Sandbox 666

From Proteopedia

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Hello, this Sandbox is reserved for a student project. It describes the structure of the endonuclese restriction enzyme Eco RI.

Presentation

EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium, which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses) in both strands. Bacterial DNA is protected by a specific methylation of EcoRI recognition sequences.

Reaction

EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor, which is the divalent ion Mg²⁺. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends, which are complementary.

Structure

EcoRI is composed of two homodimers, so it has two identical subunits (Representation of one ) of 31 kDa, but it is possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core, which is a α/β domain. There are several non-contiguous structural elements, whose are involved in DNA recognition^[1]. The constitutive subunits of EcoRI are organized into a single α/β domain (five strands sheet, which is surrounded by ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others^[2]. Four helices (two of each subunit) recognize the major groove and bring residues, whose interact with DNA bases and backbones.

The topology of EcoRI restriction endonuclease^[1].× catalytically important amino acid residues, • amino acid residues in interactions with DNA, pink regions: dimerization contacts

In the old model, the N-terminal section of each subunit forms the inner arm, which wraps around the DNA molecule (the arm brings the DNA molecule to the catalytic cleft). In the recent model, the extended chain motif (Met¹³⁷ to Ala¹⁴²) is a segment of the extended polypeptide chain, which runs through the major groove of the DNA, roughly parallel to the DNA backbone^[2] and forms the specific contacts of the enzymes to the DNA. The outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).

The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition: Arg²⁰⁰, Glu¹⁴⁴ and Arg¹⁴⁵. These amino acids are shown in red . Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. There are also two arms, whose establish contacts with DNA backbones outside the recognition sequences.

There is one ß-strand parallel to the DNA backbone, which contains amino acid residues essential for catalysis (e.g.: residues engaged in phosphate contacts^[1]). The reaction is due to a catalytic sequence motif, which is found in most type II restriction endonucleases: the PD…(D/E)XK motif. For EcoRI, is PD⁹¹ …E¹¹¹AK and the lysine residue is essential to the catalysis, but the proline residue is not important. This motif is also responsible for Mg²⁺ binding (Asp90 and Glu111)^[3].

This binding by the major groove is due to the position of scissile phosphodiester bonds.^[1]

The catalytic core of Eco RI with the target DNA. The Amino acids responsable for the reaction are shown in red and the target phosphodiester bond between the residues G and A is shown in blue

Catalytic mechanism^[1]

The function of EcoRI is to cleave infectious DNA before it is methylated or before it begins to cause damage in the bacterial cell. That’s why EcoRI is able to bind non-specifically anywhere to the DNA and continuously scans the DNA in a linear diffusion process with an approximate rate of 7*10⁶ bp.s^-1. In this way, the protein targets very quickly its specific sequences and any recognition sites is forgotten. This linear diffusion is due to electrostatic interactions; thence the DNA is trapped by EcoRI but can move easily. Indeed during non-specific binding, there is no direct interaction with the bases of the DNA but there are only five interactions between amino acid residues from each subunit and the DNA phosphate groups. Moreover around 110 water molecules are located into this complex. It was observed that the enzyme makes pauses when the site is very similar to the recognition site (affinity of the enzyme to these sites). When one of these sites or a methylated site are cut, the reactional process is very slow and takes place only in one strand of the DNA, so the cellular DNA ligase can repair the nicked DNA.

During the formation of the specific complex, the majority of the water molecules and ions are released and more and more direct interactions between the enzyme and the DNA are formed: some water molecules are immobilized between the enzyme and the DNA and formed hydrogen bounds, whose are important for the recognition and catalysis. Metal divalent ions, Mg²⁺, play also an essential role in the recognition process. The two homodimeres of EcoRI cooperate in the binding and cleavage of their substrate and are symmetrically bound to the DNA. The specific complex formation leads to conformational changes of the protein and the DNA, thus catalytic centers of the both subunits are activated and the both DNA strands are simultaneously cleaved. Specific binding bend the DNA by about 12° and involve interactions between EcoRI and DNA bases and phosphodiester backbones over approximately 10-12 bp. Moreover the residues involve to binding could participate at the catalysis. The number of interactions between EcoRI and the recognition sequence is maximal. The restriction enzymes are highly specific and highly cooperative.

Application of EcoRI in molecular biology

Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning, which is very important if you want to insert a gene in an expression vector.

Links

[1] 1ERI in the Protein Database (PDB)

References

1. ↑ ^{1.0 1.1 1.2 1.3 1.4} Recognition and cleavage of DNA by type-II restriction endonucleases, Pingoud A. & Jeltsch A. Eur.J.Biochem. 246,1-22 (1997)
2. ↑ ^{2.0 2.1} Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing, Kim, Y.C., Grable, J.C., Love, R., Greene, P.J., Rosenberg, J.M., Journal: (1990) Science 249: 1307-1309
3. ↑ Structure and function of type II restriction endonucleases Alfred Pingoud, Albert Jeltsch Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727. PMCID: PMC55916

Contributors

Raphaël BILGER, Virginie GROSBOILLOT