Interactions of SUMO proteins

A large number of proteins have shown ability to bind to SUMO (Small Ubiquitin like Modifier) proteins through a short conserved motif called SIM (SUMO Interacting Motif). The work presented here shows that the different SUMO isoforms interact with the hydrophobic core of the SIM by forming an intermolecular b-sheet with the b2 strand of SUMO. This interaction is crucial for SUMO binding, and is modulated by interactions between SUMO and the amino acids flanking the core of the SIM. The SIM can be phosphorylated, providing a possibility for regulating the strength of SUMO binding in the lifetime of a protein. Furthermore, a concentration threshold effect is observed in the binding of the unphosphorylated SIM of PIAS (Protein inhibitor of activated STAT) to SUMO. The dependency on the amino acids flanking the hydrophobic core is stronger in binding to SUMO1 than to SUMO2, providing a mechanism for SUMO isoform discrimination

Fluorophore Labeled Kinase Detects Ligands That Bind within the MAPK Insert of p38α Kinase

The vast majority of small molecules known to modulate kinase activity, target the highly conserved ATP-pocket. Consequently, such ligands are often less specific and in case of inhibitors, this leads to the inhibition of multiple kinases. Thus, selective modulation of kinase function remains a major hurdle. One of the next great challenges in kinase research is the identification of ligands which bind to less conserved sites and target the non-catalytic functions of protein kinases. However, approaches that allow for the unambiguous identification of molecules that bind to these less conserved sites are few in number. We have previously reported the use of fluorescent labels in kinases (FLiK) to develop direct kinase binding assays that exclusively detect ligands which stabilize inactive (DFG-out) kinase conformations. Here, we present the successful application of the FLiK approach to develop a high-throughput binding assay capable of directly monitoring ligand binding to a remote site within the MAPK insert of p38α mitogen-activated protein kinase (MAPK). Guided by the crystal structure of an initially identified hit molecule in complex with p38α, we developed a tight binding ligand which may serve as an ideal starting point for further investigations of the biological function of the MAPK insert in regulating the p38α signaling pathway

Specification of SUMO1- and SUMO2-interacting motifs

SUMO proteins are ubiquitin-related modifiers implicated in the regulation of gene transcription, cell cycle, DNA repair, and protein localization. The molecular mechanisms by which the sumoylation of target proteins regulates diverse cellular functions remain poorly understood. Here we report isolation and characterization of SUMO1- and SUMO2-binding motifs. Using yeast two-hybrid system, bioinformatics, and NMR spectroscopy we define a common SUMO-interacting motif (SIM) and map its binding surfaces on SUMO1 and SUMO2. This motif forms a beta-strand that could bind in parallel or antiparallel orientation to the beta2-strand of SUMO due to the environment of the hydrophobic core. A negative charge imposed by a stretch of neighboring acidic amino acids and/or phosphorylated serine residues determines its specificity in binding to distinct SUMO paralogues and can modulate the spatial orientation of SUMO-SIM interactions

High-Throughput Screening To Identify Inhibitors Which Stabilize Inactive Kinase Conformations in p38α

Small molecule kinase inhibitors are an attractive means to modulate kinase activities in medicinal chemistry and chemical biology research. In the physiological setting of a cell, kinase function is orchestrated by a plethora of regulatory processes involving the structural transition of kinases between inactive and enzymatically competent conformations and vice versa. The development of novel kinase inhibitors is mainly fostered by high-throughput screening initiatives where the small molecule perturbation of the phosphorylation reaction is measured to identify inhibitors. Such setups require enzymatically active kinase preparations and present a risk of solely identifying classical ATP-competitive Type I inhibitors. Here we report the high-throughput screening of a library of similar to 35000 small organic molecules with an assay system that utilizes enzymatically inactive human p38 alpha MAP kinase to detect stabilizers of the pharmacologically more desirable DFG-out conformation. We used protein X-ray crystallography to characterize the binding mode of hit compounds and reveal structural features which explain how these ligands stabilize and/or induce the DFG-out conformation. Lastly, we show that although some of the hit compounds were confirmed by protein X-ray crystallography, they were not detected in classic phosphorylation assays, thus validating the unique sensitivity of the assay system used in this study and highlighting the potential of screening with inactive kinase preparations

Data Collection and Refinement Statistics for p38α crystal structures in complex with <b>1b</b> and <b>8b</b>^a.

a<p>For all p38α complex structures, diffraction data from one crystal was used to determine the structure. Values in parenthesis are for the highest resolution shell.</p