Retroviral integrase

 

Retroviral Integrase (IN) catalyses viral DNA integration into the host chromosome by performing a series of DNA cutting and joining reactions. This enzyme activity takes place after virion entry into a cell and reverse transcription of the RNA genome into dsDNA. The Integrase reaction starts by the removal of two or three nucleotides from one or both ends of the viral DNA to expose 3'-hydroxyl groups. IN then catalyses the attack of the 3'-hydroyl groups to two phosphate groups of the host cell DNA, joining the two together.

 

Reference Protein and Structure

Sequence
P14350 UniProt (2.7.7.-, 2.7.7.7, 2.7.7.49, 3.1.-.-, 3.1.26.4, 3.4.23.-) IPR000721 (Sequence Homologues) (PDB Homologues)
Biological species
Human spumaretrovirus (Virus) Uniprot
PDB
4e7k - PFV integrase Target Capture Complex (TCC-Mn), freeze-trapped prior to strand transfer, at 3.0 A resolution (3.02 Å) PDBe PDBsum 4e7k
Catalytic CATH Domains
3.30.420.10 CATHdb (see all for 4e7k)
Cofactors
Magnesium(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:2.7.7.-)

DNA polyanion
CHEBI:83828ChEBI
+
hydroxide
CHEBI:16234ChEBI
+
DNA Polymer
CHEBI:X00680X00680
→
DNA Polymer
CHEBI:X00680X00680
+
DNA polyanion
CHEBI:83828ChEBI
+
hydroxide
CHEBI:16234ChEBI

Enzyme Mechanism

Introduction

A hydroxide ion formed from aqueous solution attacks the phosphate on viral DNA (vDNA) through another water molecule. Two or three nucleotides from one or both ends of the vDNA are removed to expose 3'-hydroxyl groups. Another hydroxide ion then directly deprotonates the 3'-hydroxyl of the vDNA, which performs a nucleophilic attack on host DNA (hDNA), inserting itself into the host gene.

Catalytic Residues Roles

UniProt PDB* (4e7k)
Asp936, Asp879, Glu972 Asp185(188)A, Asp128(131)A, Glu221(224)A Asp128, Asp185 and Glu221 coordinate two Mg2+ ions which stabilise and perform the catalysis. metal ligand
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

bimolecular nucleophilic substitution, coordination to a metal ion, proton transfer, overall product formed

References

  1. Araújo AR et al. (2014), J Chem Theory Comput, 10, 5458-5466. Catalytic Mechanism of Retroviral Integrase for the Strand Transfer Reaction Explored by QM/MM Calculations. DOI:https://doi.org/10.1021/ct500570g.
  2. Hare S et al. (2012), EMBO J, 31, 3020-3028. 3'-processing and strand transfer catalysed by retroviral integrase in crystallo. DOI:10.1038/emboj.2012.118. PMID:22580823.
  3. Ribeiro AJM et al. (2012), J Am Chem Soc, 134, 13436-13447. The Catalytic Mechanism of HIV-1 Integrase for DNA 3′-End Processing Established by QM/MM Calculations. DOI:https://doi.org/10.1021/ja304601k.

Step 1. A hydroxide ion activates an adjacent water molecule which then performs a nucleophilic attack on the phosphate group of the vDNA. Before the next step, the cleaved nucleotides (on the right) leave the active site and the host DNA binds in their place.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp185(188)A metal ligand
Asp128(131)A metal ligand
Glu221(224)A metal ligand

Chemical Components

ingold: bimolecular nucleophilic substitution, coordination to a metal ion, proton transfer

Step 2. A hydroxide ion deprotonates the 3'-hydroxyl of the vDNA, activating it for nucleophilic attack.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp185(188)A metal ligand
Asp128(131)A metal ligand
Glu221(224)A metal ligand

Chemical Components

proton transfer, coordination to a metal ion

Step 3. The deprotonated 3' oxygen of vDNA performs a nucleophilic attack on the phosphate of the hDNA.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp185(188)A metal ligand
Asp128(131)A metal ligand
Glu221(224)A metal ligand

Chemical Components

ingold: bimolecular nucleophilic substitution, overall product formed, proton transfer
Download: Image, Marvin File

Step 1. A hydroxide ion activates an adjacent water molecule which then performs a nucleophilic attack on the phosphate group of the vDNA. Before the next step, the cleaved nucleotides (on the right) leave the active site and the host DNA binds in their place.

Step 2. A hydroxide ion deprotonates the 3'-hydroxyl of the vDNA, activating it for nucleophilic attack.

Step 3. The deprotonated 3' oxygen of vDNA performs a nucleophilic attack on the phosphate of the hDNA.

Products.

Introduction

Asp185 deprotonates and activates an adjacent water molecule which performs a nucleophilic attack on the phosphate of the viral DNA (vDNA), exposing a 3'-hydroxyl. The 3'-hydroxyl is deprotonated by a water molecule, activated by Glu221. The deprotonated 3' oxygen of vDNA performs a nucleophilic attack on the host DNA (hDNA), inserting itself into hDNA. This reaction is unlikely given the unfavourable geometry of Glu221 and Asp185.

Catalytic Residues Roles

UniProt PDB* (4e7k)
Asp936 Asp185(188)A Asp185 deprotonates a water molecule which nucleophilically attacks a phosphorus, breaking a phosphodiester bond of the viral DNA. activator, metal ligand, proton acceptor
Asp879, Glu972 Asp128(131)A, Glu221(224)A Asp128, Asp185 and Glu221 coordinate two Mg2+ ions which stabilise and perform the catalysis. metal ligand
Glu972 Glu221(224)A Glu221 deprotonates the 3'OH through a bridging water molecule. activator, metal ligand, proton acceptor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, coordination to a metal ion, bimolecular nucleophilic substitution, overall product formed

References

  1. Araújo AR et al. (2014), J Chem Theory Comput, 10, 5458-5466. Catalytic Mechanism of Retroviral Integrase for the Strand Transfer Reaction Explored by QM/MM Calculations. DOI:https://doi.org/10.1021/ct500570g.
  2. Hare S et al. (2012), EMBO J, 31, 3020-3028. 3'-processing and strand transfer catalysed by retroviral integrase in crystallo. DOI:10.1038/emboj.2012.118. PMID:22580823.
  3. Ribeiro AJM et al. (2012), J Am Chem Soc, 134, 13436-13447. The Catalytic Mechanism of HIV-1 Integrase for DNA 3′-End Processing Established by QM/MM Calculations. DOI:https://doi.org/10.1021/ja304601k.

Step 1. Asp185 deprotonates and activates an adjacent water molecule which performs a nucleophilic attack on the phosphate of the vDNA.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu221(224)A metal ligand
Asp128(131)A metal ligand
Asp185(188)A metal ligand, proton acceptor, activator

Chemical Components

proton transfer, coordination to a metal ion, ingold: bimolecular nucleophilic substitution

Step 2. Glu221 deprotonates a water molecule that then deprotonates the 3'OH of the vDNA activating it for nucleophilic attack.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu221(224)A metal ligand
Asp128(131)A metal ligand
Asp185(188)A metal ligand
Glu221(224)A proton acceptor, activator

Chemical Components

coordination to a metal ion, proton transfer

Step 3. The deprotonated 3' oxygen of vDNA performs nucleophilic attack on the phosphate, inserting itself into the hDNA.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu221(224)A metal ligand
Asp128(131)A metal ligand
Asp185(188)A metal ligand

Chemical Components

proton transfer, overall product formed, ingold: bimolecular nucleophilic substitution
Download: Image, Marvin File

Step 1. Asp185 deprotonates and activates an adjacent water molecule which performs a nucleophilic attack on the phosphate of the vDNA.

Step 2. Glu221 deprotonates a water molecule that then deprotonates the 3'OH of the vDNA activating it for nucleophilic attack.

Step 3. The deprotonated 3' oxygen of vDNA performs nucleophilic attack on the phosphate, inserting itself into the hDNA.

Products.

Introduction

The viral DNA (vDNA) is cleaved as a result of its phosphate deprotonating a water molecule and its subsequent nucleophilic attack on the adjacent phosphate. This reaction exposes a 3'-hydroxyl which is deprotonated by another activated water molecule (activated by an adjacent phosphate group). This reaction was proven unfavourable by QM/MM studies.

Catalytic Residues Roles

UniProt PDB* (4e7k)
Asp936, Asp879, Glu972 Asp185(188)A, Asp128(131)A, Glu221(224)A Asp128, Asp185 and Glu221 coordinate two Mg2+ ions which stabilise and perform the catalysis. metal ligand
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, coordination to a metal ion, bimolecular nucleophilic substitution, overall product formed

References

  1. Araújo AR et al. (2014), J Chem Theory Comput, 10, 5458-5466. Catalytic Mechanism of Retroviral Integrase for the Strand Transfer Reaction Explored by QM/MM Calculations. DOI:https://doi.org/10.1021/ct500570g.
  2. Hare S et al. (2012), EMBO J, 31, 3020-3028. 3'-processing and strand transfer catalysed by retroviral integrase in crystallo. DOI:10.1038/emboj.2012.118. PMID:22580823.
  3. Ribeiro AJM et al. (2012), J Am Chem Soc, 134, 13436-13447. The Catalytic Mechanism of HIV-1 Integrase for DNA 3′-End Processing Established by QM/MM Calculations. DOI:https://doi.org/10.1021/ja304601k.

Step 1. A vDNA phosphate activates an adjacent water molecule which performs a nucleophilic attack on another phosphate of the vDNA. This cleaves the vDNA, leaving an exposed 3'-hydroxyl.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu221(224)A metal ligand
Asp128(131)A metal ligand
Asp185(188)A metal ligand

Chemical Components

proton transfer, coordination to a metal ion, ingold: bimolecular nucleophilic substitution

Step 2. A vDNA phosphate deprotonates the 3'-hydroxyl, creating a nucleophile.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu221(224)A metal ligand
Asp128(131)A metal ligand
Asp185(188)A metal ligand

Chemical Components

coordination to a metal ion, proton transfer

Step 3. The deprotonated 3' oxygen of vDNA performs nucleophilic attack on the phosphate, adding itself to the hDNA.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu221(224)A metal ligand
Asp128(131)A metal ligand
Asp185(188)A metal ligand

Chemical Components

proton transfer, overall product formed, ingold: bimolecular nucleophilic substitution
Download: Image, Marvin File

Step 1. A vDNA phosphate activates an adjacent water molecule which performs a nucleophilic attack on another phosphate of the vDNA. This cleaves the vDNA, leaving an exposed 3'-hydroxyl.

Step 2. A vDNA phosphate deprotonates the 3'-hydroxyl, creating a nucleophile.

Step 3. The deprotonated 3' oxygen of vDNA performs nucleophilic attack on the phosphate, adding itself to the hDNA.

Products.

Contributors

Marko Babić, Noa Marson, Antonio Ribeiro