WWP2

WWP2 Scheme without C2 Domain

WWP2 Scheme WW2-2,3-linker-HECT

Full-length WWP2 consists of an amino-terminal C2 domain, four WW domains (labeled WW1-WW4), and a carboxy-terminal HECT domain. WW domains are one of the smallest studied protein modules, consisting of less than 40 amino acids, fold into three-stranded beta-sheets. They are characterized by two highly conserved positioned 20-22 amino acids apart and a high affinity for proline-rich motifs. Linkers of varying length and secondary structure connect the C2 domain to WW1, WW1 to WW2, WW2 to WW3, WW3 to WW4, and WW4 to the HECT domain. A chimeric of WWP2 consisting of the , the WW2-WW3 linker (), and the HECT domain is shown on the right.

The HECT domain is divided into two lobes (labeled N and C). The N-lobe serves as a binding site for the E2-ubiquitin complex and includes an exosite for non-covalent ubiquitin binding relevant to autoinhibition while the C-lobe contains an active site with a catalytic Cys residue to which the substrate ubiquitin molecule can covalently attach. A connects the N and C lobes of the HECT domain and allows for flexible movement of the lobes as ubiquitin is transferred from the E2-ubiquitin complex docked on the N-lobe to the ubiquitin binding site in the C-lobe. A transthiolation reaction in this active site results in a thioester bond between the ubiquitin and a Cys residue. The HECT domain is in an inverse T shape when inactive (autoinhibited) and takes on an L shape when active. WW2 interaction with HECT is mediated by the six C terminal residues.

The alpha-helical 2,3-linker is subject to tyrosine phosphorylation at either end of the linker at residues . Chen et. al. have shown that the phosphorylation of Tyr369 allows for allosteric activation by ubiquitination at the exosite in the N-lobe, while phosphorylation of Tyr392 residue leads to a destabilization of the T conformation of the HECT domain.

Function

Studies suggest that the non-catalytic C2 domain and WW domains play a regulatory role in the catalytic activity of the HECT domain. Genetic deletions of WW domains and linkers have shown increased autoubiquitination activity. The WW domains and linkers play a role in altering protein conformation, with the 2,3-linker playing the greatest role.

This linker plays an autoinhibitory role. The ground state conformation of the protein has the 2,3-linker close to the N-lobe, while WW1 and WW2 domains block the N lobe’s ubiquitin-binding site. Upon phosphorylation the 2,3-linker changes conformation, moving further from the N lobe, allowing the protein to bind ubiquitin in the E2 and N lobe binding sites. This binding will further open the protein up to bind possible substrates, like PTEN.

Interactions between the hinge and the 2,3-linker appear to restrict flexibility of the C-lobe which is necessary for ubiquitin transferase activity. This interaction further locks the protein into the T position. A current model of WWP2 activation by Chen et. al. proposes the following: (1) autoinhibited WWP2 gets phosphorylated at the 2,3-linker, (2) loosening of the 2,3-linker interactions with the hinge and ubiquitin binding exosite allow ubiquitin to bind in the C-lobe, (3) the target substrate protein binds with specifity towards the WW domains, (4) the substrate protein gets ubiquitinated, and (5) the 2,3-linker gets dephosphorylated, allowing WWP2 to return to the autoinhibited ground state conformation.