Alternative Strategies To Inhibit Lysine Methyltransferases And Deubiquitinases In Human Cancers

X-ray crystallography is the gold standard method for imagining macromolecules to atomic resolution. Three dimensional data is central to understanding the molecular mechanism how DNA, RNA and proteins function in biological events. Structural insights into these events provide a molecular window to visualize how biological molecules influence human health. Visualizing the architecture of these molecules set the stage for rational and selective drug design. The following dissertation utilizes biochemical and biophysical tools, including X-ray crystallography, to shed light on poorly understood mechanisms related to SMYD2 activity and regulation, USP10 architecture and function, and PDZ-RhoGEF dimerization. SMYD2 is one member of the SET and MYND domain-containing protein (SMYD) family known to play key roles in cardiac function and development, innate immunity and tumorigenesis. While the molecular pathways involved in these events have been fairly described, the molecular mechanism of substrate recognition and bilobal changes have not. In this dissertation, I review the structure and function of SMYD protein family. In addition, I demonstrate SMYD2 and SMYD3 can exist in open and closed conformations based on X-ray crystallography, small angle X-ray scattering, and molecular dynamic simulations data. Lastly, I revealed a novel binding site in SMYD2 that appears to be the first recognition site for SMYD methylation clients. USP10 is one member of the ubiquitin-specific protease family important for DNA repair and apoptosis by recycling cytosolic p53. However, in the mutant p53 environment, USP10 serves as an oncogene; thereby promoting mutant p53-dependent cancer cell growth. Additional studies found related USP10 oncogene roles in other cancers. Unfortunately the biochemistry and structure of USP10 hasn’t been thoroughly explored. My dissertation aims to understand the biochemistry and architecture of the catalytic domain of USP10 along with reported USP10 inhibitors which would be valuable for future studies to probe USP10 function and inhibition. PDZ-RhoGEF is one member of the Rho guanine exchange factors (RhoGEF) family important for modulating Rho activity and actin-based cytoskeleton remodeling. PDZ-RhoGEF possesses a PDZ domain known for complexing with the cytoplasmic tail of Plexin B serving as modulator for downstream signaling factors. In our study, we found PDZ-RhoGEF complexes with the Interleukin-8 chemokine receptor, CXCR2. This novel interaction hasn’t been reported before, and in my dissertation, I solved the crystal structure of PDZ-RhoGEF in complex with the PDZ motif of CXCR2. Unexpectedly, we identified a disulfide bond linking two PDZ-RhoGEF molecules. This disulfide bond was previously reported to be important for promoting PDZ-ligand binding between PDZ-RhoGEF and Plexin B2 peptides. Here, I describe the architecture of the disulfide-linked PDZ domain of PDZ-RhoGEF in complex with two CXCR2 PDZ-motifs

Molecular Dynamics Simulation Reveals Correlated Inter-Lobe Motion in Protein Lysine Methyltransferase SMYD2

SMYD proteins are an exciting field of study as they are linked to many types of cancer- related pathways. Cardiac and skeletal muscle development and function also depend on SMYD proteins opening a possible avenue for cardiac-related treatment. Previous crystal structure studies have revealed that this special class of protein lysine methyltransferases have a bilobal structure, and an open–closed motion may regulate substrate specificity. Here we use the molecular dynamics simulation to investigate the still-poorly-understood SMYD2 dynamics. Cross-correlation analysis reveals that SMYD2 exhibits a negative cor- related inter-lobe motion. Principle component analysis suggests that this correlated dynamic is contributed to by a twisting motion of the C-lobe with respect to the N-lobe and a clamshell-like motion between the lobes. Dynamical network analysis defines possible allo- steric paths for the correlated dynamics. There are nine communities in the dynamical net- work with six in the N-lobe and three in the C-lobe, and the communication between the lobes is mediated by a lobe-bridging β hairpin. This study provides insight into the dynam- ical nature of SMYD2 and could facilitate better understanding of SMYD2 substrate specificity

New Open Conformation of SMYD3 Implicates Conformational Selection and Allostery

SMYD3 plays a key role in cancer cell viability, adhesion, migration and invasion. SMYD3 promotes formation of inducible regulatory T cells and is involved in reducing autoimmunity. However, the nearly “closed” substrate-binding site and poor in vitro H3K4 methyltransferase activity have obscured further understanding of this oncogenically related protein. Here we reveal that SMYD3 can adopt an “open” conformation using molecular dynamics simulation and small-angle X-ray scattering. This ligand-binding-capable open state is related to the crystal structure-like closed state by a striking clamshell-like inter-lobe dynamics. The two states are characterized by many distinct structural and dynamical differences and the conformational transition pathway is mediated by a reversible twisting motion of the C-terminal domain (CTD). The spontaneous transition from the closed to open states suggests two possible, mutually non-exclusive models for SMYD3 functional regulation and the conformational selection mechanism and allostery may regulate the catalytic or ligand binding competence of SMYD3. This study provides an immediate clue to the puzzling role of SMYD3 in epigenetic gene regulation

Structure and Function of SET and MYND Domain-Containing Proteins

SET (Suppressor of variegation, Enhancer of Zeste, Trithorax) and MYND (Myeloid-Nervy-DEAF1) domain-containing proteins (SMYD) have been found to methylate a variety of histone and non-histone targets which contribute to their various roles in cell regulation including chromatin remodeling, transcription, signal transduction, and cell cycle control. During early development, SMYD proteins are believed to act as an epigenetic regulator for myogenesis and cardiomyocyte differentiation as they are abundantly expressed in cardiac and skeletal muscle. SMYD proteins are also of therapeutic interest due to the growing list of carcinomas and cardiovascular diseases linked to SMYD overexpression or dysfunction making them a putative target for drug intervention. This review will examine the biological relevance and gather all of the current structural data of SMYD proteins

Protein crystallization: Eluding the bottleneck of X-ray crystallography

To date, X-ray crystallography remains the gold standard for the determination of macromolecular structure and protein substrate interactions. However, the unpredictability of obtaining a protein crystal remains the limiting factor and continues to be the bottleneck in determining protein structures. A vast amount of research has been conducted in order to circumvent this issue with limited success. No single method has proven to guarantee the crystallization of all proteins. However, techniques using antibody fragments, lipids, carrier proteins, and even mutagenesis of crystal contacts have been implemented to increase the odds of obtaining a crystal with adequate diffraction. In addition, we review a new technique using the scaffolding ability of PDZ domains to facilitate nucleation and crystal lattice formation. Although in its infancy, such technology may be a valuable asset and another method in the crystallography toolbox to further the chances of crystallizing problematic proteins

RESEARCH ARTICLE Molecular Dynamics Simulation Reveals Correlated Inter-Lobe Motion in Protein

SMYD proteins are an exciting field of study as they are linked to many types of cancer-related pathways. Cardiac and skeletal muscle development and function also depend on SMYD proteins opening a possible avenue for cardiac-related treatment. Previous crystal structure studies have revealed that this special class of protein lysine methyltransferases have a bilobal structure, and an open–closed motion may regulate substrate specificity. Here we use the molecular dynamics simulation to investigate the still-poorly-understood SMYD2 dynamics. Cross-correlation analysis reveals that SMYD2 exhibits a negative cor-related inter-lobe motion. Principle component analysis suggests that this correlated dynamic is contributed to by a twisting motion of the C-lobe with respect to the N-lobe and a clamshell-like motion between the lobes. Dynamical network analysis defines possible allo-steric paths for the correlated dynamics. There are nine communities in the dynamical net-work with six in the N-lobe and three in the C-lobe, and the communication between the lobes is mediated by a lobe-bridging β hairpin. This study provides insight into the dynam-ical nature of SMYD2 and could facilitate better understanding of SMYD2 substrate specificity

Dynamical network analysis.

<p>(A) SMYD2 dynamical network. The network is colored according to communities. Points in the network are nodes, and lines between the nodes represent edges. The thicker lines depict the stronger edges or stronger correlations. Critical nodes are colored in purple. (B) Optimal and suboptimal paths between Y311 and G46. The optimal path is colored in red and suboptimal paths in blue. The edge thickness is weighted by the number of suboptimal paths crossing the edge. Residues along the optimal path are labeled.</p

SMYD2 dynamics.

<p>(A) Backbone RMSD during the simulation. RMSD was calculated relative to the crystal structure. (B) Root mean square fluctuation (RMSF) of Cα atoms during the simulation (black line). Red line depicts the RMSF values converted from crystallographic B-factors. The inset depicts the distribution of the simulation RMSF. (C) Ribbon diagram of SMYD2 structure at 2 ns. The structure is colored according to the simulation RMSF. Color scale from blue to red depicts low to high atomic fluctuations. Secondary structures, α-helices and β-strands are labeled and numbered according to their position in the sequence. SAH is represented by sticks and zinc ions by purple spheres. (D) Cross-correlation map of the trajectory. Blue indicates a negative correlation between residue fluctuations, and red depicts a positive correlation. Lobe and domain structures of SMYD2 are indicated on the top of the map. (E) Visualization of residue–residue cross-correlations. SMYD2 is depicted by green coils. Blue and red lines indicate negative and positive correlated motions. (F) Inter-residue distance deviation map. Color scale from blue to magenta depicts small to large distance deviations. (G) Distance fluctuation of Y311–G46 during the simulation. Color bars depict the conformer clustering results obtained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145758#pone.0145758.g002" target="_blank">Fig 2</a>.</p