Team:Washington/Gram Positive/Design

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We have two main goals when redesigning CapD.  First we want to make it easier to express and more stable.  Second we want to improve the hydrolytic activity of CapD to better cleave Poly-D-γ-glutamate, making it a more potent Anthrax treatment.  
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=Making CapD a Better Anthrax Treatment=
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There are two main obstacles limiting natural CapD as an Anthrax therapeutic.  First, natural CapD is a difficult to express dimer, requiring an auto-cleavage to activate [[#References | [1]]].  Second, CapD is a better poly-γ-D-glutamate transpeptidase than poly-γ-D-glutamate hydrolase, limiting its Anthrax decapsulating potential.[[#References | [1]]]  To solve the first problem, we created a circular permutated, monomeric version of CapD that is easy to express and quantify.  To improve hydrolysis, we used [https://2010.igem.org/Team:Washington/Project/Tools/FoldIt FoldIt], a computational toolbox, to design active site mutations aimed to increase hydrolysis over transpeptidation.  
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Naturally produced CapD auto-cleaves itself to create a dimmer. The new N terminus contains a critical catalytic threonine residue which allows the enzyme to cut Poly-D-γ-glutamate. 
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==Is CapD_CP a better version of CapD?==
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[[Image:Washington_DtoCP_Graphic2.jpg|520px|right|Schematic of how we made a circularly permutated capD]]
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To increase stability and expression, we created a circular permutation of CapD.  Typically we view the primary structure of proteins as linear, but when the N and C termini are close together after folding, the protein can be though of as a circle with a small gap.  Frequently it is possible move the gap by creating a linker between the naturally occurring N and C termini and then changing the starting and ending residues.  This works so long as the amino acid sequence remains the same relative to itself (neighboring amino acids still have the same neighbors). For CapD we made the sight that normally auto-cleaves the N terminus, and added a FoldIt designed linker between natural CapD N and C terminus.  This circular permeated CapD, aka CapD_CP, is a monomer which give it greater stability and ease of purification.   
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When natural CapD is first translated, the key catalytic threonine residue is buried in the active site, rendering it inaccessible to poly-γ-D-glutamate. After auto-cleavage, this critical threonine becomes the new N terminus, which can take its place in the active site [[#References | [1]]]. By reordering the protein so the threonine is the first residue, and putting a FoldIt designed linker between the natural N and C terminus, we make a circular permutation of CapD that we named CapD_CP.  CapD_CP is a monomer, historically easier to purify and more stable than dimers.   
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Since the first residue of any nascent protein must be Methionine, we rely on E. Coli’s naturally occurring Methionine aminopeptidase to remove the first Methionine, making CapD_CP catalytically active.  The removal of the first methionine has been verified via mass spectrometry.
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Since the first residue of any nascent protein must be methionine, we rely on   E. Coli’s naturally occurring methionine aminopeptidase to remove the first methionine, making CapD_CP catalytically active.  The removal of the first methionine has been verified via mass spectrometry.  
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[[Image:CapDSimplifiedDiagram.jpg]]
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==Using [[Team:Washington/Tools_Used/Software|FoldIt]] to Make CapD_CP a Better Hydrolase==
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[[Image:CapDCPSimplifiedDiagram.jpg]]
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[[Image:WashingtonPointMutation.png|270px|frameless|right]][[Image:WashingtonShake.png|270px|frameless|right]]
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To increase the hydrolytic ability of CapD_CP we made point mutations in the active sight. We focused our attention on two types of mutations. First we created point mutations that made hydrogen bonds to a modeled transition state of our substrate in an attempt to stabilize the transition state. Second we mutated the active sight to be more open and polar in an attempt to increase the ease with witch water could enter and participate in a hydrolysis reaction.
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To increase the hydrolytic ability of CapD_CP, we made point mutations to the active site. We focused our attention on two types of mutations. [[Image:WashingtonWiggle.png|270px|frameless|right]] First type of mutations is to increase hydrolysis by lowering the activation energy. To accomplish this, we created point mutations that can establish hydrogen bondings to a modeled transition state of our substrate. Second type of mutations concerns with the openness and polarity of the active site. To accomplish this, we mutated the active site into a more open and polar area so water molecules can enter and participate in hydrolysis easily.  
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To make these point mutations we used a program called FoldIt to predict how changes in protein structure and composition will affect protein stability.  FoldIt provides the user a 3D representation of the crystal structure of a protein that can then be manipulated. Manipulation functions include point mutations, insertions, deletions, repacking of side chains, and backbone movement, which FoldIt then assesses for stability. This allows the user to quickly interact with a protein and easily predict how mutations will effect a protein.
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To make these point mutation designs, we used a computer program named FoldIt to predict how changes in protein structure and composition will affect protein stability.  FoldIt provides a 3D representation of a protein's crystal structure that can be manipulated. Manipulation functions include point mutations, insertions, deletions, repacking of side chains (rotamer optimization), and backbone movement, which FoldIt then assesses for stability. This allows the user to quickly interact with a protein and easily predict how mutations will affect a protein.
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[[Team:Washington/Tools_Used/Software|A more in depth explanation of FoldIt here.]]
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==References==
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1. Wu R, Richter S, Zhang RG, Anderson VJ, Missiakas D, Joachimiak A. Crystal structure of Bacillus anthracis transpeptidase enzyme CapD.  J Biol Chem. 2009 Sep 4;284(36):24406-14. Epub 2009 Jun 16. PMID: 19535342
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'''&larr; [[Team:Washington/Project/Baker|Overview of Gram(+) Therapeutic]]'''
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'''[[Team:Washington/Project/Baker/Build|Building the Gram(+) Therapeutic]] &rarr;'''
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'''[[Team:Washington/Gram Positive/Build|Building the Gram(+) Therapeutic]] &rarr;'''
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Latest revision as of 06:05, 27 October 2010

Making CapD a Better Anthrax Treatment

There are two main obstacles limiting natural CapD as an Anthrax therapeutic. First, natural CapD is a difficult to express dimer, requiring an auto-cleavage to activate [1]. Second, CapD is a better poly-γ-D-glutamate transpeptidase than poly-γ-D-glutamate hydrolase, limiting its Anthrax decapsulating potential. [1] To solve the first problem, we created a circular permutated, monomeric version of CapD that is easy to express and quantify. To improve hydrolysis, we used FoldIt, a computational toolbox, to design active site mutations aimed to increase hydrolysis over transpeptidation.

Is CapD_CP a better version of CapD?

Schematic of how we made a circularly permutated capD

When natural CapD is first translated, the key catalytic threonine residue is buried in the active site, rendering it inaccessible to poly-γ-D-glutamate. After auto-cleavage, this critical threonine becomes the new N terminus, which can take its place in the active site [1]. By reordering the protein so the threonine is the first residue, and putting a FoldIt designed linker between the natural N and C terminus, we make a circular permutation of CapD that we named CapD_CP. CapD_CP is a monomer, historically easier to purify and more stable than dimers.

Since the first residue of any nascent protein must be methionine, we rely on E. Coli’s naturally occurring methionine aminopeptidase to remove the first methionine, making CapD_CP catalytically active. The removal of the first methionine has been verified via mass spectrometry.



Using FoldIt to Make CapD_CP a Better Hydrolase

WashingtonPointMutation.png
WashingtonShake.png
To increase the hydrolytic ability of CapD_CP, we made point mutations to the active site. We focused our attention on two types of mutations.
WashingtonWiggle.png
First type of mutations is to increase hydrolysis by lowering the activation energy. To accomplish this, we created point mutations that can establish hydrogen bondings to a modeled transition state of our substrate. Second type of mutations concerns with the openness and polarity of the active site. To accomplish this, we mutated the active site into a more open and polar area so water molecules can enter and participate in hydrolysis easily.


To make these point mutation designs, we used a computer program named FoldIt to predict how changes in protein structure and composition will affect protein stability. FoldIt provides a 3D representation of a protein's crystal structure that can be manipulated. Manipulation functions include point mutations, insertions, deletions, repacking of side chains (rotamer optimization), and backbone movement, which FoldIt then assesses for stability. This allows the user to quickly interact with a protein and easily predict how mutations will affect a protein.

A more in depth explanation of FoldIt here.


References

1. Wu R, Richter S, Zhang RG, Anderson VJ, Missiakas D, Joachimiak A. Crystal structure of Bacillus anthracis transpeptidase enzyme CapD. J Biol Chem. 2009 Sep 4;284(36):24406-14. Epub 2009 Jun 16. PMID: 19535342






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