Protein-ligand structure determination with the NMR molecular replacement tool, NMR2.

We recently reported on a new method called NMR Molecular Replacement that efficiently derives the structure of a protein-ligand complex at the interaction site. The method was successfully applied to high and low affinity complexes covering ligands from peptides to small molecules. The algorithm used in the NMR Molecular Replacement program has until now not been described in detail. Here, we present a complete description of the NMR Molecular Replacement implementation as well as several new features that further reduce the time required for structure elucidation.

Proteome-wide analysis of phospho-regulated PDZ domain interactions.

A key function of reversible protein phosphorylation is to regulate protein-protein interactions, many of which involve short linear motifs (3-12 amino acids). Motif-based interactions are difficult to capture because of their often low-to-moderate affinities. Here, we describe phosphomimetic proteomic peptide-phage display, a powerful method for simultaneously finding motif-based interaction and pinpointing phosphorylation switches. We computationally designed an oligonucleotide library encoding human C-terminal peptides containing known or predicted Ser/Thr phosphosites and phosphomimetic variants thereof. We incorporated these oligonucleotides into a phage library and screened the PDZ (PSD-95/Dlg/ZO-1) domains of Scribble and DLG1 for interactions potentially enabled or disabled by ligand phosphorylation. We identified known and novel binders and characterized selected interactions through microscale thermophoresis, isothermal titration calorimetry, and NMR We uncover site-specific phospho-regulation of PDZ domain interactions, provide a structural framework for how PDZ domains accomplish phosphopeptide binding, and discuss ligand phosphorylation as a switching mechanism of PDZ domain interactions. The approach is readily scalable and can be used to explore the potential phospho-regulation of motif-based interactions on a large scale.

Fast NMR-Based Determination of the 3D Structure of the Binding Site of Protein-Ligand Complexes with Weak Affinity Binders.

In early drug discovery approaches, screening hits are often weak affinity binders that are difficult to characterize in structural detail, particularly towards obtaining the 3D structure of protein-ligand complexes at atomic resolution. NMR is the outstanding technique to tackle such problems, yet suffers from a tedious structure calculation process. NMR2 was recently developed to alleviate the laborious element of routine NMR structure calculation procedures and provides the structural information at protein-ligand interaction sites orders of magnitude faster than standard procedures. The NMR2 method was extended to weak binders and applied to the oncoproteins HDM2 and MDMX. The structure of the MDMX-SJ212 complex is reported with a Kd of approximately 0.7 µm; the complex structure of HDM2 with the mm affinity ligand #845 exhibits a new scaffold.

The description of protein internal motions aids selection of ligand binding poses by the INPHARMA method.

Protein internal motions influence observables of NMR experiments. The effect of internal motions occurring at the sub-nanosecond timescale can be described by NMR order parameters. Here, we report that the use of order parameters derived from Molecular Dynamics (MD) simulations of two holo-structures of Protein Kinase A increase the discrimination power of INPHARMA, an NMR based methodology that selects docked ligand orientations by maximizing the correlation of back-calculated to experimental data. By including internal motion in the back-calculation of the INPHARMA transfer, we obtain a more realistic description of the system, which better represents the experimental data. Furthermore, we propose a set of generic order parameters, derived from MD simulations of globular proteins, which can be used in the back-calculation of INPHARMA NOEs for any protein-ligand complex, thus by-passing the need of obtaining system-specific order parameters for new protein-ligand complexes.

An NMR-based scoring function improves the accuracy of binding pose predictions by docking by two orders of magnitude.

Low-affinity ligands can be efficiently optimized into high-affinity drug leads by structure based drug design when atomic-resolution structural information on the protein/ligand complexes is available. In this work we show that the use of a few, easily obtainable, experimental restraints improves the accuracy of the docking experiments by two orders of magnitude. The experimental data are measured in nuclear magnetic resonance spectra and consist of protein-mediated NOEs between two competitively binding ligands. The methodology can be widely applied as the data are readily obtained for low-affinity ligands in the presence of non-labelled receptor at low concentration. The experimental inter-ligand NOEs are efficiently used to filter and rank complex model structures that have been pre-selected by docking protocols. This approach dramatically reduces the degeneracy and inaccuracy of the chosen model in docking experiments, is robust with respect to inaccuracy of the structural model used to represent the free receptor and is suitable for high-throughput docking campaigns.