SmyD1 is a cardiac-and muscle-specific histone methyltransferase that methylates histone H3 at lysine 4 and regulates gene transcription in early heart development. The unique domain structure characterized by a "split" SET domain, a conserved MYND zinc finger, and a novel C-terminal domain (CTD) distinguishes SmyD1 from other SET domain containing methyltransferases. Here we report the crystal structure of full-length SmyD1 in complex with the cofactor analog sinefungin at 2.3 Å . The structure reveals that SmyD1 folds into a wrench-shaped structure with two thick "grips" separated by a large, deep concave opening. Importantly, our structural and functional analysis suggests that SmyD1 appears to be regulated by an autoinhibition mechanism, and that unusually spacious target lysine-access channel and the presence of the CTD domain both negatively contribute to the regulation of this cardiovascularly relevant methyltransferase. Furthermore, our structure also provides a structural basis for the interaction between SmyD1 and cardiac transcription factor skNAC, and suggests that the MYND domain may primarily serve as a protein interaction module and cooperate SmyD1 with skNAC to regulate cardiomyocyte growth and maturation. Overall, our data provide novel insights into the mechanism of SmyD1 regulation, which would be helpful in further understanding the role of this protein in heart development and cardiovascular diseases.
We suggest that GDNF exerts its anti-allodynic effect via somatostatinergic mechanisms. Our observations suggest new approaches for treating nerve injury that may prove useful in preventing delayed complications that contribute to long-term debility.
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.
The goal of this study is to reveal the biochemical function of SET and MYND domain containing protein 3 (SmyD3), a histone methyltransferase. Histone methylation is a vital epigenetic mechanism that controls many fundamental processes such as transcription activation and repression. SmyD3 methylates histone H3 at lysine 4 and was found greatly upregulated in several cancers including colon, liver, and breast cancers. However, the biochemical function of SMYD3 remains poorly understood. To gain insight into the function of SmyD3, we overexpressed mouse SmyD3 in E.coli cells. We found that SmyD3 elutes from a gel filtration column as a monomeric protein. Circular Dichroism (CD) spectroscopy showed that SmyD3 has a large amount of alpha‐helical structures. Thermal denaturation experiments showed that SmyD3 undergoes an unfolding transition with a melting temperature of ~ 45 °C. Our future studies will focus on that: first, understanding of how DNA binding to N‐terminal MYND domain impacts the second and tertiary structures of SmyD3 using Isothermal Titration Calorimetry and fluorescence spectroscopy; second, examination of the effects of substrate histone peptide binding on the protein structure and stability; finally, investigation of the function of the conserved C‐terminal domain, especially focusing on its contribution to protein integrity.
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