Nicholas Spellmon

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.

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 correlated 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 allosteric paths for the correlated dynamics. There are nine communities in the dynamical network 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 dynamical nature of SMYD2 and could facilitate better understanding of SMYD2 substrate specificity.

Structural basis of PDZ-mediated chemokine receptor CXCR2 scaffolding by guanine nucleotide exchange factor PDZ-RhoGEF.

The CXC chemokine receptor 2 (CXCR2) is a G protein coupled receptor mediating interleukin-8 chemotactic signaling and plays an important role in neutrophil mobility and tumor migration. However, efficient CXCR2 signaling requires PDZ domain-mediated scaffolding of signaling complexes at the plasma membrane and functional coupling of the signaling to specific downstream signaling pathways, in which only one PDZ protein has been characterized to interact with CXCR2. Here, we identified five novel CXCR2-binding PDZ-containing proteins, among which PDZ-RhoGEF is of particular interest because this PDZ and RGS-containing guanine nucleotide exchange factor (GEF) is also involved in cell signaling and mobility. To reveal the molecular basis of the interaction, we solved the crystal structure of PDZ-RhoGEF PDZ domain in complex with the CXCR2 C-terminal PDZ binding motif. The structure reveals that the PDZ-CXCR2 binding specificity is achieved by numerous hydrogen bonds and hydrophobic contacts with the last four CXCR2 residues contributing to specific interactions. Structural comparison of CXCR2-binding PDZ domains and PDZ-RhoGEF PDZ bound with different ligands reveals PDZ- and ligand-specific interactions that may underlie the ability of promiscuous CXCR2 binding by different PDZ domains and PDZ binding promiscuity. The structure also reveals an unexpected asymmetric disulfide bond-linked PDZ dimer that allows simultaneous parallel binding of CXCR2 to two PDZ domains. This study provides not only the structural basis for PDZ-mediated CXCR2-PDZ-RhoGEF interaction, but also a new mode of PDZ dimerization, which both could prove valuable in understanding signaling complex scaffolding in CXCR2 signaling and coupling to specific signaling pathways.

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.

Purification of Histone Lysine Methyltransferase SMYD2 and Co-Crystallization with a Target Peptide from Estrogen Receptor α

Methylation of estrogen receptor α by the histone lysine methyltransferase SMYD2 regulates ERα chromatin recruitment and its target gene expression. This protocol describes SMYD2 purification and crystallization of SMYD2 in complex with an ERα peptide. Recombinant SMYD2 is overexpressed in Escherichia coli cells. After release from the cells by French Press, SMYD2 is purified to apparent homogeneity with multiple chromatography methods. Nickel affinity column purifies SMYD2 based on specific interaction of its 6×His tag with the bead-immobilized nickel ions. Desalting column is used for protein buffer exchange. Gel filtration column purifies SMYD2 based on molecular size. The entire purification process is monitored and analyzed by SDS-polyacrylamide gel electrophoresis. Crystallization of SMYD2 is performed with the hanging drop vapor diffusion method. Crystals of the SMYD2-ERα peptide complex are obtained by microseeding using seeding bead. This method can give rise to large size of crystals which are suitable for X-ray diffraction data collection. X-ray crystallographic study of the SMYD2-ERα complex can provide structural insight into posttranslational regulation of ERα signaling.

SMYD proteins in immunity: dawning of a new era

In the past decade, the SMYD protein family has gradually become a center of research, thanking for its essential role in heart and muscle development and cancer development. However, because of such a role, the research scope for the SMYD protein family has been fairly stereotyped, its focus largely restricted to muscle and cancer. Initial studies of SMYD1 and SMYD3 were responsible for these biased research directions. SMYD1 was originally identified as a myogenic factor and regulated cardiomyocyte maturation and proper formation of the right ventricle [1]. SMYD3 was initially identified as an oncogene and regulated expression of cell-cycle genes in colorectal and hepatocellular carcinomas [2]. These findings have clearly indicated an essential role of SMYD1 and SMYD3 in cardiac muscle development and tumorigenesis respectively. Because of this, the research directions in SMYD proteins have been strongly influenced by these two studies, and much subsequent research has biased towards muscle and cancer.

SAXS analysis of a soluble cytosolic NgBR construct including extracellular and transmembrane domains

The Nogo-B receptor (NgBR) is involved in oncogenic Ras signaling through directly binding to farnesylated Ras. It recruits farnesylated Ras to the non-lipid-raft membrane for interaction with downstream effectors. However, the cytosolic domain of NgBR itself is only partially folded. The lack of several conserved secondary structural elements makes this domain unlikely to form a complete farnesyl binding pocket. We find that inclusion of the extracellular and transmembrane domains that contain additional conserved residues to the cytosolic region results in a well folded protein with a similar size and shape to the E.coli cis-isoprenyl transferase (UPPs). Small Angle X-ray Scattering (SAXS) analysis reveals the radius of gyration (Rg) of our NgBR construct to be 18.2 Å with a maximum particle dimension (Dmax) of 61.0 Å. Ab initio shape modeling returns a globular molecular envelope with an estimated molecular weight of 23.0 kD closely correlated with the calculated molecular weight. Both Kratky plot and pair distribution function of NgBR scattering reveal a bell shaped peak which is characteristic of a single globularly folded protein. In addition, circular dichroism (CD) analysis reveals that our construct has the secondary structure contents similar to the UPPs. However, this result does not agree with the currently accepted topological orientation of NgBR which might partition this construct into three separate domains. This discrepancy suggests another possible NgBR topology and lends insight into a potential molecular basis of how NgBR facilitates farnesylated Ras recruitment.

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.

Allosterically targeting EGFR drug-resistance gatekeeper mutations

Lung cancer is by far the leading cause of cancer-related death with nearly 1.7 million deaths worldwide in 2016. The most common type of lung cancer is non-small cell lung cancer (NSCLC) accounting for 80% of lung cancer cases.