LPP binds to the PDZ domains of Scrib via its C-terminal tail
Since the pACT2-mScrib prey-construct contained four PDZ domains, and since PDZ domains are one of the most commonly found protein-protein interaction domains in organisms from bacteria to humans [23], it was most likely that Scrib would bind to LPP via its PDZ domains. The LPP-bait that was used to screen the library was pGBT9-LPPWT containing the third LIM domain and carboxy-terminus of human LPP. Although PDZ domains have been shown to bind LIM domains [24], binding to carboxy-terminal peptides appears to be the typical mode of interaction [25]. The common structure of PDZ domains comprises six β strands (βA-βF) and two α helices (αA and αB), which fold in an overall six-stranded β sandwich [25]. The binding specificity of PDZ domains is critically determined by the interaction of the first residue of helix α B (position αB1) and the side chain of the -2 residue of the C-terminal ligand. This forms the basis for PDZ classification [25]. Since all four PDZ domains of Scrib contain a histidine at position αB1, they are classified as class I PDZ domains. Therefore, based on what has been demonstrated for this subclass of PDZ domains [25,26], the carboxy-terminal sequence of Scrib target proteins is predicted to require a hydrophobic amino acid (h) at the 0 (carboxy-terminus) position, and a serine (S) or threonine (T) at the -2 position.
Theoretically, the carboxy-terminus of the LPP protein, being -STDL, thus completely fulfils the criteria for binding to the PDZ domains of Scrib. To evaluate these predictions experimentally and to demonstrate that the binding of LPP to Scrib is specific, we performed yeast two-hybrid experiments using pGBT9-LPPWT as well as pGBT9-LPPS609A, pGBT9-LPPT610A, pGBT9-LPPD611A and pGBT9-LPPL612A as bait. The last four baits are identical to pGBT9-LPPWT except for a point mutation to alanine, respectively introduced at serine609 (-3 position), threonine610 (-2 position), aspartate611 (-1 position) and leucine612 (position 0). As prey, we used pACT2-mScrib. As summarized in Table 1, this alanine-scan mutant analysis identified threonine610 (-2 position) and leucine612 (0 position) of LPP as being essential for binding to Scrib indicating a PDZ domain-mediated specific interaction between Scrib and the carboxy-terminus of LPP.
Additional yeast two-hybrid analysis showed that LPP did not interact with Erbin, PICK1, PSD-95, Syntenin, CASK, or AF6 PDZ domains, as summarized in Table 2.
Table 2  Interaction of LPP with PDZ domains of proteins different from Scrib  Yeast cells, cotransformed with pGBT9-LPPWT and a pACT2-prey as indicated, were selected on medium lacking Leu and Trp, and either containing His or no His with 5 mM 3-AT.
+ indicates growth of yeast transformants; - indicates no growth of yeast transformants.