PEX5 variant R15

PEX5

Brief introduction

PEX5 is one of two proteins that mediate most of the protein import into the peroxisomal matrix. It recognizes the very c-terminal peroxisomal targeting signal 1 (PTS1), then folds itself to a more compact version and interacts with a variety of enzymes.
The whole process is divided into five steps: binding, transport, docking, translocation and receptor recycling.

Variant

We followed a targeted mutagenesis approach to achieve an orthogonal import mechanism − our PEX5 variant should recognize a non-native PTS1 variant which is not recognized with the wildtype PEX5.

Pex 5 mutagenesis – creating an orthogonal pathway

Introduction

With the worlds population rising, biotechnological production of substances like cosmetics, fuels and pharmaceuticals is of everincreasing importance. Unfortuneatly, the production of those is often inefficient due to unknown reactions or toxic intermediates inside the cell. Therefore, we want to create an intracellular space which offers complete control of its contents.

Our approach focuses on peroxisomes, hence they are already involved in metabolic and biosynthetic processes and furthermore, they are resistant against stress conditions and have a remarkable import mechanism for fully folded proteins. Given that, we divided our teams in subgroups which try to achieve the following things:

Protein import into the peroxisomal lumen

Pex5 based import

Pex7 based import

Integration of new proteins into the peroxisomal membrane

SNARE-based secretion into the extracellular space

Control of number and size

Optogenetic switches

Biosensors

To demonstrate the potential of our concept, we relocate the Violacein and Nootkatone pathway into the peroxisome – both substances are hard to synthesize due to toxic intermediates and by-products but we want to gain higher yields than currently possible.

Our subproject deals with the Pex5 based protein import – Pex5 is next to Pex7 one of two receptors that mediate protein import into the peroxisome . It recognizes the very twelve amino acids at the C-terminus of Proteins (PTS1) , binds them and then interacts with other proteins inside the peroxisomal membrane such as Pex13 and Pex14 which finally leads to the import. Then, Pex5 gets transported back into the Cytosol (see figure [fig:shuttle]).

<figure> <img src="shuttle" alt="General model for the peroxisomal protein import" /><figcaption>General model for the peroxisomal protein import</figcaption> </figure>

We want to mutate the Pex5 receptor in a way that it recognizes a new signal peptide which does not occur in nature. As Pex5 is responsible for most of the import, we have complete control over its contents once we knock out the wild type receptor and replace it with our mutated one. As this step is necessary to achieve our goal of an artificial compartment, our subproject is a crucial and central part of the whole project.

General information about the Pex5 protein

<figure> <img src="recwt" alt="Structural prediction of the yeast’s Pex5 receptor with the signal peptide YQSKL inside the binding pocket" /><figcaption>Structural prediction of the yeast’s Pex5 receptor with the signal peptide YQSKL inside the binding pocket</figcaption> </figure> <figure> <img src="tpr" alt="Human Pex5 with Pentapeptide in the binding pocket" /><figcaption>Human Pex5 with Pentapeptide in the binding pocket</figcaption> </figure>

Pex5 is a 612 amino acid protein which contains seven tetratrico peptide (TPR) regions – the TPR is a 34 amino acid motif which formes a structure of two α-helices seperated by one turn. A whole TPR domain consists of three of those strucutres. TPR domains are often involved in protein-protein interaction and as it can be seen in figure [fig:tpr] the TPR regions mediate the binding of the peroxisomal targeting signal.

Pex5 functionality is based on redox reaction – its cystein 10 is essential for the formation of disulfide based dimers/oligomers and thereby increases the affinity to the cargo. When it undergoes the change of redox balance between cytosol and the peroxisomal lumen the affinity decreases. Moreover, interaction with Pex8 helps to regulate cargo release inside the matrix. After cargo release Pex5 gets recylced and goes back to the cytosol (see figure [fig:shuttle]).

Methods

Molecular dynamics

Molecular dynamics is a computer simulation which can be used to study physical movements such as protein-proteins interactions. We use it to simulate our experiments before we actually do it in the lab. With that we can be much more efficient than we could be with a pure random mutagenesis approach.

l5cm <img src="workflow.png" alt="image" />

Our workflow is depicted in the figure [fig:workflow]. As you can see our work starts with the mutagenesis of our receptor. For that we use the predicted three dimensional structure of the yeasts Pex5 receptor, which is based on the crystal structure of the human Pex5 (pdb entry: 1FCH)<a href="#fn1" class="footnoteRef" id="fnref1">¹</a>. The mutagenesis is done on behalf of educated guesses that can be made thanks to the polarity of the amino acids. We therefore use Pymol’s built-in function to generate protein contact potentials between the receptor and a peptide. This enables us to design amino acid interactions that are not occurring naturally to finally get receptor-peptide combinations with altered affinity.

Once this is done, we do the equilibration. That means that all atoms in the receptor-peptide structure are at the energetically most favorable position<a href="#fn2" class="footnoteRef" id="fnref2">²</a>. After that we start the MD simulation in which the temperature slightly increases until 300K and in the end we can simulate the molecular movement over a certain period of time (mostly 500ns). We then do our numerical evaluation based on the diffusion of the signal peptide – the diffusion can be calculated with the coordinates that we obtain from the MD simulation. For visualization purposes we use the software Visual Molecular Dynamics package<a href="#fn3" class="footnoteRef" id="fnref3">³</a>, a tool to map the generated MD data to a graphical representation.

To ensure, that our experiments will lead to the anticipated result, we test all different combinations.

Wild type receptor with wild type peptide

Wild type receptor with our new peptide

Mutated receptor with wild type peptide

Mutated receptor with our new peptide

A comparison of this data reveals which receptor might do the job and those then will be tested in the laboratory.

Lab work

We chose Saccharomyces cerevisiae as expression organism and Escherischia coli for cloning purposes. As we use yeast, we decided to make use of the Yeast Modular Cloning Toolkit from the Dueber lab. With that we are able to do fast and versatile cloning, which helps us to experiment with lots of different promotors, terminators and many more.

0.45 <img src="lvl2" title="fig:" alt="Plasmids used for yeast co-transformation" />

0.45 <img src="pex11" title="fig:" alt="Plasmids used for yeast co-transformation" />

Our plan is to create a multi-gene plasmid which contains our modified Pex5-Rezeptor and a fluorescent protein with the signal peptide attached to it. Additionally we create a plasmid with a peroxisomal membrane protein, e.g. Pex11 or Pex13, with another fluorescent protein. With that we can lable the peroxisomal mebrane and verify if the import works. To validate the efficiency and ensure comparability to the wild type import, we plan to do quantitaive microscopy and select the best receptor peptide combination for our toolbox.

After the preparatory work with the MDs, we select the best receptor and order the DNA-sequence, provided with the specific overhangs for a part 3, by IDT. Once the order arrives, we use Golden Gate cloning to bring our gene into a level 0 vector. After validation by sequencing, we use this receptor to create a level 1 plasmid. Additionally we create level 1 plasmid with a certain peroxisomal targeting signal attached to a fluorescent protein, e.g. mTurqouise. Those two level 1 plasmids were designed to fit in one level 2 plasmid as it can bee seen in figure [fig:lvl2]. We then use

[a)]

our level 2 plasmid with the receptor-peptide combination and

the level 1 plasmid with Pex11-mRuby

for co-transformation of yeast.

Plan B – random mutagenesis

Since time is a bottleneck in this competition and computational simulations take a long time, we plan to do an alternative approach with random mutagenesis. For that, we want to use level 2 plasmids and randomly change bases within the essential TPR regions of the Pex5 receptor. With that, we can try lots of different variants and also safe time, because yeast can be transformed immediately without further cloning.

Outlook

The golden goal of our subproject would be to obtain a new receptor that does detect our PTS but not the wild type one. With that, complete control of the peroxisomes enzyme contents would be ensured and we would take a huge step forward to an artificial compartment. If we can understand the mechanisms how the development of peroxisomes really takes place, we could imagine future projects that work on the generation of two distinct peroxsiome based compartments – one that performs the role of a wild type peroxisome and one that is tailored to fulfill the specific requirements of the intended applications.

A biological system like that would be perfect for all kinds of synthetic biology applications – in general, unknown and uncontrollable reactions can affect the engineered design but as a result of compartmentalization those would not matter anymore.

This was done by Daniel Mulnaes and Markus Dick who is working for Professor Gohlke who kindly gave us the possibility to use his infrastructre and expertise for our project.<a href="#fnref1">↩</a>
This is under the assumption of 0K.<a href="#fnref2">↩</a>
http://www.ks.uiuc.edu/Research/vmd/<a href="#fnref3">↩</a>

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Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 409
1000
COMPATIBLE WITH RFC[1000]

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