Student Projects for UMass Chemistry 423 Spring 2012-3

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Contents 1 EcoRV endonuclease 1.1 Introduction 1.2 Overall Structure 1.3 Binding Interactions 1.4 Additional Features 2 References 3 Credits

EcoRV endonuclease

spinqualitylabelsall models EcoRV is a restriction enzyme found in escherichia coli bacteria. It can be thought of as scissors for cutting DNA and applications are being researched in gene analysis and cloning. Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Introduction

EcoRV endonuclease is a type II restriction enzyme, or restriction endonuclease, found in e. coli bacteria. The main biological function of restriction endonucleases is to protect the cell's genome against any foreign DNA. Restriction enzymes recognize and cleave specific sequences of DNA. In the figure to the right, the enzyme is shown with the AAAGATATCTT, which includes the recognition site GATATC. The recognition site is shown . A type II restriction enzymes cleave at a short distance from the recognition site and often use Mg(2+) as a cofactor, as does this enzyme. They are commonly found in bacteria and shared structural features indicate that they are evolutionarily related.^[1] Type II endonucleases have been the site of much research because of applications in gene analysis and cloning and because they are great at modeling protein-DNA interactions.^[2]

Specifically, EcoRV is an orthodox restriction endonuclease. This means that the DNA sequence recognized is palindromic, meaning each strand contains the same sequence.^[3] The DNA duplex is cleaved at the phosphodiester bond located at 5'-GAT*ATC-3'. The other DNA strand will also be cleaved in the same location, producing blunt ends. The cleavage site is shown . This is relatively unique among restriction enzymes, as many cleave each DNA stand at a different location, leaving what are known as sticky ends. Cleavage occurs by the breaking of the bond between a 3' oxygen and the phosphorus by nucleophilic attack by water.^[4] Mg(2+) acts as a catalyst for this reaction, however it is not shown in the representation to the right.^[5] The process of DNA cleavage is covered in more detail in the Binding Interactions section.

The steps of DNA cleavage are as follows. EcoRV binds to the DNA without specificity, which is followed by a diffusional walking "search" down the DNA molecule. If the protein encounters its recognition site, conformational changes occur in the enzyme-DNA complex.^[6] These changes are discussed in more depth in the additional features section.

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Overall Structure

spinqualitylabelsall models EcoRV Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

EcoRV endonuclease is functional as a consisting of two monomers; monomer B and monomer A are in a U shape. Each monomer consists of 245 amino acids arranged in alpha/beta secondary structures. These monomers are identical in their sequencing but not their structure.

The monomer's structure are identical in all but two sets of residue numbers. There are 9 identical alpha helices shown and 10 identical beta strands shown .

The structures at residue number 144-150, where monomer A has an additional alpha helix and a residue numbers 150-153, where monomer B has an additional beta strand.

At the point on the U dimer where the monomers meet there is a 5 strand . This anti-parallel beta sheet is made of 3 stands from monomer B and 2 strands from monomer A. This beta sheet also has three alpha helices packed against it shown . Two of these alpha helicies are from monomer A and the other is from monomer B This structure assists in the structure and stability of the dimer as a whole. Once you , you can see how these beta strands form one anti-parallel beta sheet connecting the two monomers at the bottom of the U shape.

There are two structural sub-domains. The first, called the dimerization sub-domain,shown , is the smallest of the sub-domains, residue numbers 19-32, 150-160, and 144-150 of monomer A. This section forms all of the dimer interface interactions and stabilizes the dimer. This sub-domain is unique to EcoRV and only one other restriction endonuclease. The second is called the DNA binding sub-domain shown , residue numbers 2-18 38-140 and 167-243. This sub-domain is where the majority of the dimer's function occurs. This sub-domain contains the loops that are responsible for the interactions with DNA which include binding to and breaking it's bonds. The remaining segments of amino acids make up the flexible linkage between the two sub-domains, residue numbers 33-37,141-143, and 161-165 shown . This flexibility allows for the enzyme to open up and the monomers to slightly separate during the free enzyme form. ^[7]

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Binding Interactions

spinqualitylabelsall models EcoRV Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

The EcoRV molecule is a type II restriction endonuclease, which means that its purpose is to cleave DNA at the center of its , which is the site that the enzyme recognizes on the DNA to be cleaved. DNA recognition sites on the EcoRV molecule, called R-loops, bind to the major grooves of the double stranded DNA at its recognition sequence, which is GATATC, by hydrogen bonding. These hydrogen bonds make the DNA form a kinked conformation that is later stabilized by the addition of the Mg2+ ion. The Mg2+ ion is a catalyst that causes the DNA to shift in a way that increases the rate necessary for the DNA cleavage. It does not bind to EcoRV until the enzyme is prepared to cleave the DNA helix. When the enzyme is ready to cleave, the affinity for the Mg2+ ion increases and then it binds to the enzyme. The recognition sequence of the DNA is shown in yellow and it is cleaved at the center between the T and A base pairs on the D chain when interacted with a Mg2+ ion, leaving a blunt end in the DNA.

The of the enzyme is shown here in pink. It consists of four residues, Asp74, Asp90, Ile91, and Lys92, which participate in the binding of the Mg2+ ion along with binding interactions of the Adenine, A, base pair. Mg2+ binding only occurs in along one side of the DNA double helix. The Mg2+ binding site is formed when ionic interactions cause the slightly acidic Asp90 residue and the slightly negatively charged scissile phosphodiester group on the DNA strand to approach each other. This allows the Mg2+ ion to bind to this enzyme, also with ionic interactions between the positively charged Mg2+ and the partially negative charged . These molecules that bind to the Mg2+ ion are the carboxylate oxygen atoms from the Asp74 and Asp90 residues, the nonesterified oxygen from the scissile phosphodiester group, and three additional oxygen atoms from three water molecules. These six ionic bonds form an octahedral shape in the active site of this enzyme.

These six ionic interactions all have about the same binding distance except for one bond between the oxygen from the and the Mg2+ ion that is significantly longer. The five similar bond lengths are all about 2.08 Å, but the bond between Mg2+ and the Asp74 oxygen spans a distance of 2.9 Å. This is noted because the Asp90 and scissile phosphodiester molecules that bind to this Mg2+ ion change their bonding interactions with hydrogen to accommodate the addition of the Mg2+ ion. The Asp74 residue maintains its hydrogen bond interactions on its side chain with the main chain of the Ile91 residue and the water molecule, which is why it keeps a greater distance between itself and the Mg2+ ion.^[8]

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Additional Features

spinqualitylabelsall models EcoRV Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

As a prerequisite for efficient target site recognition, the enzyme is capable of nonspecific binding with a DNA molecule's Phosphate backbone. The non-specific Enzyme(PDB: 2RVE) slides along a DNA molecule with movement characterized by a helical diffusion due to hydrogen bonding along the minor groove . The biological significance of this process is accelerated target site location, increased processivity, and dissociation of the enzyme upon cleavage. Specific binding on the other hand is precisely the process of recognition which involves an interplay between interaction with the bases of the recognition sequence as well as indirect interaction with the respective phosphate backbone. Recognition precedes conformational changes in both DNA and the protein, thereby bending the DNA and activating the catalytic centers. In both the and complexes, the DNA is oriented so that the minor groove faces the floor of the Binding site.^[9]

Unlike R-loops (Recognition Loops, Residues 182-187) which are disordered in both the and complex(PDB: 1RVE). Q-Loops (Residues 68-71) are ordered in the , forming a beta turn. They are disordered in the . Hydrogen bond interactions in the non-specific complex are indicated in crystal structures of the between the two Glutamine, Aspargarine, and Histidine Residues of the Q-Loops and the DNA Phosphates. While Histidine and the two Glutamine residues contribute less to hydrogen bonding due to their high conformational entropy, Aspargarine forms the strongest hydrogen bonds with the Phosphates because of its short alkyl chain, and thus is a strong contributor to the ability of the Enzyme to diffuse Linearly along the DNA molecule.^[10]

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References

1. ↑ Kostrewa D, Winkler FK. Mg2+ binding to the active site of EcoRV endonuclease: a crystallographic study of complexes with substrate and product DNA at 2 A resolution. Biochemistry. 1995 Jan 17;34(2):683-96. PMID:7819264
2. ↑ Pingoud A, Jeltsch A. Structure and function of type II restriction endonucleases. Nucleic Acids Res. 2001 Sep 15;29(18):3705-27. PMID:11557805
3. ↑ Pingoud A, Jeltsch A. Structure and function of type II restriction endonucleases. Nucleic Acids Res. 2001 Sep 15;29(18):3705-27. PMID:11557805
4. ↑ Pingoud A, Jeltsch A. Structure and function of type II restriction endonucleases. Nucleic Acids Res. 2001 Sep 15;29(18):3705-27. PMID:11557805
5. ↑ Kostrewa D, Winkler FK. Mg2+ binding to the active site of EcoRV endonuclease: a crystallographic study of complexes with substrate and product DNA at 2 A resolution. Biochemistry. 1995 Jan 17;34(2):683-96. PMID:7819264
6. ↑ Pingoud A, Jeltsch A. Structure and function of type II restriction endonucleases. Nucleic Acids Res. 2001 Sep 15;29(18):3705-27. PMID:11557805
7. ↑ Winkler FK, Banner DW, Oefner C, Tsernoglou D, Brown RS, Heathman SP, Bryan RK, Martin PD, Petratos K, Wilson KS. The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments. EMBO J. 1993 May;12(5):1781-95. PMID:8491171
8. ↑ Kostrewa D, Winkler FK. Mg2+ binding to the active site of EcoRV endonuclease: a crystallographic study of complexes with substrate and product DNA at 2 A resolution. Biochemistry. 1995 Jan 17;34(2):683-96. PMID:7819264
9. ↑ Pingoud A, Jeltsch A. Structure and function of type II restriction endonucleases. Nucleic Acids Res. 2001 Sep 15;29(18):3705-27. PMID:11557805
10. ↑ Winkler FK, Banner DW, Oefner C, Tsernoglou D, Brown RS, Heathman SP, Bryan RK, Martin PD, Petratos K, Wilson KS. The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments. EMBO J. 1993 May;12(5):1781-95. PMID:8491171

Credits

Introduction - Jesse Guillet

Overall Structure - Nicole Bundy

Binding Interactions - Julia Tomaszewski

Additional Features - Sam Kmail