
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Figure 1
a
NH
O
O
O
H
H
H N
N
N
2
N
N
FQAKDVENASAPV
H
H
O
O
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P gB1
Pr er es es egetalinB1
PCY1
O
O
O
NH HN
NH
+
FQAKDVENASAPV
O
NH
HN
SegB1
TailB1
H
N
Peptide bond
formed
O
b
PresegF1
PresegD1
PresegA1
PresegB1
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