NMR studies of molecular interactions involved in the type III secretion system, SUMOylation, and the RNA binding protein HuR

Abstract

Proteins are one of the most intriguing, versatile, and complex macromolecules in living systems. Proteins rarely function independently and perform their activities through a multitude of interactions with other proteins or molecules. Such molecular interactions are fundamental to almost all biological processes and their disruption is often associated with cellular irregularities and disease states. It is therefore of immense importance and interest to identify and characterize the binding interfaces of these biologically relevant molecular interactions. NMR spectroscopy is unparalleled in its ability to monitor molecular interactions in solution at atomic level over a wide range of affinities. In this body of work, various NMR methods were successfully used to study and characterize the protein-ligand interactions of three discrete systems: the bacterial type III secretion system (T3SS), the post-translational SUMO modification system, and post-transcriptional regulation by the RNA binding protein HuR. The T3SS is a macromolecular structure assembled by many Gram-negative bacterial pathogens, such as, Shigella flexneri, Yersinia pestis, and multi-drug resistant Pseudomonas aeruginosa, to cause infectious diseases. The structural component of the T3SS, the needle apparatus, consists of a base, a needle, a tip complex, and a translocon. Because the needle apparatus is exposed on the bacterial surface, present only among the pathogens, and essential for the virulence, disrupting the needle assembly is an attractive strategy for the development of novel anti-virulence drugs. However, this approach demands a detailed understanding of the protein-protein interactions involved in the needle assembly. Here, NMR methods were used to characterize the protein-protein interactions that are important in the assembly of the tip-translocon complex in the Shigella T3SS. Additionally, fragment-based screening was performed to identify small molecule binders of the tip proteins from Yersinia and Pseudomonas T3SS. The hits were subsequently validated and characterized using NMR spectroscopy. Our results provide novel insight into the assembly of the needle apparatus and reveal the first small molecules that directly bind to the tip proteins of Yersinia and Pseudomonas T3SS. Small ubiquitin-like modifier (SUMO) conjugation is a reversible post-translational modification process that can modulate biochemical and cell biological functions of the target protein substrate. SUMO E3 ligases are the enzymes that carry out the final step in SUMO conjugation pathway and facilitate the transfer of SUMO to the target protein. Prior to this work, SUMO binding by the PIAS family of E3 ligases was poorly understood. Here, using NMR spectroscopy, the protein-protein interactions involved in the SUMO-PIASy binding were characterized. The NMR binding studies surprisingly uncovered a novel SUMO-interacting motif in the E3 ligase PIASy, which was found to be essential for the ligase activity of PIASy. The RNA binding protein HuR binds to adenine- and uridine-rich elements (AREs) located in the untranslated region of target mRNAs, regulating their stability and translation. HuR-ARE interaction contributes to carcinogenesis by stabilization of oncogenic mRNAs. HuR is overexpressed in a broad range of human cancers and associated with poor clinical outcome. In vitro and in vivo studies have demonstrated that HuR is an attractive therapeutic target. Drugs that disrupt HuR-ARE interaction could potentially inhibit cancer growth and persistence. Here, a fungal natural product azaphilone (AZA-9) was identified as a novel disruptor of HuR-ARE interaction using fluorescence polarization based screening. AZA-9 binding to HuR was validated and characterized by NMR methods. Results of NMR studies suggest that AZA-9 binds in the ARE-binding cleft of HuR, and thus competitively inhibits the HuR-ARE interaction. The work presented in this dissertation illustrates the strength of NMR spectroscopy and its wide applicability as a tool to characterize and understand diverse interactions of proteins.