Elucidating Substrate and Inhibitor Binding Sites on the Surface of GSK-3β and the Refinement of a Competitive Inhibitor

A molecular understanding of substrate recognition of protein kinases provides an important basis for the development of substrate competitive inhibitors. Here, we explored substrate recognition and competitive inhibition of glycogen synthase kinase (GSK)-3 beta using molecular and computational tools. In previous work, we described Gln89 and Asn95 within GSK-3 beta as important substrates binding sites. Here, we show that the cavity bordered by loop 89-QDKRFICN-95, located in the vicinity of the GSK-3 beta catalytic core, is a promiscuous substrate binding subsite. Mutations within this segment highlighted Phe93 as an additional essential contact residue for substrates' recognition. However, unlike Gln89 and Asn95, Phe93 was also important for the binding of our previously described substrate competitive inhibitor, L803 [KEAPPAPPQS(p)P], and its cell-permeable variant L803-mts. The effects of the substitution of charged or polar residues within L803 further suggested that binding to GSK-3 beta is governed by hydrophobic interactions. Our computational model of GSK-3 beta bound to L803 was in agreement with the experimental data. It revealed L803 binding with a hydrophobic surface patch and identified interactions between Pro8 (L803) and Phe93 (GSK-3 beta). Computational modeling of new L803 variants predicted that inhibition would be strengthened by adding contacts with Phe93 or by increasing the hydrophobic content of the peptide. Indeed, the newly designed L803 variants showed improved inhibition. Our study identified different and overlapping elements in GSK-3 beta substrate and inhibitor recognition and provides a novel example for model-based rational design of substrate competitive inhibitors for GSK-3. (C) 2011 Elsevier Ltd. All rights reserved.