Translation with D-amino acids

179 Pages·2015·28.71 MB·English

by  John Achenbach

Through the mirror: Translation with D-amino acids

vorgelegt von
Dipl. Ing.
John-Philipp Achenbach
geb. in Henstedt-Ulzburg

von der Fakultät III – Prozesswissenschaften
der Technischen Universität Berlin
zur Erlangung des akademischen Grades
Doktor der Ingenieurwissenschaften
- Dr.-Ing. -

genehmigte Dissertation

Promotionsausschuss:
Vorsitzender: Prof. Dr. Jens Kurreck
Gutachter: Prof. Dr. Roland Lauster
Gutachter: Prof. Dr. Knud H. Nierhaus
Gutachter: Prof. Dr. Juri Rappsilber

Tag der wissenschaftlichen Aussprache: 15.07.2015

Berlin 2015
1

1 Abstract
In a living cell, peptides or proteins produced by ribosomal translation of a messenger RNA
consist exclusively of L-amino acids (L-aa), although D-amino acids (D-aa) are prevalent in
nature. The bacterial translation apparatus holds several mechanisms to counteract the
incorporation of D-aa into peptides or proteins by ribosomal synthesis, because the
accidental incorporation of D-aa may hamper the folding of a nascent protein into its active
conformation. Accumulation of such misincorporations would have lethal consequences for a
cell.
The chirality of the protein building blocks is controlled at four sequent checkpoints:
1. Aminoacyl-tRNA synthetases (aaRS) esterify amino acids to tRNA molecules, giving
rise to aminoacyl-tRNA. Most of the enzymes are stereoselective and do not charge
D-amino acids.
2. D-tyrosyl-tRNA deacylase controls the formed aminoacyl-tRNAs and selectively
deacylates D-amino acids.
3. Elongation factor Tu (EF-Tu) is a translation factor that binds to aminoacyl-tRNA and
shuttles it to the ribosome, but strongly discriminates against D-aminoacyl-tRNA.
4. The ribosome uses aminoacyl-tRNA as a substrate to build up peptide chains in an
mRNA sequence dependent manner. The ribosome is also stereoselective and can
reject D-aminoacyl-tRNA.
This work focuses on the bypassing of stereoselectivity of the E. coli translation apparatus to
achieve efficient site-directed incorporation of D-amino acids, which can be useful both for a
number of applications such as drug discovery by in vitro translation based techniques as
well as to deepen the understanding how stereoselectivity is established on a molecular
level. This goal has been achieved by the following means:
1. D-amino acids are attached to tRNA molecules using ribozymes (Flexizymes) instead
of aminoacyl-tRNA synthetases.
2. A defined cell-free in vitro translation system made of individually purified translation
factors and ribosomes or mutants thereof was prepared that does not contain
D-tyrosyl-tRNA deacylase.
3. To enable EF-Tu mediated delivery of D-aa-tRNA to the ribosome, the D-aa were
attached to a tRNA that shows a high intrinsic affinity for EF-Tu.
4. The equilibrium between D-aa-tRNA rejection and D-aa incorporation by the ribosome
was shifted towards incorporation by using a tRNA that also shows a high intrinsic
affinity to the ribosomal A-site.
The efficient incorporation of 17 different D-aa out of 18 D-aa tested could be demonstrated
and in some cases also the incorporation of consecutive D-aa.
2

2 Kurzzusammenfassung
Peptide und Proteine, die in lebenden Zellen durch ribosomale Translation einer messenger
RNA hergestellt werden, bestehen ausschließlich aus L-Aminosäuren, obwohl auch
D-Aminosäuren in der Natur verbreitet vorkommen. Der prokaryotische Translationsapparat
hält etliche Mechanismen bereit, um den Einbau von D-Aminosäuren in Peptide oder
Proteine durch ribosomale Synthese zu verhindern, da der zufällige Fehleinbau von
D-Aminosäuren die Faltung eines Proteins in seine aktivie Konformation behindern kann. Die
Anhäufung derartiger Fehleinbauten hätte letale Konsequenzen für eine Zelle.
Die Chiralität der Proteinbausteine wird an vier aufeinanderfolgenden Kontrollstellen
überwacht:
1. Aminoacyl-tRNA Synthetasen verestern Aminosäuren mit tRNA Molekülen, wobei
Aminoacyl-tRNA entsteht. Die meisten dieser Enzyme sind stereoselektiv und
beladen tRNA nicht mit D-Aminosäuren.
2. D-Tyrosyl-tRNA Deacylase kontrolliert die gebildete Aminoacyl-tRNA und deacyliert
selektiv D-Aminosäuren.
3. Elongationsfaktor Tu ist ein Translationsfaktor, der Aminoacyl-tRNA bindet und zum
Ribosom transportiert, aber D-Aminoacyl-tRNA dabei stark benachteiligt.
4. Das Ribosom verwendet Aminoacyl-tRNA als Substrat, um Peptidketten gemäß der
Sequenz der codierenden messenger RNA zu bilden. Das Ribosom ist ebenfalls
stereoselektiv und kann D-Aminoacyl-tRNA aussortieren.
Diese Arbeit beschäftigt sich damit, wie die Stereoselektivität des Translationsapparats von
E. coli in einem definierten, zellfreien in vitro Translationssystem bestehend aus individuell
aufgereinigten Translationsfaktoren und Ribosomen oder Mutanten derselben umgangen
werden kann. Das Ziel war dabei, effizienten, gerichteten Einbau von D-Aminosäuren zu
erreichen, was sich sowohl für einige Anwendungen wie z.B. der Identifikation neuer
Wirkstoffe mit Hilfe von auf in vitro Translation basierenden Techniken als sinnvoll erweisen
kann, wie auch generell das Verständnis dafür verbessert, wie Stereoselektivität auf
molekularer Ebene funktioniert. Das Ziel wurde durch die folgenden Maßnahmen erreicht:
1. D-Aminosäuren wurden mit Hilfe von Ribozymen (Flexizymes) an tRNA gekoppelt.
2. Das Translationssystem besteht nur aus einzeln aufgereinigten Komponenten und
enthält keine D-Tyrosyl-tRNA Deacylase.
3. Um den Transport von D-Aminoacyl-tRNA zum Ribosom durch EF-Tu zu
gewährleisten, wurde eine tRNA gewählt, die eine besonders hohe, intrinsische
Affiniät zu EF-Tu aufweist.
4. Das Gleichgewicht zwischen Aussortierung von D-Aminoacyl-tRNA und Einbau von
D-Aminosäuren durch das Ribosom wurde durch Verwendung einer tRNA, die eine
besonders hohe intrinsische Affinität zur ribosomalen A-Stelle hat, in Richtung des
Einbaus verschoben
Zusammenfassend konnte der effiziente Einbau 17 verschiedener D-Aminosäuren von 18
getesteten gezeigt werden, und in einigen Fällen sogar der Einbau aufeinanderfolgender D-
Aminosäuren.
3

3 Table of Contents
1 Abstract .......................................................................................................................... 1
2 Kurzzusammenfassung .................................................................................................. 2
3 Table of Contents ........................................................................................................... 3
4 Introduction .................................................................................................................... 5
4.1 Motivation and Aim of the Project ............................................................................ 6
4.2 Translation in Bacteria ............................................................................................. 8
4.3 Stereoselectivity of the Translational Apparatus .....................................................49
4.4 In vitro Translation Systems ...................................................................................51
4.5 Misacylation of tRNA ..............................................................................................52
5 Methods ........................................................................................................................54
5.1 Gel Electrophoresis ................................................................................................54
5.2 Cloning ...................................................................................................................55
5.3 Protein Expression .................................................................................................56
5.4 Protein Purification .................................................................................................57
5.5 3’-32P-labeling of tRNA ...........................................................................................59
5.6 Synthesis of Substrates for Flexizyme ....................................................................60
5.7 Acylation of tRNA by Flexizyme..............................................................................60
5.8 Precipitation of Nucleic Acids .................................................................................61
5.9 Quantification of Aminoacylation Ratio ...................................................................61
5.10 Activity Tests of Recombinant Proteins ..................................................................62
5.11 Production of Fully Modified tRNAGly by Ligation of Fragments ...............................63
5.12 EF-Tu Electromobility Shift Assay ..........................................................................64
5.13 Analytical Expression and Purification of Mutant Ribosomes ..................................64
5.14 Large-Scale Expression and Purification of Mutant Ribosomes ..............................65
5.15 5’ 32P Labeling of Oligonucleotides .........................................................................67
5.16 Relative Quantification of Mutant versus Wildtype Ribosomes ...............................67
5.17 Assembly of the Translation System ......................................................................68
5.18 Activity Test of the Translation System ...................................................................70
5.19 Translation and Analysis of 35S-Met Labeled Peptides ...........................................70
5.20 Enantioselective LC-MS Analysis of Translated Peptides .......................................71
4

6 Results ..........................................................................................................................72
6.1 Getting started ........................................................................................................72
6.2 Establishing the in vitro Translation System ...........................................................73
6.3 Mutant Ribosomes .................................................................................................76
6.4 tRNA Syntheses .....................................................................................................79
6.5 Enzymatical Charging of D-Amino Acids ................................................................82
6.6 Synthesis, Enantiopurity and Aminoacylation of Flexizyme Substrates...................84
6.7 Formation of Ternary Complexes with D-Aminoacyl-tRNA .....................................86
6.8 Translation of Peptides Containing D-Amino Acids.................................................95
7 Discussion ...................................................................................................................1 05
7.2 Charging tRNA with D-Amino Acids ..................................................................... 109
7.3 Bypassing the Stereoselectivity of EF-Tu .............................................................1 13
7.4 Translation with D-amino acids.............................................................................1 23
7.5 Summary and Outlook ..........................................................................................1 28
8 Acknowledgements .....................................................................................................1 31
9 Declaration of Originality .............................................................................................1 32
10 Abbreviations ...........................................................................................................1 32
11 Materials ..................................................................................................................1 35
11.1 Bacterial Strains ...................................................................................................1 35
11.2 Equipment ............................................................................................................1 35
11.3 Consumables .......................................................................................................1 36
11.4 Reagents ..............................................................................................................1 38
11.5 Primer ..................................................................................................................1 41
12 Tables ......................................................................................................................1 43
13 Figures ....................................................................................................................1 44
14 Publications .............................................................................................................1 46
15 Literature .................................................................................................................1 47
5

4 Introduction
All naturally occurring nucleic acids consist of D-configured nucleotides, whereas all peptides
or proteins arising from translation of a gene exclusively consist of L-configured amino acids.
This phenomenon is known as the homochirality of life (1). However, D-amino acids are
found in peptides or proteins from all kingdoms of life, e.g. in bacterial cell walls, antibiotics,
crustacean peptide hormones, venoms from snails, funnel web spiders or the enigmatic
platypus (2-4). They add structural features that cannot be constituted by L-amino acids
alone (5,6). While D-amino acids do occur in peptides or proteins, they are never
incorporated by the ribosome in a site-directed manner. D-amino acid containing peptides or
proteins arise from either post-translational isomerization (2,5,6) or non-ribosomal peptide
synthesis (2,7). The translational apparatus tightly controls the chirality of its substrates in
order to exclude D-amino acids from translation. This is of vital importance, since accidental
misincorporation of a D-amino acid into a protein may hamper its folding into an active
conformation, thus perturbing its activity (4,8). Protein synthesis belongs to the most energy-
consuming processes in a cell (9). If misincorporation-events accumulate, thus a
considerably high ratio of inactive proteins are produced at the full cost of energy, the
consequences may be lethal. In this respect, it is worthy to mention that especially highly
processed or fermented foods show a high content of D-aa, which can be as high as 10% of
the total amino acid content (10,11). The presence of D-amino acids in living cells
presumably upholds the selective pressure that forces maintainance of a rigid defense of the
translational apparatus against D-amino acids.
Stereoselectivity of the translational apparatus arises from four sequent check points. On its
path into a protein, an amino acid is first selected and charged onto a tRNA molecule by an
aminoacyl-tRNA synthetase (aaRS) via formation of an aminoacyl-ester bond. Aminoacyl-
tRNA synthetases are specific for both amino acid and tRNA species, thus pairing their
cognate substrates. The majority of aaRS do not recognize the optical antipodes of their
cognate L-amino acid as a substrate and some even discharge erroneously esterified
D-amino acids (12-14). D-tyrosyl-tRNA deacylase marks the second checkpoint, this enzyme
can deacylate several D-amino acids from tRNA (4,12,14-20). The third checkpoint is
elongation factor Tu (EF-Tu), a translational GTPase serving one vitally important task:
Binding aminoacyl-tRNA in a ternary complex (aminoacyl-tRNA•EF-Tu•GTP) and delivering
it to the ribosome, which uses aminoacyl-tRNA as a substrate for protein synthesis. EF-Tu is
also stereoselective. It has been demonstrated for one exemplary D-aminoacyl-tRNA that
EF-Tu shows a substantially decreased affinity in comparison to the corresponding
L-aminoacyl-tRNA (21). It is assumed that it acts as a bouncer, denying D-aminoacyl-tRNAs
the access to the ribosome (21-24). The ribosome itself is also stereoselective, so even if a
6

D-aminoacyl-tRNA is formed and delivered to the ribosome, it can still be rejected instead of
being used as a substrate for protein synthesis (21,25-27). For the incorporation of
D-tyrosine into a protein in presence of L-tyrosine, Yamane et al. calculated the factors by
which L-tyrosine is favored over D-tyrosine as follows: 25 (aminoacylation step) × 25 (ternary
complex formation) × 10 (EF-Tu mediated delivery to the ribosome) × 5 (peptide bond
formation), totaling in an overall stereoselectivity factor of ~3 × 104, virtually excluding
D-tyrosine from translation as long as L-tyrosine is present (21). Notably, tyrosyl-tRNA
synthetase belongs to the aaRS with markedly reduced stereoselectivity (15,28-31), thus the
selectivity against other D-amino acids could be stronger by orders of magnitude. For
comparison, misincorporation of canonical L-amino acids occurs at a rate of one in 103 – 104
(32).
4.1 Motivation and Aim of the Project
In the past decades, the interest in peptides and proteins containing D-aa or entirely
composed of D-aa has grown significantly for various reasons. For example, deltorphins are
heptameric peptides isolated from the skin of the South American leaf frog Phyllomedusa
sauvagei containing one D-amino acid (D-Met, D-Leu or D-Ala) in position 2 and have been
shown to be potent opioids, whereas the corresponding all-L-peptides are virtually inactive
on the δ-opioid receptor (33,34). The same applies analogously for many other D-aa
containing peptides. Combining L-aa and D-aa in a peptide gives access to structural motifs
that cannot be constituted by amino acids of only one chirality. This can be reconciled in the
following light: the dihedral φ-angle used to describe the torsion of a peptide bond is negative
for L-amino acids and conversely, it is positive for D-amino acids. Glycine, which has no
stereocenter, is the only canonical amino acid that can adapt both positive and negative
φ-angles. The co-crystal structure of a serine protease and a peptide ligand that was
developed as an inhibitor to this protease, revealed a positive φ-angle for one of the
inhibitor’s glycyl-residues, prompting the authors to replace it by a D-amino acid. Indeed, this
measure improved both target inhibition and resistance to proteolytic cleavage (35). These
are just two examples for why one should consider D-amino acids in the development of new
peptidic pharmacological modalities.
Peptides composed entirely of D-aa also pose an interesting substance class for drug
development. Their unnatural chirality protects them from proteases such that their
biostability is strongly increased and they might even be orally administered. Furthermore,
assumptions are that D-peptides are less immunogenic.
One possible way to identify new drugs composed of D-aa is the so-called mirror-image
phage display. Using a phage library, peptides composed of regular L-aa are selected that
7

bind to a mirror-image of the dedicated target molecule – in most cases a protein that has to
be chemically synthesized from D-amino acids. The key aspect of the phage display method
is linking the active peptide together with the encoding nucleic acid, which is amplifiable and
its sequence is easy to determine. The necessity of mirroring the target molecule arises from
the stereoselectivity of the translational apparatus; the peptide library is expressed by
ribosomes of phage-infected bacteria and thus must consist of L-aa. The mirror image of the
identified L-peptide, the corresponding D-peptide, then binds to the natural target molecule
composed of L-amino acids (36,37). This method has been applied e.g. to generate inhibitors
of HIV-1 cell entry (37). Chemical synthesis of the mirror-image target molecule as a bait for
the selection procedure is often challenging or not possible with presently available
techniques, although much progress has been achieved in chemical protein synthesis. Most
importantly, peptide ligation techniques emerged (native chemical ligation (38,39), segment
condensation, KAHA ligation (40-42), ligation mediated by proteases such as clostripain (43-
45)), which give access to the chemical synthesis of peptides and proteins of about 200
amino acids length. Mirror image baits are also required for the selection of Spiegelmers,
which are L-nucleic acid (‘mirror-image’) aptamers that are resistant to nuclease degradation
and virtually invisible to the immune system due to their non-natural chirality (46-48).
Further methods for the identification of pharmacologically interesting peptide ligands include
ribosome display (49,50), mRNA display (51) and TRAP display (52), which share a common
technique: a cell-free in vitro translation system is used to translate an mRNA library into a
peptide library, followed by a selection step to separate target-binding and non-binding
peptides along with their encoding mRNAs. This allows to amplify mRNA sequences coding
for target-binding peptides after each round of selection and to obtain the sequence
information after the final selection round.
The armamentarium of these techniques may be greatly expanded by including D-amino
acids in the translation procecces for reasons as given above. However, it was almost seen
as a dogma that the translational apparatus is unable to incorporate D-amino acids when this
work was started. For decades, many renowned workgroups had attempted to demonstrate
D-amino acid incorporation by the ribosome, but the results were disappointing and will be
discussed in more detail in chapter 7 (p. 105). Two publications stood out of the crowd: The
Hecht group demonstrated the incorporation of either one D-Met- or one D-Phe-residue into
a protein (53), whereby the authors used mutant ribosomes with an impaired accuracy (54);
wildtype ribosomes barely showed any incorporation of the respective D-aa. The other report
showed the incorporation of D-amino acids at the N-terminus of a peptide (55).
8

The aim of the project is to find a way to efficiently incorporate D-amino acids into peptides
by ribosomal synthesis in a site-directed manner. The mechanisms of stereoselectivity of
both EF-Tu and the ribosome are poorly understood. Current knowledge is mostly based on
hypotheses and isolated experiments rather than systematic testing of putative parameters.
The challenge is to identify the molecular determinants for stereoselectivity and to find ways
to bypass or overcome stereoselectivity of the translational apparatus, which requires an in-
depth understanding of translational processes.
Starting with a brief overview of translational processes to provide some initial orientation
and an introduction to the general structure of the ribosome, the chapter below gives a
comprehensive review on the whole process of translation and describes the mechanisms of
stereoselectivity as far as they have been elucidated. This is followed by an introduction to
in vitro translation systems and strategies to incorporate non-canonical amino acids using
these systems.
4.2 Translation in Bacteria
The process of translation in a nutshell: ribosomal translation of a genetic message into a
protein can be divided into four phases: Initiation, elongation, termination and recycling (56).
As shown in Figure 1, a large number of proteins are involved in translation. In brief: Before
translation begins, a DNA-dependent RNA polymerase transcribes a DNA encoded gene into
a messenger RNA (mRNA), providing the molecular blueprint for the protein to be translated
(see 4.2.2, p. 14). 20 aminoacyl-tRNA synthetases (aaRS) and methionyl-tRNA
formyltransferase (MTF) assemble aminoacyl-tRNA, the substrates for translation (see 4.2.3,
p. 15). Three initiation factors (IF1, IF2, IF3) help to prepare the ribosome for the translation
of a genetic message (see chapter 4.2.4, p. 19). Assisted by elongation factor EF-Ts, the
elongation factors EF-Tu and EF-G direct the growth of a nascent peptide chain, which is
catalyzed by the ribosome (see chapter 4.2.5, p. 25). When the translation of a protein is
finished, three termination factors (RF1, RF2, RF3) guide the release of the translated
protein from the ribosome (see chapter 4.2.6, p. 44). Afterwards, joint action of the ribosome
recycling factor (RRF) and EF-G can split the subunits and leave the ribosome in a state, in
which the initiation factors can again prepare it for the translation of the next message (see
chapter 4.2.7, p. 46). In an alternative pathway termed “70S scanning mode”, the 70S
ribosome can re-initiate without splitting into subunits (see chapter 4.2.4.3, p. 24).
All of the processes described in the following text proceed in the experiments described in
this work. Some of the processes, i.e. DNA transcription (p. 14), translation initiation (p. 19-
24), forward translocation (p. 37-42), termination (p. 44-46) and recycling (p. 46-48) are not
essential to understand this work but rather serve review purposes.
9

Figure 1 | Bacterial translation at a glance. 30S binding initiation: Initiation factors IF1, IF2 and
IF3, the initiatior-tRNA (fMet-tRNAfMet) and the mRNA bind to the small ribosomal subunit (30S). The
large ribosomal subunit then joins and the initiation factors dissociate. Elongation: Elongation factor
EF-Tu delivers an aminoacyl-tRNA to the ribosomal A-site. If the mRNA codon exposed at the A-site
and the anticodon of the tRNA match, the tRNA is accommodated in the A-site and EF-Tu dissociates.
The ribosome facilitates peptide bond formation, transferring the amino acid or peptide attached to the
P-site-tRNA to the amino acid attached to the A-site tRNA, prolonging the nascent peptide chain by
one amino acid. Elongation factor EF-G then promotes translocation of the two ribosome bound tRNA
molecules from the A- to the P-site and from the P- to the E-site, respectively. Simultaneously, the
mRNA is translocated and EF-G dissociates. EF-Tu can now deliver the next tRNA to the A-site and
the E-site tRNA is ejected. In case a ribosome is stalled after a faulty translocation, EF4 can back-
translocate the tRNA from P- and E-site to A- and P-site. Termination: If a stop-codon is exposed at
the A-site, a class I release factor (RF1 or RF2) releases the translated protein and is subsequently
removed from the ribosome by the help of RF3, a class 2 release factor. The ribosome can now enter
either the recycling phase or the 70S scanning initiation mode. Recycling: By combined action of the
ribosome recycling factor (RRF), EF-G and IF3, the ribosomal subunits are dissociated and tRNA and
mRNA are removed, thus preparing the ribosome for a new 30S binding initiation. 70S scanning
initiation: Bacterial mRNAs are often polycistronic, encoding several genes in a row. Following
termination, the ribosome can scan the mRNA for another initiation signal in proximity to the stop
codon and re-initiate without subunit dissociation. This figure has been prepared by Dr. Jaroslaw Kijek
and Prof. Dr. Knud H. Nierhaus, used with kind permission.