MMGBSA as a tool to understand the binding affinities of filamin-peptide interactions.

Filamins (FLN) are large dimeric proteins that cross-link actin and work as important scaffolds in human cells. FLNs consist of an N-terminal actin-binding domain followed by 24 immunoglobulin-like domains (FLN1-24). FLN domains are divided into four subgroups based on their amino acid sequences. One of these subgroups, including domains 4, 9, 12, 17, 19, 21, and 23, shares a similar ligand-binding site between the ß strands C and D. Several proteins, such as integrins ß2 and ß7, glycoprotein Ibα (GPIbα), and migfilin, have been shown to bind to this site. Here, we computationally estimated the binding free energies of filamin A (FLNa) subunits with bound peptides using the molecular mechanics-generalized Born surface area (MMGBSA) method. The obtained computational results correlated well with the experimental data, and they ranked efficiently both the binding of one ligand to all used FLNa-domains and the binding of all used ligands to FLNa21. Furthermore, the steered molecular dynamics (SMD) simulations pinpointed the binding hot spots for these complexes. These results demonstrate that molecular dynamics combined with free energy calculations are applicable to estimating the energetics of protein-protein interactions and can be used to direct the development of novel FLN function modulators.

Molecular mechanism of T-cell protein tyrosine phosphatase (TCPTP) activation by mitoxantrone.

T-cell protein tyrosine phosphatase (TCPTP) is a ubiquitously expressed non-receptor protein tyrosine phosphatase. It is involved in the negative regulation of many cellular signaling pathways. Thus, activation of TCPTP could have important therapeutic applications in diseases such as cancer and inflammation. We have previously shown that the α-cytoplasmic tail of integrin α1ß1 directly binds and activates TCPTP. In addition, we have identified in a large-scale high-throughput screen six small molecules that activate TCPTP. These small molecule activators include mitoxantrone and spermidine. In this study, we have investigated the molecular mechanism behind agonist-induced TCPTP activation. By combining several molecular modeling and biochemical techniques, we demonstrate that α1-peptide and mitoxantrone activate TCPTP via direct binding to the catalytic domain, whereas spermidine does not interact with the catalytic domain of TCPTP in vitro. Furthermore, we have identified a hydrophobic groove surrounded by negatively charged residues on the surface of TCPTP as a putative binding site for the α1-peptide and mitoxantrone. Importantly, these data have allowed us to identify a new molecule that binds to TCPTP, but interestingly cannot activate its phosphatase activity. Accordingly, we describe here mechanism of TCPTP activation by mitoxantrone, the cytoplasmic tail of α1-integrin, and a mitoxantrone-like molecule at the atomic level. These data provide invaluable insight into the development of novel TCPTP activators, and may facilitate the rational discovery of small-molecule cancer therapeutics.