X-ray crystallography remains a powerful method to gain atomistic insights into the catalytic and regulatory functions of RNA molecules. However, the technique requires the preparation of diffraction-quality crystals. This is often a resource- and time-consuming venture because RNA crystallization is hindered by the conformational heterogeneity of RNA, as well as the limited opportunities for stereospecific intermolecular interactions between RNA molecules. The limited success at crystallization explains in part the smaller number of RNA-only structures in the Protein Data Bank. Several approaches have been developed to aid the formation of well-ordered RNA crystals. The majority of these are construct-engineering techniques that aim to introduce crystal contacts to favor the formation of well-diffracting crystals. A typical example is the insertion of tetraloop–tetraloop receptor pairs into non-essential RNA segments to promote intermolecular association. Other methods of promoting crystallization involve chaperones and crystallization-friendly molecules that increase RNA stability and improve crystal packing. In this review, we discuss the various techniques that have been successfully used to facilitate crystal packing of RNA molecules, recent advances in construct engineering, and directions for future research in this vital aspect of RNA crystallography.

Glycogen Synthase Kinase 3-beta (GSK3β) is a critical regulator of several cellular pathways involved in neuroplasticity and is a potential target for neurotherapeutic development in the treatment of neuropsychiatric and neurodegenerative diseases. The majority of efforts to develop inhibitors of GSK3β have been focused on developing small molecule inhibitors that compete with ATP through direct interaction with the ATP binding site. This strategy has presented selectivity challenges due to the evolutionary conservation of this domain within the kinome. The Disrupted in Schizophrenia (DISC1) protein, has previously been shown to bind and inhibit GSK3β activity. Here, we report the characterization of a 44-mer peptide derived from human DISC1 (hDISCtide) that is sufficient to both bind and inhibit GSK3β in a non-competitive mode that is distinct from classical ATP competitive inhibitors. Based on multiple independent biochemical and biophysical assays, we propose that hDISCtide interacts at two distinct regions of GSK3β: an inhibitory region that partially overlaps with the binding site of FRATide, a well-known GSK3b binding peptide, and a specific binding region that is unique to hDISCtide. Taken together, our findings present a novel avenue for developing a peptide-based selective inhibitor of GSK3b.

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Glycogen Synthase Kinase 3-beta (GSK3β) is a critical regulator of several cellular pathways involved in neuroplasticity and is a potential target for neurotherapeutic development in the treatment of neuropsychiatric and neurodegenerative diseases. The majority of efforts to develop inhibitors of GSK3β have been focused on developing small molecule inhibitors that compete with ATP through direct interaction with the ATP binding site. This strategy has presented selectivity challenges due to the evolutionary conservation of this domain within the kinome. The Disrupted in Schizophrenia (DISC1) protein, has previously been shown to bind and inhibit GSK3β activity. Here, we report the characterization of a 44-mer peptide derived from human DISC1 (hDISCtide) that is sufficient to both bind and inhibit GSK3β in a non-competitive mode that is distinct from classical ATP competitive inhibitors. Based on multiple independent biochemical and biophysical assays, we propose that hDISCtide interacts at two distinct regions of GSK3β: an inhibitory region that partially overlaps with the binding site of FRATide, a well-known GSK3b binding peptide, and a specific binding region that is unique to hDISCtide. Taken together, our findings present a novel avenue for developing a peptide-based selective inhibitor of GSK3b.

Disrupted in Schizophrenia 1(DISC1) is a candidate risk gene in a number of major mental illnesses, e.g. depression, bipolar disorder and schizophrenia. DISC1 is a scaffold protein that interacts with a myriad of proteins, forming a large protein-protein-interaction network that coordinates various stages of brain development. Our lab is interested to understand how the structure of DISC1 facilitates its function in brain development. One of DISC1's interactors is the enzyme glycogen synthase kinase 3β (GSK-3β). As a target for lithium, GSK3 is itself implicated in bipolar disorder. Through physical interactions, DISC1 inhibits GSK3 's function in the canonical Wnt/β-catenin signalling pathway, which controls the proliferation of neural progenitors. The full length DISC1 protein is composed of 854 amino acids. The most potent GSK3 inhibitory region has been mapped to a short region in the N-terminus (residue 195-238). This 44 amino acid region (DISC1-44mer) can inhibit and bind GSK3 in in vitro experiments. To understand the mechanism of how DISC1 inhibits GSK3 , we are pursuing structural studies of GSK3 in complex with different truncations of DISC1. We will present our progress towards the structural studies of this important complex that is relevant to the pathophysiology of psychiatric diseases.