12 research outputs found
Characterizing Protein-Protein Interactions with the Fragment Molecular Orbital Method
Proteins are vital components of living systems, serving as building blocks, molecular machines, enzymes, receptors, ion channels, sensors, and transporters. Protein-protein interactions (PPIs) are a key part of their function. There are more than 645,000 reported disease-relevant PPIs in the human interactome, but drugs have been developed for only 2% of these targets. The advances in PPI-focused drug discovery are highly dependent on the availability of structural data and accurate computational tools for analysis of this data. Quantum mechanical approaches are often too expensive computationally, but the fragment molecular orbital (FMO) method offers an excellent solution that combines accuracy, speed and the ability to reveal key interactions that would otherwise be hard to detect. FMO provides essential information for PPI drug discovery, namely, identification of key interactions formed between residues of two proteins, including their strength (in kcal/mol) and their chemical nature (electrostatic or hydrophobic). In this chapter, we have demonstrated how three different FMO-based approaches (pair interaction energy analysis (PIE analysis), subsystem analysis (SA) and analysis of protein residue networks (PRNs)) have been applied to study PPI in three protein-protein complexes
Small molecules, peptides and natural products:getting a grip on 14-3-3 protein-protein modulation
\u3cp\u3eOne of the proteins that is found in a diverse range of eukaryotic protein-protein interactions is the adaptor protein 14-3-3. As 14-3-3 is a hub protein with very diverse interactions, it is a good model to study various protein-protein interactions. A wide range of classes of molecules, peptides, small molecules or natural products, has been used to modify the protein interactions, providing both stabilization or inhibition of the interactions of 14-3-3 with its binding partners. The first protein crystal structures were solved in 1995 and gave molecular insights for further research. The plant analog of 14-3-3 binds to a plant plasma membrane H(+)-ATPase and this protein complex is stabilized by the fungal phytotoxin fusicoccin A. The knowledge gained from the process in plants was transferred to and applied in human models to find stabilizers or inhibitors of 14-3-3 interaction in human cellular pathways. \u3c/p\u3
A thermodynamic model for multivalency in 14-3-3 protein-protein interactions
\u3cp\u3eProtein-protein interactions (PPIs) are at the core of molecular control over cellular function. Multivalency in PPI formation, such as via proteins with multiple binding sites and different valencies, requires fundamental understanding to address correlated challenges in pathologies and drug development. Thermodynamic binding models are needed to provide frameworks for describing multivalent PPIs. We established a model based on ditopic host-guest systems featuring the effective molarity, a hallmark property of multivalency, as a prime parameter governing the intramolecular binding in divalent interactions. By way of illustration, we study the interaction of the bivalent 14-3-3 protein scaffold with both the nonavalent CFTR and the hexavalent LRRK2 proteins, determining the underlying thermodynamics and providing insights into the role of individual sites in the context of the multivalent platform. Fitting of binding data reveals enthalpy-entropy correlation in both systems. Simulations of speciations for the entire phosphorylated protein domains reveal that the CFTR protein preferably binds to 14-3-3 by combinations including the strongest binding site pS768, but that other binding sites take over when this site is eliminated, leading to only a minor decrease in total affinity for 14-3-3. For LRRK2, two binding sites dominate the complex formation with 14-3-3, but the distantly located pS1444 site also plays a role in complex formation. Thermodynamic modeling of these multivalent PPIs allowed analyzing and predicting the effects of individual sites regarding their modulation via, for example, (de)phosphorylation or small-molecule targeting. The results specifically bring forward the potential of PPI stabilization, as an entry for drug discovery for multivalent PPIs.\u3c/p\u3
Structural interface between LRRK2 and 14-3-3 protein
Binding of 14-3-3 proteins to leucine-rich repeat protein kinase 2 (LRRK2) is known to be impaired by many Parkinson's disease (PD)-relevant mutations. Abrogation of this interaction is connected to enhanced LRRK2 kinase activity, which in turn is implicated in increased ubiquitination of LRRK2, accumulation of LRRK2 into inclusion bodies and reduction in neurite length. Hence, the interaction between 14-3-3 and LRRK2 is of significant interest as a possible drug target for the treatment of PD. However, LRRK2 possesses multiple sites that, upon phosphorylation, can bind to 14-3-3, thus rendering the interaction relatively complex. Using biochemical assays and crystal structures, we characterize the multivalent interaction between these two proteins
Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR
\u3cp\u3eCystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein-protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3-CFTR interface might offer an approach for cystic fibrosis therapeutics.\u3c/p\u3